Chapter33,EssentialstoGlycobiology(3rdedition)P-typeLectinsChapter44,EssentialstoGlycobiology(3rdedition)GeneticDisordersofGlycanDegradationArticle,LysosomalStorageDiseases:FromPathophysiologytoTherapy

Chapter33,EssentialstoGlycobiology(3rdedition)

P-typeLectins

CHAPTER 33. P-type Lectins

Ajit Varki and Stuart Kornfeld

Lysosomes are intracellular membrane-bound organelles that turnover and degrade many types of macromolecules, via the action of lysosomal enzymes (also called acid hydrolases because of the low pH characteristic of lysosomes). These enzymes are synthesized in the endoplasmic reticulum (ER) on membrane-bound ribosomes and traverse the ER-Golgi pathway along with other newly synthesized proteins. At the terminal Golgi compartment they are segregated from other glycoproteins and selectively delivered to lysosomes. In most “higher” animal cells, this specialized trafficking is achieved primarily through the recognition of N-glycans containing mannose 6-phosphate by “P-type” lectins. As the first clear-cut example of a biological role for glycans on mammalian glycoproteins and the first demonstrated link between glycoprotein biosynthesis and human disease, the interesting history of its discovery is described in some detail. More recent data on other proteins with “P-type” lectin domains are also mentioned.

HISTORICAL BACKGROUND

I-Cell Disease and the Common Recognition Marker of Lysosomal Enzymes Early studies of human genetic storage disorders by Elizabeth Neufeld indicated a failure to degrade cellular components, which therefore accumulated in lysosomes (Chapter 44). Soluble corrective factors from normal cells could reverse these defects when added to the culture media. These factors were later identified as lysosomal enzymes that were found to be deficient in patients with storage disorder diseases. These enzymes, secreted in small quantities by normal cells in culture, or by cells from patients with a different “complementary” defect (Figure 33.1), exist in two forms: a high-uptake form, recognized by saturable, high-affinity receptors that could correct the defect in deficient cells, and an inactive low-uptake form that could not correct the defect. Fibroblasts from patients with a rare genetic disease exhibiting prominent inclusion bodies in cultured cells (therefore termed I-cell disease) were found to be deficient in almost all lysosomal enzymes. Interestingly, all of these enzymes are actually synthesized by I-cells, but are mostly secreted into the medium, instead of being retained in lysosomes. Neufeld showed that while I-cells incorporate the high-uptake enzymes secreted by normal cells, enzymes secreted by I-cells are not taken up by other cells (Figure 33.1). She therefore proposed that I-cell disease results from a failure to add a common recognition marker to lysosomal enzymes. This marker was assumed to be responsible for the proper trafficking of newly synthesized enzymes to lysosomes in normal cells. Because the high-uptake property was destroyed by strong periodate treatment, which oxidizes vicinal hydroxyl groups in sugars, it was predicted that the recognition marker was a glycan.

Discovery of the M6P Recognition Marker High uptake of lysosomal enzymes was next found by William Sly to be blocked by mannose 6-phosphate (M6P) and its stereoisomer fructose 1-phosphate, but not by other sugar phosphates. Treatment of lysosomal enzymes with alkaline phosphatase also abolished high-uptake activity. By this time, the general pathway for N-glycan

processing had been defined (see Chapter 9). Because oligomannosyl N-glycans are rich in mannose residues, these were predicted to be phosphorylated on lysosomal enzymes. Indeed, the M6P moiety detected on oligomannosyl N-glycans was released by endo-β-N-acetylglucosaminidase H (endo H) from high-uptake forms of lysosomal enzymes, confirming the hypothesis. Surprisingly, the groups of Stuart Kornfeld and Kurt von Figura then found that a portion of the M6P moieties were “blocked” by α-linked N-acetylglucosamine residues attached to the phosphate residue, creating a phosphodiester.

Enzymatic Mechanism for Generation of the M6P Recognition Marker Comparison of glycans with phosphodiesters and phosphomonoesters predicted that the metabolic precursor of the M6P determinant was a phosphodiester and that phosphorylation was mediated not by an ATP-dependent kinase, but by a UDP-GlcNAc-dependent GlcNAc-1-phosphotransferase. This was proven using a double-labeled donor and Golgi extracts: Reactants: Uridine-P-32P-[6-3H]GlcNAc + Manα1-(N-glycan)-lysosomal enzyme Products: Uridine-P + [6-3H]GlcNAcα1-32P-6-Manα1-(N-glycan)-lysosomal enzyme Another Golgi enzyme was shown to remove the outer N-acetylglucosamine residue and uncover the M6P moiety. Pulse-chase studies confirmed the order of events, documenting that multiple glycans on a given lysosomal enzyme could acquire one or two phosphate residues and that removal of some mannose residues on the N-glycan by processing Golgi mannosidases was also required (Figure 33.2). In most cell types, the phosphate is eventually lost from the M6P, presumably after exposure to acid phosphatase in lysosomes. Thus, the overall biochemical pathway is as follows:

Uridine−P−P-GlcNAc+Manα1−(N−glycan)−Lysosomal enzyme ↓ Phosphotransferase

Uridine−P+GlcNAcα1−P−6−Manα1−(N−glycan)−Lysosomal enzyme ↓ Phosphodiester glycosidase

GlcNAc+P−6−Manα1−(N−glycan)−Lysosomal enzyme ↓ Lysosomal phosphatase

P+Manα1−(N−glycan)−Lysosomal enzyme The genes encoding the first two enzymes that mediate these reactions were then purified, cloned and characterized. UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (GlcNAc-P-T) is an α2β2γ2 complex encoded by two genes. The α and β subunits are encoded by the GNPTAB gene, whose 1256 amino acid type-III transmembrane product undergoes proteolysis in the Golgi to give rise to the two subunits, while the 305 amino acid γ subunit is encoded by the GNPTG gene (Figure 33.3). The α/β subunits contain the catalytic function of the enzyme (Stealth domains) as well as elements (Notch1,2 and DMAP) that mediate binding to protein determinants present on the lysosomal enzymes. The γ subunit facilitates the phosphorylation of a subset of the lysosomal enzymes. It contains an M6P receptor homology (MRH) domain that is thought to bind oligomannosyl N-glycans on the lysosomal enzymes and present them to the catalytic site for phosphorylation, and a DMAP (DNA methyltransferase-

associated protein) recognition domain. The second enzyme, α-N-acetylglucosamine-1-phosphodiester glycosidase encoded by the NAGPA gene, is a 272-kD complex of four identical 68-kD subunits, arranged as two disulfide-linked homodimers. Unlike other Golgi enzymes, this is a type-I membrane-spanning glycoprotein with its amino terminus in the lumen of the Golgi.

The oligomannosyl N-glycans of lysosomal enzymes are identical to those of many other glycoproteins passing through the ER-Golgi pathway. Thus, specific recognition by GlcNAc-P-T is crucial to achieve selective trafficking. This recognition is not explained by any similarities in the primary polypeptide sequences of lysosomal enzymes. Indeed, denatured lysosomal enzymes lose their specialized GlcNAc-P-T acceptor activity, indicating that features of secondary or tertiary structure are critical for recognition. Two complementary approaches have been used to define elements of this recognition marker. In loss-of-function studies, various amino acids of the lysosomal enzyme have been replaced with alanine, with the effect on phosphorylation determined. In gain-of-function experiments, residues of the lysosomal protease cathepsin D have been substituted into the homologous secretory protease glycopepsinogen. These studies revealed that selected lysine residues have a critical role in the interaction with GlcNAc-P-T. In fact, as few as two lysines in the correct orientation to each other and to an N-glycan can serve as minimal elements of the recognition domain. However, additional amino acid residues function to enhance the interaction with GlcNAc-P-T. In some instances (e.g., cathepsin D), the enzyme may contain a very extended determinant, or perhaps, more than one recognition domain.

The GlcNAc phosphodiester glycosidase that catalyzes the exposure of the M6P recognition marker is found primarily in the trans-Golgi network (TGN), and it cycles between this compartment and the plasma membrane. Thus, uncovering the recognition marker is a late event in the Golgi apparatus, occurring just before loading of the enzymes onto the mannose 6-phosphate receptors (MPRs).

Enzymatic Basis for I-Cell Disease and Pseudo-Hurler Polydystrophy Analysis of fibroblasts from patients with I-cell disease (also called mucolipidosis-

II; ML-II) revealed a deficiency in GlcNAc-P-T enzyme activity. A milder variant called pseudo-Hurler polydystrophy (mucolipidosis-III; ML-III) showed a less severe deficiency of enzyme activity. Metabolic radiolabeling of fibroblasts corroborated the failure to phosphorylate mannose residues in these diseases, and asymptomatic obligate heterozygotes showed a partial deficiency, with slightly elevated levels of serum lysosomal enzymes. Mutations of various types in the two GlcNAc-P-T genes have since been detected in all examined patients with ML-II and -III, indicating that deficiency of this enzyme is the primary genetic cause of the disorder.

COMMON FEATURES OF P-TYPE LECTINS (M6P RECEPTORS) The first candidate (~275-kD) receptor for the M6P recognition marker was isolated by affinity chromatography and was found to bind M6P in the absence of cations (CI-MPR, cation-independent mannose 6-phoshate receptor). Certain cells deficient in this receptor still showed M6P-inhibitable binding of lysosomal enzymes, leading to the discovery of a second MPR of approximately 45 kD, which required divalent cations for optimal binding (CD-MPR, cation-dependent MPR). Both receptors bind with highest affinity to glycans carrying two M6P residues (Figure 33.2, structure C) and poorly to

molecules bearing GlcNAc-P-Man phosphodiesters (Figure 33.2, structures A and B). Binding to molecules carrying one M6P (Figure 33.2, structure D) is intermediate in affinity. Removal of outer mannose residues by processing Golgi mannosidases enhances binding. Genes encoding both MPRs have been cloned and extensively characterized. Both are type I transmembrane glycoproteins with large extracytoplasmic domains, transmembrane regions and carboxy-terminal cytoplasmic domains. The CI-MPR has 15 contiguous repetitive units of approximately 145 amino acids with partial identity to one another. The CD-MPR has a single extracellular domain, showing homology to the repeating domains of the CI-MPR. Together with conservation of certain intron–exon boundaries, this homology suggests that the two genes evolved from a common ancestor. On the basis of their sequence relationships and unique binding properties to M6P, the two MPRs have been formally classified as P-type lectins. Structural homologs of the MPRs are present in yeast and Drosophila, but these proteins lack M6P-binding ability.

The CD-MPR exists mainly as a dimer, with each monomer binding one M6P residue. However, monomeric and tetrameric forms of the CD-MPR exist, and the equilibrium between forms is affected by temperature, pH, and the presence of ligands. The CI-MPR also seems to be a dimer in the membrane, although it readily dissociates upon solubilization. Two of its repeating units (3 and 9) bind M6P phosphomonoesters with high affinity, a third (5) binds M6P phosphodiesters with low affinity, and a fourth (15) binds both M6P phosphomonoesters and diesters with equal affinity. Mutagenesis studies have identified the specific residues of these receptors involved in M6P binding, and crystal structures of several M6P binding domains have been obtained in a complex with M6P. The CD-MPR has been crystallized as a dimer, with each monomer folded into a nine-stranded flattened β-barrel that has a striking resemblance to the protein folds in avidin (Figure 33.4). The distance between the two ligand-binding sites of the dimer provides a good explanation for the differences in binding affinity shown by the CD-MPR toward various lysosomal enzymes. The crystal structure of the amino-terminal 432 residues of the CI-MPR, encompassing domains 1–3 (domain 3 is one of the M6P-binding domains), has also been solved. Each domain exhibits a topology similar to that of the CD-MPR, and the three domains assemble into a compact structure that provides insight into the arrangement of the entire extracellular region of the CI-MPR. The proposed model does not position the two M6P-binding domains (3 and 9) sufficiently close to bind a single, diphosphorylated N-glycan. This suggests that the high-affinity binding of this N-glycan is due to the spanning of binding sites located on different CI-MPR dimers. An interesting possibility is that the receptor is dynamic, with the spacing between the two M6P-binding sites being flexible, to enhance interactions with lysosomal enzymes containing phosphorylated glycans at various positions on their protein backbones.

INDUCED GENETIC DEFECTS IN THE MPRS Targeted disruption of the CD-MPR gene in mice is associated with normal or only slightly elevated levels of lysosomal enzymes in the circulation and an otherwise grossly normal phenotype. However, thymocytes or primary cultured fibroblasts from such mice show an increase in the amount of phosphorylated lysosomal enzymes secreted into the medium. This indicates that there are mechanisms that compensate for the deficiency in vivo. Intravenous injection of inhibitors of other glycan-specific receptors capable of

mediating endocytosis (e.g., the mannose receptor of macrophages and the asialoglycoprotein receptor of hepatocytes; see Chapter 34) give rise to a marked increase in lysosomal enzymes in the serum of the deficient mice. Thus, such receptors are likely part of the compensatory mechanisms in vivo. Like fibroblasts that lack only CD-MPR, fibroblasts that lack only CI-MPR have a partial impairment in sorting. Fibroblasts from embryos that lack both receptors show a massive missorting of multiple lysosomal enzymes. Thus, both receptors are required for efficient intracellular targeting of lysosomal enzymes. Comparison of lysosomal enzymes secreted by the different cell types indicates that the two receptors may interact preferentially with different subgroups of enzymes. Thus, the structural heterogeneity of the M6P recognition marker within a single lysosomal enzyme and between different enzymes is one explanation for the evolution of two MPRs with complementary binding properties: that is, to provide an efficient but varied targeting of lysosomal proteins in different cell types or tissues. In the final analysis, what initially appeared to be a precise lock-and-key mechanism turns out to be a far more complex and flexible system, with functionally useful biological flexibility.

SUBCELLULAR TRAFFICKING OF THE MPRS At steady state, MPRs are concentrated in the TGN and late endosomes, but they cycle constitutively between these organelles, early (sorting) endosomes, recycling endosomes, and the plasma membrane (Figure 33.5). MPRs avoid delivery to lysosomes, where they would be degraded. This trafficking is governed by a number of short amino acid sorting signals in the cytoplasmic tails of the receptors. The TGN is the site where newly synthesized lysosomal enzymes bind to MPRs that are then collected into clathrin-coated pits and budded into clathrin-coated vesicles for delivery to the early endosome. This process involves interaction of the MPRs with two types of coat proteins: the GGAs (Golgi-localized, γ-ear-containing, ADP-ribosylation factor binding) and AP1 (adapter protein 1). In addition to binding MPRs, the coat proteins recruit clathrin for the assembly of clathrin-coated vesicles. Following delivery to early endosomes, lysosomal enzymes are released from MPRs as the endosomes mature to late endosomes and the pH decreases. Late endosomes then undergo dynamic fusion/fission with lysosomes, allowing selective transfer of lysosomal enzymes to the lysosomes and leaving the MPRs behind in subdomains of the late endosomes. These MPRs may then either return to the TGN or move to the plasma membrane, where internalization via clathrin-coated pits occurs, mediated by the coat protein AP2. There are several pathways for the MPRs to be returned to the TGN from the various endosomal compartments, although the relative importance of the different pathways is unclear.

The CI-MPR mediates uptake of phosphorylated lysosomal enzymes – Implications for Enzyme Replacement Therapy The original experiments of Neufeld demonstrated that a portion of newly synthesized lysosomal enzyme molecules are secreted into the medium, but may be recaptured by the same cell or by adjacent cells expressing cell-surface CI-MPRs (Figure 33.1). Enzyme molecules that bind to such cell-surface MPRs are endocytosed via clathrin-coated pits and vesicles, eventually reaching the same late endosomal compartments where newly synthesized molecules arrive from the Golgi. This secretion-recapture

pathway is a minor one in most cells, but plays a critical role in enzyme replacement therapy.

As described in Chapter 44, there are many genetic disorders in glycan degradation that result from decreased activity of a given lysosomal enzyme. Some of these enzymes that are targeted to lysosomes via the M6P pathway have been prepared in large quantities as recombinant soluble proteins and used in enzyme replacement therapy. To date the benefits have been variable but less than optimal. There are a number of potential reasons for this. First, some of the preparations may not contain the physiologic complement of the phosphomannosyl recognition marker. It is reasonable to suggest that a greater M6P content may improve the efficacy of enzyme replacement in these patients. This has been shown to be the case in mouse and dog model systems. However, even with fully phosphorylated enzymes there may be obstacles that are difficult to overcome. For example, some cell types in the body may not express adequate levels of the CI-MPR on their surfaces to endocytose sufficient enzyme to restore normal lysosomal function. Also, the organ that is most seriously affected in many of these diseases (the brain) is inaccessible because of the blood–brain barrier. This is an area where further studies are needed.

EVOLUTIONARY ORIGINS OF THE M6P RECOGNITION SYSTEM Although the MPR pathway has a major role in vertebrate lysosomal enzyme trafficking, its contribution in invertebrate systems is not prominent. Lysosomal enzymes are targeted in organisms such as Saccharomyces, Trypanosoma, and Dictyostelium, without the aid of identifiable MPRs. The slime mold Dictyostelium discoideum produces a novel methylphosphomannose structure on some of its lysosomal enzymes that can be recognized in vitro by the mammalian CI-MPR (not the CD-MPR). However, despite the presence of a GlcNAc-P-T that recognizes α1-2-linked mannose residues, the enzyme does not specifically recognize lysosomal enzymes, and no receptor for M6P has been found in this organism. The protozoan Acanthamoeba produces a GlcNAc-P-T that specifically recognizes lysosomal enzymes. However, this organism lacks a gene that could encode an “uncovering” enzyme, and so would not be expected to form M6P available to an MPR. Although some additional lower organisms do show evidence for an uncovering enzyme, no MPR activity has yet been found. The evolutionary divergence point at which the complete MPR system became established remains to be defined.

THE CI-MPR BINDS MANY OTHER LIGANDS Although originally discovered as a receptor for lysosomal enzyme trafficking, the CI-MPR turns out to be a remarkably multifunctional molecule. It binds IGF-II with high affinity even though this polypeptide lacks M6P residues. Many studies have explored potential interactions between these two disparate ligands. Although these two ligands bind to distinct sites on the CI-MPR receptor, there are conflicting reports regarding synergistic or antagonistic interactions between the two activities. Regardless, it appears that the CI-MPR acts primarily as a general sink for excess IGF-II in the extra-cellular fluid, carrying it to the lysosome for degradation, and reducing the amount available to bind to the IGF-I receptor. Several reports indicate that binding of IGF-II to the CI-MPR regulates motility and growth in some cell types. It has also been found that the CI-MPR binds retinoic acid with high affinity at a site that is distinct from those for M6P and IGF-

II. This binding of retinoic acid seems to enhance the primary functions of the CI-MPR receptor, and the biological consequence appears to be the suppression of cell proliferation and/or induction of apoptosis. The significance of this unexpected observation is still being explored. Other ligands include urokinase-type plasminogen activator receptor (uPAR) and plasminogen. There are also some unexplained changes in CI-MPR expression in relation to malignancy. Loss of heterozygosity at the CI-MPR locus occurs in dysplastic liver lesions and in hepatocellular carcinomas associated with the high-risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in about 50% of these tumors, which also seem to frequently develop from clonal expansions of phenotypically normal, CI-MPR-mutated hepatocytes. Thus, the CI-MPR fulfills many of the classic criteria to be classified as a liver “tumor-suppressor” gene.

SIGNIFICANCE OF M6P ON NONLYSOSOMAL PROTEINS Interestingly, M6P-containing N-glycans have been found on a variety of nonlysosomal proteins. Some are hydrolytic enzymes that seem to have taken on a predominantly secretory route, for example, uteroferrin and DNase I. In the first case, the failure of removal of the blocking N-acetylglucosamine residues may be the cause for secretion. With DNase I, the native level of phosphorylation simply appears to be low. M6P has been found on the transforming growth factor β (TGF-β) precursor and the phosphate is lost from the mature form. It appears that M6P may serve to target the precursor to CI-MPR for activation. Other nonlysosomal proteins reported to carry M6P include proliferin, CREG (cellular repressor of E1A-stimulated genes), LIF (leukemia inhibitory factor), and thyroglobulin. In the last case, M6P-containing N-glycans are suggested as a mechanism to target the protein for degradation and release of thyroid hormones. But one should not necessarily assume that M6P-containing N-glycans on all of these proteins are involved in intracellular trafficking. Just as phosphorylation of serine residues has diverse biological roles, M6P might be used for more than one purpose in a complex multicellular organism. Herpes simplex virus (HSV) and varicella zoster virus (VZV) glycoproteins have also been shown to carry N-glycans with M6P. In these cases, M6P is on complex N-glycans, suggesting that it originates from a distinct biosynthetic pathway. Regardless of its mode of synthesis, interaction of cell-free VZV with CI-MPR at the cell surface is required for viral entry into endosomes. Interestingly, intracellular CI-MPR can also divert newly synthesized enveloped VZV to late endosomes where the virions are inactivated before exocytosis. This is suggested as the mechanism by which this successful parasite limits immediate excessive spread and avoids killing the host or possibly, a form of host defense. Biopsies of VZV-infected human skin showed that CI-MPR expression is lost in maturing superficial epidermal cells, preventing diversion of VZV to endosomes and allowing constitutive secretion of infectious VZV. These data implicate CI-MPR in the complex biology of VZV infection.

OTHER PATHWAYS FOR TRAFFICKING LYSOSOMAL ENZYMES Although the M6P recognition marker has a crucial role in trafficking newly synthesized lysosomal enzymes to vertebrate lysosomes, alternate mechanisms exist in some cell types. In patients with I-cell disease some cells and tissues, such as the liver and circulating granulocytes, have essentially normal levels of enzymes. B-lymphoblast lines derived from these patients also do not show the complete phenotype of enzyme

deficiency seen in fibroblasts. One possible explanation is that the M6P pathway for trafficking of lysosomal enzymes is a specialized form of targeting, superimposed on some other basic mechanisms that more primitive organisms use, some of which remain undefined. In this regard, two lysosomal enzymes, acid phosphatase and β-glucocerebrosidase, are not at all affected in their distribution in I-cell disease fibroblasts. With acid phosphatase, the enzyme is synthesized initially as a membrane-bound protein, and once in the lysosome, it is proteolytically cleaved to generate the mature soluble form. Glucocerebrosidase is targeted to lysosomes independently of the MPR pathway by forming a complex with LIMP-2, a lysosomal membrane protein that contains a targeting signal in its cytoplasmic tail. Likewise, other integral membrane proteins of the lysosome use similar targeting motifs in their cytoplasmic tails to traffic to the lysosome.

Mannose 6-phosphate Receptor Homology (MRH) Domain-Containing Proteins Function in the Secretory Pathway. A number of proteins have now been identified as P-type lectins due to the presence of M6P receptor homology (MRH) domains. These MRH domains contain the residues that bind mannose, but lack the residues that bind phosphate. In several instances the proteins have been shown to bind to oligomannosyl structures. Most of these proteins reside in the ER where they function as lectins in the ER quality control pathway. Interestingly, the gamma subunit of GlcNAc-P-T has an MRH domain that plays a critical role in the phosphorylation of a subset of lysosomal enzymes. The precise biologic role of these MRH domains remains to be elucidated.

ACKNOWLEDGMENTS

The authors appreciate helpful comments and suggestions from Sally Riewe Boyd, Sumit Rai and Patience Sanderson.

FURTHER READING

Fratantoni JC, Hall CW, Neufeld EF. 1968. Hurler and Hunter syndromes: mutual correction of the defect in cultured fibroblasts. Science 162: 570–572.

Hickman S, Neufeld EF. 1972. A hypothesis for I-cell disease: defective hydrolases that do not enter lysosomes. Biochem. Biophys. Res. Commun. 49: 992–999.

Kaplan A, Achord DT, Sly WS. 1977. Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc. Natl. Acad. Sci. U S A 74: 2026–2030.

Tabas I, Kornfeld S. 1980. Biosynthetic intermediates of beta-glucuronidase contain high mannose oligosaccharides with blocked phosphate residues. J. Biol. Chem. 255: 6633–6639.

Hasilik A, Klein U, Waheed A, Strecker G, von Figura K. 1980. Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups. Proc. Natl. Acad. Sci. U S A 77: 7074–7078.

Reitman ML, Varki A, Kornfeld S. 1981. Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5’-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity. J. Clin. Invest. 67: 1574–1579.

Varki A, Kornfeld S. 1983. The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor. J. Biol. Chem. 258: 2808–2818.

von Figura K, Hasilik A. 1986. Lysosomal enzymes and their receptors. Annu Rev Biochem 55: 167–193.

Kornfeld S. 1986. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest 77: 1–6.

Kornfeld S, Mellman I. 1989. The biogenesis of lysosomes. Annu Rev Cell Biol 5: 483–525.

Munier-Lehmann H, Mauxion F, Hoflack B. 1996. Function of the two mannose 6-phosphate receptors in lysosomal enzyme transport. Biochem Soc Trans 24: 133–136.

Ghosh P, Dahms NM, Kornfeld S. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol 4: 202–212.

Bonifacino JS, Traub LM. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 72: 395–447.

Vogel P, Payne BJ, Read R, Lee WS, Gelfman CM, Kornfeld S. Comparative pathology of murine mucolipidosis types II and IIIC. 2009. Vet Pathol 46: 313-324.

Braulke T, Pohl S, Storch S. 2008. Molecular analysis of the GlcNac-1-phosphotransferase. J Inherit Metab Dis 31: 253–257.

Dahms NM, Olson LJ, Kim JJ. 2008. Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18: 664–678.

Castonguay AC, Olson LJ, Dahms NM. 2011. Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway. Biochim Biophys Acta 1810:

815–826.

van Meel E, Lee WS, Liu L, Qian Y, Doray B, Kornfeld S. 2016. Multiple domains of GlcNAc-1-phosphotransferase mediate recognition of lysosomal enzymes. J. Biol. Chem. 291: 8295–8307.

Figure Legends

FIGURE 33.1. Historical background regarding cross-correction of lysosomal enzyme deficiencies in cultured cells. Small amounts of high-uptake lysosomal enzymes secreted by normal fibroblasts (thin arrows) were found to be taken up by fibroblasts from a patient with a genetic lack of a single lysosomal enzyme, correcting that deficiency. In contrast, I-cell disease fibroblasts secrete large amounts of multiple lysosomal enzymes of the low-uptake variety (thick arrows). The latter forms cannot correct lysosomal enzyme deficiencies in other cells. However, I-cells retain the capability to accept high-uptake enzymes secreted by other cells (thin arrows). Uptake was blocked by the addition of mannose 6-phosphate (M6P) to the media.

FIGURE 33.2. Pathways for biosynthesis of N-glycans bearing the mannose 6-phosphate (M6P) recognition marker. Following early N-glycan processing (Chapter 9), a single GlcNAc phosphodiester is added to the N-glycans of lysosomal enzymes on one of three mannose (Man) residues on the arm with the α1–6 Man linked to the core mannose (structure A). A second phosphodiester can then be added to the other side of the N-glycan (structure B) or onto other mannose residues (alternate sites for phosphorylation are denoted by asterisks). Removal of the outer N-acetylglucosamine residues and further processing of mannose residues give structures C and D. Further mannose removal is restricted by the phosphomonoesters. Thus, C and D represent only two of several possible structures bearing one or two phosphomonoesters. Glycans that are not phosphorylated become typical complex or hybrid N-glycans. Some hybrid N-glycans with M6P are also found (structure E). Binding studies of these N-glycans with purified mannose 6-phosphate receptors (MPRs) have shown the relative affinities indicated in the figure ([++] strong; [+] moderate; [+/−] weak; [−] no binding).

FIGURE 33.3. GlcNAc-P-T is an α2β2γ2 hexamer encoded by two genes. The GNPTAB gene encodes a catalytically inactive type 3 transmembrane precursor that undergoes a proteolytic cleavage between Lys-928 and Asp-929 in the Golgi to generate the active α and β subunits. These subunits mediate the catalytic function via the Stealth domains (lime-green color). These domains are similar to bacterial genes that function as sugar-phosphate transferases in the biosynthesis of cell wall polysaccharides. The Notch modules (N1,N2) and the DMAP domain participate in lysosomal enzyme recognition. The GNPTG gene encodes the γ subunit that contains a DMAP lysosomal enzyme binding domain and an MRH domain that is postulated to bind oligomannosyl N-glycans on lysosomal enzymes and present them to the catalytic site of the transferase. SS, signal sequence.

N1

DMAP N2

γ

DMAP SS

FIGURE 33.4. Ribbon diagram of the bovine cation-dependent M6P receptor (CD-MPR). Shown are the two monomers (magenta and cyan ribbons) of the dimer as well as the ligand M6P (gold ball-and-stick model). (Modified, with permission, from Roberts et al. 1998. Cell 93: 639–648, ©Cell Press.)

FIGURE 33.5. Subcellular trafficking pathways of glycoproteins, lysosomal enzymes, and MPRs. Newly synthesized glycoproteins originating from the rough ER pass through the Golgi stacks and are then sorted to various destinations. Along this route, lysosomal

enzymes are recognized by GlcNAc-P-T and phosphorylated in the intermediate compartment (cis-Golgi network) and then acted upon by the uncovering enzyme (phosphodiester glycosidase) in the TGN. Beyond the TGN, trafficking of lysosomal enzymes is primarily mediated by the MPRs through early endosomes to late endosomes, in which their lysosomal enzyme cargo is released. Smaller amounts of lysosomal enzymes can escape capture by MPRs and be secreted into the extracellular fluid. Lysosomal enzymes can also re-enter the cell by binding to cell-surface MPRs and subsequent endocytosis. Once in the endocytic pathway, internalized lysosomal enzymes can intermingle with those following the biosynthetic route, as depicted. (Open arrowheads) Pathways general to many nonlysosomal glycoproteins; (red arrows) specific itineraries of lysosomal enzymes; (green arrows) pathways of MPRs. Additional pathways for MPRs include recycling from the early endosome to the cell surface and back to the TGN, and from the recycling endosome and the late endosome, following release of their lysosomal enzyme cargo.

Chapter44,EssentialstoGlycobiology(3rdedition)

GeneticDisordersofGlycanDegradation

CHAPTER 44. Genetic Disorders of Glycan Degradation

Hudson H. Freeze, Taroh Kinoshita and Ronald L. Schnaar This chapter explores degradation and turnover of glycans in lysosomes, especially with respect to human genetic disorders, with representative glycans illustrating features unique to different pathways. Degradation of oligomannosyl N-glycans removed from misfolded, newly synthesized glycoproteins is covered in Chapters 39 and 45.

LYSOSOMAL ENZYMES

Most glycans are degraded in lysosomes by highly ordered pathways employing endo- and exoglycosidases, sometimes aided by noncatalytic proteins. Insights that unraveled these complex pathways emerged from studies of rare human genetic disorders called lysosomal storage diseases. In each disease, undigested molecules accumulate in lysosomes. Clever experiments combining enzymology with glycan structural analyses revealed the steps of the pathways and also unlocked the mechanisms of lysosomal enzyme targeting (Chapter 33). Lysosomes contain approximately 50–60 soluble hydrolases that degrade various macromolecules. Most of the glycan-degrading enzymes (endo- and exoglycosidases and sulfatases) have pH optima between 4 and 5.5, but a few have higher pH optima, nearing neutral. Exoglycosidases cleave the glycosidic linkage of terminal sugars from the nonreducing end of glycans (the outermost left end of glycans for figures in this book, e.g., FIGURE 44.1). Exoglycosidases recognize only one monosaccharide (rarely two) in a specific anomeric linkage, and are much less particular about the structure of the molecule beyond that glycosidic linkage. This lack of specificity allows these enzymes to act on a broad range of substrates. However, exoglycosidases do not usually work unless all of the hydroxyl groups of the terminal sugar are unmodified. Acetate, sulfate, or phosphate groups usually have to be removed prior to action of the glycosidases. Esterases cleave acetyl groups and specific sulfatases remove the sulfate groups on glycosaminoglycans and N- or O-linked glycans. Endoglycosidases cleave internal glycosidic linkages of larger chains. These enzymes are often more tolerant of modifications of the glycan; in some cases, they require a modified sugar for optimal cleavage. Even though the lysosomal glycosidases perform similar reactions, their amino acid sequences are only about 15–20% identical to each other. Thus, there are no highly conserved glycosidase catalytic domains. Lysosomal enzymes are all N-glycosylated and most are targeted to the lysosome by the mannose-6-phosphate pathway (Chapter 33), share aspects of the recognition marker for assembly of mannose-6-phosphate on N-linked glycans, and have affinity for mannose-6-phosphate receptors. The concentration of enzymes within the lysosome is difficult to determine. Proteinases such as cathepsins are estimated to be ~1 mM; glycosidases are probably present at much lower concentrations.

GENETIC DEFECTS IN LYSOSOMAL DEGRADATION OF

GLYCANS

About 50 known inherited diseases impair lysosomal degradation of macromolecules. Although each one is rare, together their occurrence is about one in every 5-10,000 births. Loss of a single lysosomal hydrolase leads to the accumulation of its substrate as undegraded fragments in tissues and the appearance of related fragments in urine. Many of the human disorders have animal models. Tables 44.1, 44.2 and 44.3 show some of the major clinical symptoms of diseases associated with the degradation of three classes of glycans. Many of the diseases share overlapping symptoms, and yet each disease has unique clinical features that allow it to be diagnosed. The advent of exome and genome sequencing has shortened the diagnostic waiting period for concerned families. Many of the diseases also present with a range of severities. Usually, an infantile onset is the most severe and the juvenile or adult onsets have milder symptoms. The later onset forms may even affect organ systems distinct from those affected by early onset forms. Hundreds of mutations have been mapped in the different disorders. The severity usually depends on the combination of mutated alleles. Predicting the disease severity (prognosis) from the specific mutation is generally difficult. Complete absence of a lysosomal hydrolase is uniformly severe. Hypomorphic alleles have variable residual glycosidase activity making their prognosis is difficult. It is not clear whether accumulating different types of undegraded glycans leads to the different symptoms characteristic of each disease. There is no evidence that the stored material causes lysosomes to burst and spew their contents into the cytoplasm. Some leakage may occur into the cytoplasm or even out of cells, or the cell may sense an “engorged” lysosome. The pathology likely depends on the cell type and the cellular balance of synthesis and turnover rates. For instance, dermatan sulfate, a glycosaminoglycan (Chapter 17), predominates in connective tissue, which might explain the bone, joint, and skin problems in mucopolysaccharidosis (MPS) I, II, VI, and VII. Keratan sulfate, another glycosaminoglycan, is present in cartilage; therefore, MPS IV is largely a skeletal disease. Gangliosides (Chapter 11) are most abundant in neurons; therefore, gangliosidoses are predominantly brain disorders. The importance of glycogen for muscle explains the impact of Pompe disease on the heart and diaphragm, leading to rapid lethality in that disease. Given the balance between synthesis and degradation of multiple glycans, reducing synthesis somewhat by the use of an inhibitor may help to retard the accumulation of undegraded material and reduces the pathology of some diseases.

GLYCOPROTEIN DEGRADATION

The great majority of N- and O-glycans reaching the lysosome contain only six sugars linked in one or two anomeric configurations: β-N-acetylglucosamine (βGlcNAc), α/β-N-acetylgalactosamine (α/βGalNAc), α/β-galactose (α/βGal), α/β-mannose (α/βMan), α-fucose (αFuc), and α-sialic acid (αSia). Each linkage should theoretically require only one anomer-specific glycosidase, assuming that each glycosidase ignores the underlying glycan. This number is close to the known number of enzymes in most glycan degradation pathways. However, some linkages require a specific enzyme outside of

this group. For example, β-N-acetylhexosaminidase cleaves both βGlcNAc and βGalNAc residues. Degradation of the GlcNAcβAsn and GalNAcαSer/Thr linkages also requires specific enzymes.

Lysosomal Degradation of Complex N-Glycans

Much of what we know about this pathway comes from analysis of products that accumulate in patients’ tissues or urine due to the absence of one of the degradative enzymes (Table 44.1). Structural analyses of monosaccharide-labeled glycoproteins during degradation in perfused rat liver aided elucidation of the pathway. By conducting the latter studies in the presence of inhibitors of different lysosomal enzymes, a picture of simultaneous and independent bidirectional degradation of the protein and carbohydrate chains emerged (Figure 44.1). The relative degradation rates vary depending on structural and steric factors of the protein and the sugar chains. The accumulation of GlcNAcβ1-4GlcNAcβAsn in cells that cannot cleave the GlcNAcβAsn linkage clearly shows that degradation of the sugar chain does not require prior cleavage of the Asn. Much of the protein is probably degraded before N-glycan catabolism begins. Removal of core fucose (Fucα1–6GlcNAc) and probably any peripheral fucose residues linked to the outer branches of the chain (e.g., Fucα1–3GlcNAc) appears to be the first step in degradation because patients lacking α-fucosidase still have intact N-glycans bound to asparagine. Glycosylasparaginase (aspartyl-N-acetyl-β-D-glucosaminidase) then cleaves the GlcNAcβAsn bond, producing a glycosylamine +Asp, not Asn. In rodents and primates, chitobiase (an endo-β-N-acetylglucosaminidase) removes the reducing N-acetylglucosamine, leaving the oligosaccharide with only one terminal GlcNAc. In many other species, splitting of the chitobiose linkage (GlcNAcβ1–4GlcNAc) uses the β-N-acetylhexosaminidase mentioned below as the last step in degradation. Either pathway appears to be effective, leaving the presence of chitobiase in some species unexplained. The oligosaccharide chain is then sequentially degraded by sialidases and/or α-galactosidase, followed by β-galactosidase, β-N-acetylhexosaminidase, and α-mannosidases. The remaining Manβ1–4GlcNAc (or Manβ1-4GlcNAcβ1-4GlcNAc in species lacking chitobiase) is cleaved by β-mannosidase to mannose and GlcNAc (or chitobiose, which is then cleaved to GlcNAc by β-N-acetylhexosaminidase). Lysosomal sialidase (neuraminidase), β-galactosidase, and a serine carboxypeptidase called protective protein/cathepsin A form a complex in the lysosome that is required for efficient degradation of sialylated glycoconjugates. Cathepsin A protects β-galactosidase from rapid degradation and also activates the sialidase precursor, but the protection does not depend on the catalytic activity of cathepsin A. Mutations in this protective protein lead to galactosialidosis in which the simultaneous deficiencies in β-galactosidase and sialidase are secondary effects of defective cathepsin A. Glycans that have GalNAcβ1–4GlcNAc, GlcAβ1–3Gal, or Galα1–3Gal on the outer branches must first have these residues removed by β-N-acetylhexosaminidase, β-glucuronidase, and α-galactosidase, respectively, prior to any further digestion of the underlying oligosaccharide chains.

Lysosomal Degradation of Oligomannosyl N-Glycans

Oligomannosyl N-glycans that enter the lysosome are hydrolyzed by an α-mannosidase to yield Manα1–6Manβ1–4GlcNAc, a common intermediate of hybrid and complex N-glycans. A second α1–6-specific mannosidase can cleave this linkage in humans and rats, but only on molecules that have a single core region N-acetylglucosamine (i.e., those generated by chitobiase cleavage). Finally, β-mannosidase completes the degradation. Oligomannosyl N-glycans derived from dolichol-linked precursors or misfolded glycoproteins are handled differently (Chapter 39).

Degradation of O-Glycans

Degradation of typical αGalNAc-initiated O-glycans has not been systematically studied. Many of the outer structures of N-glycans are also found on O-glycans (Chapter 14); therefore, degradation of these glycans probably uses the same group of exoglycosidases as discussed above. An exception to this is the linkage region, GalNAcα-O-Ser/Thr. Patients with Schindler disease lack the α-N-acetylgalactosaminidase specific for αGalNAc and will not cleave αGlcNAc. This same enzyme probably removes terminal αGalNAc from blood group A–containing glycans (GalNAcα1–3Gal) and some glycolipids such as the Forsmann antigen (GalNAcα1–3GalNAcβ1–3Galα1–4Galβ1–4GlcβCer). Patients lacking α-N-acetylgalactosaminidase accumulate GalNAc-containing glycopeptides in their urine, but curiously they also accumulate more complex, extended glycopeptides containing N-acetylglucosamine, galactose, and sialic acid. The structures are the same as those found on some native glycoconjugates. Their production could result from a general slowdown of oligosaccharide degradation, or may arise by reassembly of glycans on accumulated GalNAcα-O-Ser/Thr glycopeptides

GLYCOSAMINOGLYCAN DEGRADATION

Glycosaminoglycans (GAGs), including heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan, are degraded in a highly ordered fashion. The first three are O-xylose-linked to core proteins (Chapter 17), KS is N- or O-linked , and hyaluronan is a free glycan (Chapter 16). Some proteoglycans are internalized from the cell surface and the protein portion is degraded. The GAG chains are then partially cleaved by enzymes such as endo-β-glucuronidases or endohexosaminidases that clip at a few specific sites. Endoglycosidase cleavage creates multiple terminal residues that can be degraded by unique or overlapping sets of sulfatases and exoglycosidases. Structural analyses of partially degraded fragments in the lysosomes of cells from patients with mucopolysaccharidoses (MPS) revealed both the degradation pathways and genetic defects responsible (Table 44.2). The clinical severities and manifestations vary even with mutations in the same gene. For instance, MPS I is clinically subdivided into Hurler (most severe), Hurler/Scheie, and Scheie syndromes, although the three disorders represent a continuum of the same disease (Table 44.2). Hurler/Scheie patients progress more slowly and die in early adulthood, whereas Scheie patients can survive to middle or old age. The milder forms of this disease do not cause intellectural disabity.

Hyaluronan

Hyaluronan (Chapter 16) is the largest and most abundant GAG: A 70-kg person degrades 5 g of hyaluronan (molecular mass 107 daltons) per day. Degradation of hyaluronan involves a series of two endo-β-N-acetylhexosaminidases called hyaluronidases (Hyal-1 and Hyal-2), β-glucuronidase, and finally β-N-acetylhexosaminidase. Hyal-2 is active at low pH and is GPI-anchored on the cell surface. This enzyme associates both with hyaluronan in lipid rafts and with an Na+/H+ exchanger to create an acidic microenvironment. Cleavage generates fragments of approximately 20 kD (about 50 disaccharides), which are internalized, delivered to endosomes, and then finally to lysosomes, where the fragments are degraded by Hyal-1 into tetra- and disaccharides (Figure 44.2). The exoglycosidases are thought to participate in the degradation of the larger fragments as well as that of the di-and tetrasaccharide units. Mutations in HYAL-1 cause MPS IX.

Heparan Sulfate

HS (Chapter 17) is first degraded by an endoglucuronidase, heparanase, followed by a well-ordered sequential degradation. An example is shown in Figure 44.2. A terminal iduronic acid-2-sulfate must be desulfated by iduronic acid-2-sulfatase to make the modified sugar a substrate for α-iduronidase. If glucuronic acid (GlcA)-2-sulfate is at this position, GlcA-2-sulfatase removes the sulfate prior to β-glucuronidase cleavage. The new terminal glucosamine sulfate (GlcNSO4) is the next sugar for cleavage, requiring three steps. Sulfate is first removed by N-sulfatase forming glucosamine, which cannot be cleaved by α-N-acetylglucosaminidase. The free amino group is N-acetylated by an N-acetyltransferase embedded in the lysosomal membrane, converting glucosamine to N-acetylglucosamine, which is a substrate of α-N-acetylglucosaminidase. In the second step, acetyl CoA donates the acetyl group to a histidine residue in the cytoplasmic domain of the N-acetyltransferase. The acetyl group then becomes available on the luminal side of the lysosomal membrane and is transferred at low pH to the amino group of glucosamine. If cleavage of glucuronic acid reveals a 6-O-sulfated αGlcNAc, the sulfate is removed by a specific GlcNAc-6-sulfatase prior to GlcNAc removal. Table 44.2 provides a list of the enzymatic defects in HS degradation and the disorders that result.

Dermatan Sulfate and Chondroitin Sulfate

DS and CS are related GAG chains based on a repeating polymer of βGlcA and βGalNAc (Chapter 17). Figure 44.3 shows that a combination of endoglycosidases, sulfatases, and exoglycosidases degrades DS in the lysosome. Iduronic acid-2-sulfatase is followed by α-iduronidase. The terminal GalNAc-4-SO4 can be removed by either of two pathways. In the first pathway, a GalNAc-4-SO4 sulfatase acts followed by β-N-acetylhexosaminidase A or B to remove N-acetylgalactosamine. In the second, β-N-acetylhexosaminidase A removes the entire GalNAc-4-SO4 unit followed by sulfatase cleavage. Absence of the GalNAc-4-SO4 sulfatase (MPS VI) is unique to the DS pathway. β-Glucuronidase cleaves the β-glucuronic acid residue and the process is repeated on the rest of the molecule.

To degrade CS, GalNAc-6-SO4 sulfatase and GalNAc-4-SO4 sulfatase work in combination with β-N-acetylhexosaminidase A or B and β-glucuronidase. Hyaluronidases can also degrade CS, but no CS-specific endoglycosidases have been found.

Keratan Sulfate

KS is an N- or O-glycan with a heavily sulfated poly-N-acetyllactosamine chain. Mammalian cells do not have an endoglycosidase to break down KS (Figure 44.3). The sequential action of sulfatases and exoglycosidases is needed. Galactose-6-SO4 sulfatase is the same enzyme that desulfates GalNAc-6-SO4 in CS degradation. This hydrolysis is followed by β-galactosidase digestion, leaving a terminal GlcNAc-6-SO4. Desulfation by the sulfatase followed by cleavage with β-N-acetylhexosaminidase A or B eliminates GlcNAc-6-SO4. Alternatively, β-N-acetylhexosaminidase A can directly release GlcNAc-6-SO4, followed by desulfation of the monosaccharide.

Linkage Region

Degradation of the core region of O-linked KS (skeletal type; type II) probably occurs by the same route as other O-glycans. The N-glycan corneal-type KS (type I) is probably degraded by the same set of enzymes used for N-glycan degradation. The O-xylose-linked GAG chains (DS, CS, HS) all share a common core tetrasaccharide: GlcAβ1-3Galβ1–3Galβ1–4Xylβ-O-Ser. An endo-β-xylosidase has been detected in rabbit liver. How the linkage regions are degraded is not established.

Multiple Sulfatase Deficiency

A very rare human disorder is multiple sulfatase deficiency (MSD). All sulfatases are casualties of this defect since they all undergo a posttranslational conversion of an active site cysteine residue to a Cα-Formylglycine (2-amino-3-oxopropionic acid) catalyzed by the Cα-Formylglycine generating enzyme (FGE) which is essential for activity. In essence, the SH group of cysteine is replaced by a double-bonded oxygen atom that probably acts as an acceptor for cleaved sulfate groups. Loss of function mutations in the FGE gene lead to inactive sulfatases. This deficit affects GAG degradation and any other sulfated glycan such as sulfatides.

GLYCOSPHINGOLIPID DEGRADATION

Glycosphingolipids (Chapter 11) are degraded from the nonreducing end by exoglycosidases while they are still bound to the lipid moiety ceramide. Since glycosphingolipids share some of the same outer sugar sequences found in N- and O-glycans (Chapter 14), many of the same glycosidases are used for their degradation (Figure 44.4). However, specific hydrolases cleave the glucose-ceramide and galactose-ceramide bonds. Besides specific enzymes, noncatalytic sphingolipid activator proteins

(SAPs or saposins) help to present the lipid substrates to enzymes for cleavage. Endoglycoceramidases from leeches and earthworms release the entire glycan chain from the lipid, much like PNGase releases N-glycans from proteins.

Specialized Issues for Glycolipid Degradation

Some exoglycosidases are unique to glycolipid degradation, and their absence causes glycolipid storage diseases (Table 44.3). Glucocerebrosidase, also called β-glucoceramidase, is specific for the degradation of the GlcβCer bond. The loss of this enzyme causes Gaucher’s disease. A specialized β-galactosidase, β-galactoceramidase, hydrolyzes the bond between galactose and ceramide and can also cleave the terminal galactose from lactosylceramide. Loss of this enzyme produces Krabbe disease. Galactosylceramide is often found with a 3-sulfate ester (sulfatide) and a specific sulfatase (arylsulfatase A) is needed for its removal prior to β-galactosylceramidase action. Loss of this sulfatase causes metachromatic leukodystrophy and the accumulation of sulfatide. Glycolipids terminated with α-galactose residues (globo-series, Chapter 11) are degraded by a specific α-galactosidase, the loss of which causes Fabry disease.

Activator Proteins

Sugars that lie too close to the lipid bilayer apparently give limited access to soluble lysosomal enzymes, and additional proteins called saposins are required to present the substrates to the enzymes. Saposins (SAPs), also called “liftases”, form complexes with multiple degradative enzymes for more efficient hydrolysis of short glycolipids close to the membrane. SAPs are derived from a 524-amino-acid precursor called prosaposin, which is processed into four homologous activator proteins each comprising about 80 amino acids: saposins A, B, C, and D. Despite their homology, each saposin has different properties. SAP-A and SAP-C both help β-glucosyl- and β-galactosylceramidase degradation. SAP-B assists arylsulfatase A, α-galactosidase, sialidase, and β-galactosidase. The activation mechanism of SAPs is thought to be similar to that of the GM2 activator described below. SAP-D and SAP-B also assist in sphingomyelin degradation by sphingomyelinase. Complete absence of prosaposin is lethal in humans, and deficiencies in SAP-B and SAP-C lead to defects that clinically resemble arylsulfatase A deficiency (metachromatic leukodystrophy) and Gaucher’s disease. These symptoms might be predicted based on the enzymes that these saposins assist. GM2 activator protein forms a complex with either GM2 or GA2 and presents them to β-N-acetylhexosaminidase A for cleavage of β-GalNAc. The activator protein binds a molecule of glycolipid, forming a soluble complex, which can be cleaved by the hexosaminidase. The resulting product is then inserted back into the membrane, and the activator presents the next GM2 molecule, and so on. Genetic loss of this activator protein causes the accumulation of GM2 and GA2, resulting in the AB variant of GM2 gangliosidosis.

Topology for Degradation and Roles of Lipids

Endocytic vesicles deliver membrane components to the lysosome for degradation and are often seen as intralysosomal multivesicular storage bodies (MVB). They appear like vesicles within the lysosome and are especially prominent in patients with glycolipid storage disorders. How do “vesicles within vesicles” form and what is their function? The internal surface of the lysosomal membrane has a thick, degradation-resistant glycocalyx of integral and peripheral membrane proteins decorated with poly-N-acetyllactosamines, which protect the lysosomal membrane against destruction. However, these proteins would also shield incoming membranes from degradation if vesicle fusion simply occurred. By creating multiple internal membranes seen in typical MVBs, the target molecules are exposed to the soluble lysosomal enzymes, and digestion proceeds efficiently on the membrane surfaces of these internal vesicles. MVB formation starts with inward budding of the limiting endosomal membrane. Lipids and proteins are sorted to either the internal or the limiting membrane. Ubiquitination of cargo proteins targets them to internal vesicles of MVBs, but ubiquitin-independent routes also exist. Intra-endosomal membranes and limiting endosomal membrane have different lipid and protein compositions. Membrane segregation and lipid sorting prepare the internal membranes for lysosomal degradation. During maturation of the internal membranes, cholesterol is continually stripped away (to <1%) and a negatively charged lipid bis(monoacylglycero)phosphate (BMP) increases up to 45%. This molecule is highly resistant to phospholipases. BMP also stimulates sphingolipid degradation on the inner membranes of acidic compartments. Since BMP is not present in the lysosomal outer membrane, the unique lipid profile ensures that degradation by hydrolases and membrane-disrupting SAPs occurs without digesting the lysosomal outer membrane.

DEGRADATION AND RESYNTHESIS

Degradation of glycans is not always complete. Partially degraded or incomplete glycans on glycoproteins, glycopeptides and glycosphingolipids can be internalized within a functional Golgi compartment containing sugar nucleotides and glycosyltransferases and then elongated. In the case of glycosphingolipids, this pathway makes a substantial contribution to total cellular synthesis, but in glycoproteins it probably makes a relatively small contribution. These processes can be salvage and repair mechanisms or may play an integral part in an unidentified physiological pathway.

Reglycosylation and Recycling of Glycoproteins

The half-life of certain membrane proteins is longer than the half-life of their sugar chains. Terminal monosaccharides turn over faster than those near the reducing end of the glycan, suggesting that terminal sugars are removed by exoglycosidases. Cleavage may occur at the cell surface or when proteins are endocytosed in the course of normal membrane recycling. Because the mildly acidic late endosomes contain lysosomal enzymes with a fairly broad pH range, terminal sugars such as sialic acid can be cleaved. If the endocytosed proteins are not degraded in lysosomes, the proteins may re-encounter sialyltransferases in the Golgi, become resialylated, and appear again on the cell surface. A similar situation may occur if a protein has lost both sialic acid and

galactose residues from its glycans. Some membrane proteins synthesized in the presence of oligosaccharide processing inhibitors can still reach the cell surface in an unprocessed form. Subsequent incubation in the absence of inhibitors leads to normal processing over time. The extent of processing and the kinetics depend on the protein and the cell type. Because Golgi enzymes are not distributed identically in all cells, the extent of reprocessing is variable. Most studies have monitored N-glycans, but membrane proteins with O-glycans probably behave similarly.

Glycosphingolipid Recycling

The majority of the newly synthesized glucosylceramide arrives at the cell surface by a Golgi-independent cytoplasmic pathway. Some of this material, as well as glucosylceramide generated by partial degradation of complex glycosphingolipids, can recycle to the Golgi. There, like glycoproteins, simple glycosphingolipids can serve as acceptors for the synthesis of longer sugar chains. In the lysosome, sphingosine is produced by complete degradation of glycosphingolipids and reutilized. Together, these pathways, especially the latter, may account for the majority of complex glycolipid synthesis in many cells. Depending on the physiological state of the cell and synthetic demands, the de novo pathway starting from serine and palmitoyl CoA may account for only 20–30% of the total synthesis. Shuttling the recycled components through and among the various organelles appears to involve vimentin intermediate filaments. Mechanisms and details of this process are presently lacking.

SALVAGE OF MONOSACCHARIDES

Monosaccharide salvage is discussed in Chapter 5. Monosaccharides derived from glycan degradation are transported back into the cytoplasm using transporters specific for neutral sugars, N-acetylated hexoses, anionic sugars, sialic acid, and glucuronic acid. Turnover of glycans is high and the salvage of sugars can be quite substantial. There have been few studies comparing the actual contributions of exogenous monosaccharides, those salvaged from glycan turnover, and those generated from de novo synthesis. Cells probably differ in their preference for each source of monosaccharides. These differences may explain why monosaccharide therapy used to treat some glycosylation disorders is effective in certain cells and not others.

BLOCKING DEGRADATION

Lysosomal degradation of glycans can be blocked by raising the intralysosomal pH, by inactivating a particular glycosidase, by mutation, or by protein-specific inhibitors. Lysosomal enzymes usually have acidic pH optima and adding ammonium chloride or chloroquine slows degradation by increasing the intralysosomal pH. However, some lysosomal enzymes have relatively broad pH optima, higher than the lysosome. Some active “lysosomal” enzymes can be found in early and late endosomes, which have a higher pH than lysosomes. Glycosylation inhibitors are covered in Chapter 55 but some of these agents also inhibit specific lysosomal enzymes. For example, swainsonine blocks lysosomal α-

mannosidase as well as the α-mannosidase II involved in glycoprotein processing. Sheep and cattle become neurologically deranged by eating food rich in swainsonine, which is also called locoweed. These temporary symptoms are likely induced because lysosomal α-mannosidase is inhibited. Undegraded oligosaccharides probably accumulate in affected animals. Although many inhibitors block various enzymes in vitro, they may not be effective in cells or whole animals because they may not enter the lysosome at sufficient concentrations.

THERAPY FOR LYSOSOMAL ENZYME DEFICIENCIES

Study of lysosomal enzyme defects provided major insights into catabolic pathways and showed their importance to human health. Mannose-6-phosphate targeting of lysosomal enzymes was discovered by showing that cocultivation of fibroblasts from patients with different storage diseases led to the disappearance of stored material in both types of cells. Each supplied the corrective factors (i.e., lysosomal enzymes) that the other cell lacked by delivery of secreted enzymes through cell-surface mannose-6-phosphate receptors (Chapter 33). Cross-correction highlights the fact that only a relatively small amount of enzyme may be needed to prevent accumulation of storage products and the resulting pathology. Some estimates suggest that less than 5% of normal β-N-acetylhexosaminidase activity may be sufficient to prevent pathological symptoms of Tay–Sachs disease. In fact, the index Scheie patient with α-L-iduronidase deficiency had less than 1% normal activity in fibroblasts and lived 77 years. Current therapeutic approaches include enzyme replacement therapy (ERT), substrate reduction therapy (SRT) and enzyme enhancement therapy (EET). Gene replacement therapy is a promising future approach, but not yet clinically tested. Accumulation of stored undegraded material occurs because the rate of glycan synthesis is greater than its degradation at steady state. SRT reduces the glycan’s synthetic rate to counter low glycosidase activity. This hypothesis was tested in a mouse model of Sandhoff disease by using N-butyldeoxynojirimycin, an inhibitor of glucosylceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. When wild-type mice were treated with this compound, the amount of glycosphingolipids fell 50–70% in all tissues without obvious pathological effects. When this compound was given to Sandhoff disease mice, the accumulation of GM2 in the brain was blocked and the amount of stored ganglioside reduced. Thus, reducing the synthesis of the primary precursor reduced the load of GM2 to levels that were degradable by the glycosidase-deficient mice. Because this compound inhibits the first biosynthetic step, theoretically, this drug could reduce the accumulation of storage products in any glycolipid storage disorder. In clinical trials, N-butyldeoxynojirimycin (miglustat, Zavesca®) was effective for many patients with mild-to-moderate Gaucher’s disease, although improvement varied as did side effects. Small trials in infantile Tay-Sachs disease patients did not arrest neurological deterioration, but prevented macrocephaly. Further clinical studies are likely to reveal the potential and limitations of SRT. Enzyme enhancement therapy (EET) or pharmacological chaperone therapy (PCT) is based on the concept that inhibitors of an enzyme can act as molecular chaperones to stabilize the mutated enzymes in the endoplasmic reticulum. When the enzyme reaches the lysosome, the inhibitor dissociates at low pH and the abundant stored material is gradually digested. The overall increase in activity is small, but it can have significant clinical benefits. Clinical trials are in progress for treating Fabry disease, Gaucher Type I, and Pompe disease, while preclinical trials are being done for GM1 and GM2

gangliosidosis. A potential problem is that some inhibitors may not cross the blood-brain barrier, limiting their effectiveness in the CNS. The advent of high throughput screening methods and large chemical libraries increase the potential of this approach. In addition, allosteric site binders can be selected that remain bound to the enzyme and any successful compound could be used in combination with ERT to further enhance activity. ERT has been quite successful for treating Gaucher’s disease. Injection of glucocerebrosidase carrying mannose-terminated N-glycans targets the enzyme to the macrophage/monocytes, which are the primary sites of substrate accumulation. In use now for many years, the added enzyme consistently improves patients’ clinical features with minimal side effects. The cost of treatment is high, and high doses are no more effective than lower doses for improving visceral and hematological pathologies. The proven effectiveness makes glucocerebrosidase the favorite therapy for these patients, but terminal mannose residues cannot be used to target the enzyme to other cells or organs or to enable treatment of other lysosomal storage disorders. Insufficient infused enzyme crosses the blood-brain barrier, thus, the enzyme (Cerezyme®) works very well for type I disease without neurological involvement but not for the less common Gaucher’s with neurological involvement. In addition, patients sometimes develop antibodies against the injected human protein. ERT trials with other recombinant lysosomal enzymes have progressed. Enzyme replacement therapy is available for Fabry disease, Pompe disease, and mucopolysaccharidosis types I, II, IVA, and VI and is under development for others. Recombinant α-galactosidase for Fabry disease works well and recombinant α-L-iduronidase for MPS I works very well with the intermediate severity form (Hurler/Scheie), but its effect is unclear for patients with the more common neurological form (Hurler syndrome). Recombinant N-acetylgalactosamine 4-sulfatase (arylsulfatase B; Naglazyme®) was approved for MPS VI, as was α-glucosidase (Myozyme®) for treating Pompe disease patients. Iduronic acid-2-sulfatase (Elaprase®) is approved for treating Hunter syndrome. These results clearly validate this approach. The efficacy of most of these enzymes relies on the engineering of Man-6-P-containing glycans. This is one of the foremost examples of how the clinically motivated exploration of a disorder (I-cell disease) led to the discovery of Man-6-P-based targeting of lysosomal enzymes (Chapter 33)and the development of an entire industry targeting therapy for multiple diseases. The availability of animals deficient in specific lysosomal enzymes offers appropriate systems to test the efficacy of corrective genes, compounds and enzymes.

ACKNOWLEDGMENTS The authors and appreciate helpful comments and suggestions from Michelle Dookwah, Chrissa Dwyer and Bastian Ramms.

FURTHER READING Neufeld EF, Lim TW, Shapiro LJ. 1975. Inherited disorders of lysosomal metabolism.

Annu Rev Biochem 44: 357–376. Winchester B. 2005. Lysosomal metabolism of glycoproteins. Glycobiology 15: 1R–15R. Winchester B. 2014. Lysosomal diseases: diagnostic update. J Inherit Metab Dis 37:

599-608. Deng H, Xiu X, Jankovic J. 2015. Genetic convergence of Parkinson's disease and

lysosomal storage disorders. Mol Neurobiol 51: 1554-1568. Coutinho MF, Matos L, Alves S. 2015. From bedside to cell biology: a century of history

on lysosomal dysfunction. Gene 555: 50-58. ClarkeLA,HollakCE.2015.Theclinicalspectrumandpathophysiologyofskeletal

complicationsinlysosomalstoragedisorders.BestPractResClinEndocrinolMetab29:219-235.

RastallDP,AmalfitanoA.2015.Recentadvancesingenetherapyforlysosomalstoragedisorders.ApplClinGenet8:157-169.

Parenti G, Andria G, Valenzano KJ. 2015. Pharmacological chaperone therapy: Preclinical development, clinical translation, and prospects for the treatment of lysosomal storage disorders. Mol Ther 23: 1138-1148.

Oh DB. 2015. Glyco-engineering strategies for development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases. BMB Rep 48: 438-444.

Espejo-Mojica ÁJ, Alméciga-Díaz CJ, Rodríguez A, Mosquera Á, Díaz D, Beltrán L, Díaz S, Pimentel N, Moreno J, Sánchez J, Sánchez OF, Córdoba H, Poutou-Piñales RA, Barrera LA. 2015. Human recombinant lysosomal enzymes produced in microorganisms. Mol Genet Metab 116: 13-23.

FIGURE LEGENDS

FIGURE 44.1. Degradation of complex N-glycans. The lysosomal degradation pathway of glycoproteins carrying complex-type glycans proceeds simultaneously on both the protein and glycan moieties. The N-glycans are sequentially degraded by the indicated exoglycosidases in a specific order as discussed in the text.

FIGURE 44.2. Degradation of hyaluronan and heparan sulfate. (Left) Hyaluronidase (an endoglycosidase) cleaves large chains into smaller fragments, each of which is then sequentially degraded from the nonreducing end. (Right) Degradation of heparan sulfate (HS). An endo-β-glucuronidase first cleaves large chains into smaller fragments and each monosaccharide is then removed from the nonreducing end as described in the text. N- and O-sulfate groups must first be removed before exoglycosidases can act. An unusual feature of HS degradation is that this process also involves a synthetic step. After removal of the N-sulfate residue on GlcNSO4, the nonacetylated glucosamine must first be N-acetylated using acetyl-CoA before α-N-acetylglucosaminidase can cleave this residue.

FIGURE 44.3. Degradation of chondroitin/dermatan sulfates and keratan sulfate. (Left) Only DS degradation is shown. After removal of sulfated iduronic acid in two steps, further sequential degradation can proceed by two different routes. One route (straight down) uses GalNAc-4-SO4 sulfatase followed by cleavage with β-N-acetylhexosaminidase A or B. The other route uses only β-N-acetylhexosaminidase A, which is one of the few exoglycosidases that can cleave sulfated amino sugars at low pH. These two routes of sequential degradation also works in CS degradation. (Right) Degradation of keratan sulfate. Sequential degradation of KS occurs from the nonreducing end like the degradation of DS and CS. The terminal GlcNAc-6-SO4 can be cleaved sequentially by a sulfatase and then by β-N-acetylhexosaminidase A or B or, alternatively, β-N-acetylhexosaminidase A can cleave GlcNAc-6-SO4 directly at low pH.

FIGURE 44.4. Degradation of glycosphingolipids. Required activator proteins are shown in parentheses and individual enzymes are as shown in previous figures in this chapter. (Sap) saposin.

TABLE 44.1 Defects in glycoprotein degradation Effects on degradation of Disorder Defect Glycoprotein Glycolipid Clinical symptoms α-Mannosidosis (types I and II)

α-mannosidase major none type I: infantile onset, progressive intellectual disability, hepatomegaly, death between 3 and 12 years type II: juvenile/adult onset, milder, slowly progressive

β-Mannosidosis β-mannosidase major none severe quadriplegia, death by 15 months in most severe cases; mild cases have intellectual disability, angiokeratoma, facial dysmorphism

Aspartylglucosaminuria aspartyl-glucosaminidase

major none progressive, coarse facies, intellectual disability

Sialidosis (mucolipidosis I)

sialidase major minor progressive, severe mucopolysaccharidosis-like features, intellectual disability

Schindler (types I and II)

α-N-acetyl-galactosaminidase

yes ? type I: infantile onset, neuroaxonal dystrophy. severe psychomotor delay and intellectual disability, cortical blindness, neurodegeneration type II: mild intellectual impairment, angiokeratoma, corpus diffusum

Galactosialidosis protective protein/cathepsin A

major minor coarse facies, skeletal dysplasia, early death

Fucosidosis α-fucosidase major minor spectrum of severities includes psychomotor delay, coarse facies, growth retardation

GM1 gangliosidosis β-galactosidase minor major progressive neurological disease and skeletal dysplasia in severe infantile form

GM2 gangliosidosis β-hexosaminidase minor major severe form: neurodegeneration with death by 4 years less severe form: slower onset of symptoms and variable symptoms, all relating to various parts of the central nervous

system

TABLE 44.2 Defects in glycosaminoglycan degradation—the mucopolysaccharidosesa)

Number Common name

Enzyme (Gene) deficiency

Glycosamino-glycan affected Clinical symptoms

MPS I H Hurler, Hurler/Scheie, Scheie

α-L-iduronidase (IDUA) DS, HS Hurler: corneal clouding, organomegaly, heart disease, intellectual disability, death in childhood Hurler/Scheie and Scheie: less severe, individuals survive longer

MPS II Hunter iduronic acid-2-sulfatase (IDS)

DS, HS severe: organomegaly, no corneal clouding, intellectual disability, death before 15 years less severe: normal intelligence, short stature, survival age 20–60

MPS III A

Sanfilippo A heparan N-sulfatase (SGSH)

HS profound mental deterioration, hyperactivity, relatively mild somatic manifestations

MPS III B

Sanfilippo B α-N-acetylglucosaminidase (NAGLU)

HS similar to III A

MPS III C

Sanfilippo C acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT)

HS similar to III A

MPS III D

Sanfilippo D N-acetylglucosamine 6-sulfatase (GNS)

HS similar to III A

MPS IV A

Morquio A N-acetylgalactosamine-6-sulfatase (GALNS)

KS, CS distinctive skeletal abnormalities, corneal clouding, odontoid hypoplasia, milder forms known to exist

MPS IV B

Morquio B β-galactosidase (GLB1) KS same as IV A

MPS VI Maroteaux-Lamy

N-acetylgalactosamine 4-sulfatase (ARSB)

DS corneal clouding, normal intelligence, survival to teens in severe form; milder forms known to exist

MPS VII Sly β-glucuronidase (GUSB) DS, HS, CS wide spectrum of severity, including

hydrops fetalis and neonatal form

MPS IX Natowicz Hyaluronidase (HYAL-1) Hyaluronan Periarticular soft tissues masses, short stature, mild facial changes

multiple sulfatase deficiency

FGE converts cysteine→formyl glycine (SUMF1)

all sulfated glycans

hypotonia, developmental delay, neurodegeneration

(MPS)Mucopolysaccharidoses;(DS)dermatansulfate;(HS)heparansulfate;(KS)keratansulfate;(CS)chondroitinsulfate.a)NeufeldE,MuenzerJ.Themucopolysaccharidoses.In:ScriverCR,BeaudetAL,SlyWS,ValleD,ChildsB,KinzlerKW,VogelsteinB,eds.TheMetabolicandMolecularBasisofInheritedDisease.8ed.NewYork.NY:McGraw-Hill;2001:3421-52.

TABLE 44.3 Defects in glycolipid degradation

Disease name Enzyme or protein deficiency Clinical symptoms

Tay–Sachs β-hexosaminidase A severe: neurodegeneration, death by 4 years less severe: slower onset of symptoms, variable symptoms all relating to parts of the nervous system

Sandhoff β-hexosaminidase A and B

same as Tay–Sachs

GM1 gangliosidosis β-galactosidase see Table 44.1 Sialidosis sialidase see Table 44.1 Fabry α-galactosidase severe pain, angiokeratoma, corneal opacities,

death from renal or cerebrovascular disease Gaucher’s β-glucoceramidase severe: childhood or infancy onset,

hepatosplenomegaly, neurodegeneration mild: child/adult onset, no neurodegenerative course

Krabbe β-galactoceramidase early onset with progression to severe mental and motor deterioration

Metachromatic leukodystrophy

arylsulfatase A (cerebroside sulfatase)

infantile, juvenile, and adult forms can include mental regression, peripheral neuropathy, seizures, dementia

Saposin deficiency saposin precursor similar to Tay–Sachs and Sandhoff

Article

LysosomalStorageDiseases:FromPathophysiologytoTherapy

ME66CH31-Ballabio ARI 17 December 2014 10:28

Lysosomal Storage Diseases:From Pathophysiology toTherapyGiancarlo Parenti,1,2 Generoso Andria,2

and Andrea Ballabio1,2,3,4

1Telethon Institute of Genetics and Medicine, Pozzuoli 80078, Italy2Department of Translational Medical Sciences, Section of Pediatrics, Federico II University,80131 Naples, Italy3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston,Texas 770304Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston,Texas 77030; email: ballabio@tigem.it

Annu. Rev. Med. 2015. 66:471–86

The Annual Review of Medicine is online atmed.annualreviews.org

This article’s doi:10.1146/annurev-med-122313-085916

Copyright c© 2015 by Annual Reviews.All rights reserved

Keywords

enzyme replacement therapy, pharmacological chaperone therapy,proteostasis regulators, substrate reduction therapy, gene therapy

Abstract

Lysosomal storage diseases are a group of rare, inborn, metabolic errorscharacterized by deficiencies in normal lysosomal function and by intralyso-somal accumulation of undegraded substrates. The past 25 years have beencharacterized by remarkable progress in the treatment of these diseases andby the development of multiple therapeutic approaches. These approachesinclude strategies aimed at increasing the residual activity of a missing en-zyme (enzyme replacement therapy, hematopoietic stem cell transplantation,pharmacological chaperone therapy and gene therapy) and approaches basedon reducing the flux of substrates to lysosomes. As knowledge has improvedabout the pathophysiology of lysosomal storage diseases, novel targets fortherapy have been identified, and innovative treatment approaches are beingdeveloped.

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THE BIOLOGY OF LYSOSOMES

Lysosomes are catabolic organelles that break down and recycle a range of complex substrates,including glycosaminoglycans, sphingolipids, glycogen, and proteins. The catabolic function isperformed through the concerted action of approximately 60 different acidic hydrolases that be-long to different protein families, such as glycosidases, sulfatases, peptidases, phosphatases, lipases,and nucleases. Several lysosomal and nonlysosomal proteins participate in lysosomal functioning(e.g., by modulating the activity of lysosome-resident proteins).

Lysosomal catabolic function depends on direct interaction between hydrolases and substratesin the lysosomal lumen. Lysosomal enzymes are synthesized in the endoplasmic reticulum,where they adopt their native conformations and translocate to lysosomes via specializedpathways. The majority of these hydrolases are targeted to lysosomes through the additionof mannose-6-phosphate (M6P) residues onto the oligosaccharide moieties of the enzymes, amodification that takes place in the late Golgi compartment, and through the M6P receptor(MPR) pathway (1). Some specific enzymes do not depend on MPR for lysosomal delivery, suchas β-glucocerebrosidase, which is transported to lysosomes by lysosomal integral membraneprotein 2 (LIMP-2) (2).

Substrates are transported to lysosomes through different routes. Specialized endocytic mech-anisms (phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endo-cytosis, and clathrin- and caveolin-independent endocytosis) are preferentially exploited accordingto the nature of the cargo. Intracellular materials are transported to the lysosomal compartmentmainly through autophagy, a process by which cells capture and convey their own cytoplasmiccomponents and organelles to lysosomal degradation and recycling (3).

Recent studies have expanded our perspective on lysosomal function. They have shown thatlysosomes are not only catabolic organelles, but also involved in fundamental cellular functions,including nutrient sensing, signaling, vesicle trafficking, and cellular growth, to name a few.

It has become evident that the lysosome plays an important part in nutrient sensing and insignaling pathways involved in cell metabolism and growth. The recent discovery that the mam-malian target of rapamycin complex 1 (mTORC1) kinase complex, the master controller of cellgrowth, exerts its function on the lysosomal surface suggests that cell growth and cell catabolismare coregulated (4, 5). Recent studies have shown that the level of amino acids in the lysosomallumen controls mTORC1 localization on the lysosomal surface. During starvation, mTORC1inhibition potently activates autophagy.

Lysosomal function requires the concerted action of hydrolases, acidification machinery, andmembrane proteins. It has been recently discovered that genes involved in lysosomal func-tion belong to a gene network—the coordinated lysosomal expression and regulation (CLEAR)network—and are transcriptionally regulated by the lysosomal master gene TFEB (6). TFEB (tran-scription factor EB) positively regulates the expression of lysosomal genes, controls the numberof lysosomes, and promotes degradation of lysosomal substrates.

TFEB-mediated regulation allows lysosomal function to adapt to different physiological andpathological conditions (e.g., starvation or storage). Interestingly, phosphorylation of TFEBby mTORC1 negatively regulates its activity by retaining it in the cytoplasm, thus blocking itsnuclear translocation. During starvation, mTORC1 inhibition allows TFEB to translocate to thenucleus and perform its transcriptional activity. This is the first example of a lysosome-to-nucleussignaling mechanism (7).

LYSOSOMAL STORAGE DISEASES

Lysosomal storage diseases (LSDs) are a group of more than 50 inherited metabolic disorderscharacterized by the intralysosomal accumulation of undegraded substrates. LSDs are traditionally

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classified according to the chemical properties of the accumulated substrate. Individually, each ofthese disorders is rare. However, their cumulative prevalence is relatively high when comparedwith other groups of rare diseases, and prevalence is estimated to be approximately 1 in 8,000 livebirths (8).

The clinical consequence of substrate storage in multiple organs and systems is the variableassociation of visceral, ocular, hematological, skeletal, and neurological manifestations, and thereis partial phenotypic overlap among different disorders. Symptoms may emerge at variable ages,in some cases starting in utero or during the newborn period (such as fetal hydrops), or becomingevident in late adulthood. In general, the diseases progress and evolve over time.

In some cases the manifestations of LSDs are highly peculiar and thus allow for a gestaltdiagnosis. One of these peculiar features is the facial dysmorphism that gives patients a typicalHurler-like or gargoyle-like appearance. Visceral manifestations (hepatosplenomegaly) and hema-tological abnormalities (enlarged, substrate-filled vacuoles visible in lymphocytes or histiocytes)are also typical of LSDs. Skeletal involvement is characterized by generalized bone dysplasia, com-monly referred to as dysostosis multiplex; joint limitations; abnormalities of bone density; areas ofbone infarction; and osteonecrosis. Ocular manifestations include a range of abnormalities, such ascorneal or lenticular opacities, retinal involvement (cherry red spot, retinal dystrophy), optic nerveatrophy, glaucoma, and blindness. About two-thirds of patients with LSDs display a significantneurological component, which is extremely variable and ranges from progressive neurodegener-ation and severe cognitive impairment to epileptic, behavioral, and psychiatric disorders.

However, any other organ or system may be affected in LSDs, including the heart (car-diomegaly, valvular thickening, arrhythmias, and cardiac failure), skin (angiokeratoma, hypo-hidrosis), kidneys, upper respiratory tract, lungs, and intestine.

LSDs are often responsible for physical and neurological disabilities, and they have impacts onpatients’ health and life expectancy. For all these reasons, caring for patients with LSDs requires amultidisciplinary approach. Although remarkable progress in treatment has been made in recentyears, these disorders remain associated with important unmet medical needs and heavy burdensin terms of public health and economic costs.

LSDs are caused by mutations in genes encoding soluble acidic hydrolases, integral membraneproteins, activator proteins, transporter proteins, or nonlysosomal proteins that are necessaryfor lysosomal function. Functional deficiencies in these proteins trigger a pathogenetic cascadethat leads to intralysosomal accumulation of undegraded substrates in multiple tissues and organs(Figure 1).

Secondary impairment of other lysosome-related pathways may contribute to the pathologyof LSDs. A prominent pathological feature of Pompe disease, a progressive myopathy caused byacid α-glucosidase (GAA) deficiency, is an expansion of the autophagic compartment in muscles(9, 10). In multiple sulfatase deficiency (MSD), which is caused by defective posttranslationalactivation of sulfatase-modifying factor 1 (SUMF-1) and simultaneous deficiency of all sulfatases,aberrant autophagy results from impaired fusion between autophagosomes and lysosomes (11).In Niemann–Pick disease type C1, a neurodegenerative disorder caused by mutations in theNPC1 gene, sphingosine storage in lysosomes causes calcium depletion in these organelles,which results in secondary storage of cholesterol, sphingomyelin and glycosphingolipid (12). Theabnormal composition of lysosomal membranes affects vesicle fusion and trafficking in MSD andin mucopolysaccharidosis (MPS) type IIIA, which is caused by heparan sulfamidase deficiency(13).

Perturbation of lysosomal function may also lead to less obvious consequences. A link hasnow been well established between impairment of lysosomal–autophagic pathways and Parkinsondisease, which is the most prevalent neurodegenerative disorder; histopathologically, Parkinsondisease is characterized by the accumulation of insoluble aggregates of the presynaptic protein

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Nucleus

Endoplasmicreticulum

Golgiapparatus

Autophagy

TGN

Lysosomes

Endocytic pathway

Exocytosis

Synthesis of amutant protein

Gene mutationCorrection of genetic information:

Gene therapy

Enhancing lysosomalexocytosis

Substrate storage

Secondary cellularabnormalities

Correction ofsecondary cellular

abnormalities

Inhibition ofsubstrate synthesis

Improving folding and stability ofmutant enzymes

Pharmacological chaperonesProteostasis regulators

Degradation ofmutant protein

Deficiency of lysosomalenzyme activities

Enzyme therapies

Cross-correctionby HSCT andgene therapy

Figure 1The pathogenetic cascade of lysosomal storage diseases and the therapeutic approaches to treating these disorders. Lysosomal storagediseases are caused by mutations in genes encoding proteins involved in the lysosomal functions. Missense mutations may causedegradation and retention in the endoplasmic reticulum, abnormal glycosylation, and mistrafficking of the mutant protein through theGolgi apparatus, trans-Golgi network (TGN), and to the lysosomes. Deficiency of lysosomal enzymes causes intra-lysosomalaccumulation of undegraded substrates and secondary abnormalities of cellular pathways. Approaches developed to treat lysosomalstorage diseases are based on different strategies, each targeted to a specific event in the pathogenetic cascade. The gene mutation maybe corrected by delivering a wild-type copy of the mutated gene, which can direct the synthesis of the normal enzyme in the patient’scells. The mutant enzyme may be stabilized and protected from degradation, thus increasing its residual activity, with pharmacologicalchaperones or proteostasis regulators. The normal enzyme may be provided as a recombinant protein through the endocytic pathway ofthe patients’ cells by using intravenous administration (enzyme replacement therapy, ERT). Alternatively, the normal enzyme can beprovided as a precursor or secreted into the circulation by engineered patient cells or by allograft-transplanted cells (hematopoieticstem cell transplantation; HSCT). Other strategies are directed toward reducing substrate synthesis by enhancing the clearance ofsubstrates from cells and tissues, or by manipulating specific cellular pathways (such as those involved in vesicle trafficking).

α-synuclein in typical intraneuronal inclusions (Lewy bodies) by the selective loss of dopaminergicneurons in the substantia nigra, and clinically by movement and postural defects. Although themechanisms underlying this connection have not been fully elucidated, dysfunctions in severallysosomal proteins (and lysosomal gene mutations) have been implicated in the pathogenesis ofParkinson disease, with either loss-of-function or gain-of-function mechanisms. Examples of the

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dysfunctional proteins (or mutated genes) implicated in Parkinson disease are β-glucocerebrosidase (14, 15) and lysosomal type 5 P-type ATPase (ATP13A2) (16, 17).

Mutations in genes encoding essential components of the endolysosomal–autophagic path-way have also been described in other neurodegenerative diseases, including Alzheimer disease,Huntington disease, frontotemporal dementia, and Charcot-Marie-Tooth type 2B (18).

THE EVOLUTION IN PATIENT CARE

Until about two decades ago, the treatment of LSDs was exclusively based on multidisciplinarypalliative therapy, and this approach remains a cornerstone in the care of LSD patients. Supporttherapies include managing neurological complications (such as neurosurgical decompression ofthe cervical spine and treatment of hydrocephalus, the use of anticonvulsant medication, and assis-tance for patients with learning disability); providing noninvasive or invasive ventilatory support;managing the increased risks of anesthesia; offering orthopedic interventions to alleviate spinaldeformities, joint limitations, and retractions; providing nutritional support; treating cardiac in-volvement; and several others.

The past 25 years have been characterized by intensive and continual efforts to develop therapiesspecifically aimed at correcting the metabolic defects of these disorders. The approaches developedto treat LSDs are based on different strategies, each targeting a specific event in the pathogeneticcascade (Figure 1).

Most of these approaches are directed toward increasing the activity of the defective enzymeor protein. This can be achieved in different ways. The normal enzyme may be provided to thepatient’s cells through the endocytic pathway, by intravenous administration, as a recombinantprotein. Alternatively, the normal enzyme can be provided as a precursor secreted into the cir-culation by engineered cells from the patient or by an allograft of transplanted cells. The genemutation may be corrected by delivering a wild-type copy of the mutated gene that will direct thesynthesis of the normal enzyme in the patient’s cells. The mutant enzyme may be stabilized andprotected from degradation, thus increasing its residual activity.

Other strategies are directed towards restoring the equilibrium between the synthesis ofsubstrates and their degradation by lysosomal enzymes (the so-called storage equation of LSDs).This can be achieved by reducing substrate synthesis, by enhancing the clearance of substratesfrom cells and tissues, or by manipulating specific cellular pathways, such as those involved invesicle trafficking.

In this review we focus on some of the therapies that are currently approved and in clinical useor that appear to hold promise for future advancements in the treatment of LSDs.

Enzyme Replacement Therapy

Enzyme replacement therapy (ERT), considered the standard of care for several LSDs, is anexcellent example of how progress in the field of lysosomal biology prompted the developmentof novel therapeutic approaches. The logic behind ERT evolved from pivotal studies on themechanisms implicated in the sorting of newly synthesized lysosomal enzymes by way of theM6P and MPR pathways (19). Since the first studies in the early 1990s demonstrated the efficacyof ERT in Gaucher disease (20, 21), this approach has been extended to several other LSDs,including Fabry disease (22, 23), Pompe disease (24), and MPS I (25), II (26), and VI (27). Novelrecombinant enzymes are currently being tested for the treatment of MPS IVA, MPS VII, MPSIIIA, metachromatic leukodystrophy, and acid lipase deficiency (Table 1). Comprehensive reviewsof ERT are available (28, 29).

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Table 1 Approved treatments and selected clinical trials for lysosomal storage diseasesa

DiseaseApproved treatment; clinical trialtype of treatment, route of administration: therapeutic agent (company, sponsor)

Fabry disease Approved treatmentERT, IV: Agalsidase alfa (Shire)b; Agalsidase beta (Genzyme)Clinical trialERT, IV: PRX-102 (Protalix Biotherapeutics);SRT, O: Genz682452 (Genzyme, a Sanofi Company);ChT, O: Migalastat HCl (Amicus Therapeutics);ChT, O and ERT, IV: Migalastat HCl and either Agalsidase alfa or Agalsidase beta (Amicus Therapeutics);GT, IV: RV-GLA (National Institute of Neurological Disorders and Stroke, United States)

Gaucher disease type I Approved treatmentERT, IV: Imiglucerase (Genzyme); Velaglucerase alfa (Shire); Taliglucerase alfa (ProtalixBioTherapeutics)c

SRT, O: Miglustat (Actelion Pharmaceuticals); Eliglustat Tartrate (Genzyme Corp)c

Clinical trialChT, O: afegostat tartrate (Amicus Therapeutics); Ambroxol (Exsar Corporation);GT, IV: RV-GBA (National Institute of Neurological Disorders and Stroke, United States)

GM1 and GM2gangliosidoses

Clinical trialSRT, O: Miglustat and ketogenic diet (University of Minnesota - Clinical and TranslationalScience Institute, United States)

GM2 gangliosidosis Clinical trialChT, O: Pyrimethamine (The Hospital for Sick Children, Canada)

Lysosomal acid lipasedeficiency

Clinical trialERT, IV: Sebelipase alfa (Synageva BioPharma)

Alpha-mannosidosis Clinical trialERT, IV: Lamazym (Zymenex A/S)

Metachromaticleukodystrophy(late infantile)

Clinical trialERT, IV: Metazym (Shire)ERT, IT: HGT-1110 (Shire)GT, IV: LV-ARSA in CD34+ cells (IRCCS Ospedale San Raffaele/Fondazione Telethon, Italy)GT, IC: AAVrh.10-ARSA (Institut National de la Sante et de la Recherche Medicale, France)

MucopolysaccharidosisI

Approved treatmentERT, IV: Laronidase (Genzyme)Clinical trialERT, IT: Laronidase (Los Angeles Biomedical Research Institute, United States)

MucopolysaccharidosisII

Approved treatmentERT, IV: Idursulfase (Shire)Clinical trialERT, IV: Idursulfase beta (Green Cross Corporation, Republic of Korea)ERT, IV and IT: Idursulfase (Shire)GT, IV: RV-IDS (National Institute of Child Health and Human Development, United States)

MucopolysaccharidosisIIIA

Clinical trialERT, IT: HGT 1410 (Shire)GT, IC: AAVrh.10-SGHS and SUMF1 (Lysogene)

MucopolysaccharidosisIIIB

Clinical trialGT, IC: AAV5- NAGLU (Institut Pasteur, France)

(Continued )

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Table 1 (Continued)

DiseaseApproved treatment; clinical trialtype of treatment, route of administration: therapeutic agent (company, sponsor)

MucopolysaccharidosesIII A, B, C

Clinical trialSRT, O: Genistein aglycone (Central Manchester University Hospitals, United Kingdom)

MucopolysaccharidosisIVA

Approved treatmentERT, IV: Elosulfase alfa (BioMarin Pharmaceutical)

MucopolysaccharidosisVI

Approved treatmentERT, IV: Galsulfase (BioMarin Pharmaceutical)

MucopolysaccharidosisVII

Clinical trialERT, IV: recombinant human beta-glucuronidase (Ultragenyx Pharmaceutical)

Neuronal ceroidlipofuscinosis, lateinfantile (CLN2)

Clinical trialGT, IC: AAVrh.10-CLN2 (Weill Medical College, Cornell University, United States)ERT, IC: BMN 190 (BioMarin Pharmaceutical)

Niemann-Pick diseasetype B

Clinical trialERT, IV: recombinant human acid sphingomyelinase (Genzyme, a Sanofi Company)

Niemann-Pick diseasetype C

Approved treatmentSRT, O: Miglustat (Actelion Pharmaceuticals)b

Clinical trialOther, IC: 2-hydroxypropyl-beta-cyclodextrin (National Institute of Child Health and HumanDevelopment, United States)

Other, O: Vorinostat (National Institute of Child Health and Human Development, United States)Pompe disease Approved treatment

ERT, IV: Alglucosidase alfa (Genzyme)Clinical trialERT, IV: neoGAA (Genzyme, a Sanofi Company)ERT, IV: GILT-tagged recombinant human (BioMarin Pharmaceutical)ERT, IV: Alglucosidase alfa and albuterol (Duke University, United States)ERT, IV: Alglucosidase alfa and clenbuterol (Duke University, United States)ChT, O: duvoglustat HCl (Amicus Therapeutics)ERT, IV and ChT, O: Alglucosidase alfa and miglustat (Universita degli Studi di Napoli Federico II,Italy)

ERT, IV and ChT, O: Alglucosidase alfa and duvoglustat HCl (Amicus Therapeutics)GT, ID: AAV1-GAA (University of Florida, United States)

Abbreviations: AAV, adeno-associated viral vector; ARSA, arylsulfatase A gene; ChT, chaperone therapy; CLN2, neuronal ceroid lipofuscinosis 2 gene;ERT, enzyme replacement therapy; GILT, glycosylation-independent lysosomal targeting; GAA, alpha-glucosidase gene; GBA, beta-glucocerebrosidasegene; GLA,alpha-galactosidase A gene; GT, gene therapy; IC, intracerebral; ID, intradiaphragmatic; IDS, iduronate-2-sulfatase gene; IT, intrathecal; IV,intravenous; LV lentiviral vector; NAGLU, N-acetylglucosaminidase gene; O, oral; RV, retroviral vector; SGHS, N-sulfoglucosamine sulfohydrolasegene; SRT, substrate reduction therapy; SUMF-1, sulfatase-modifying factor 1 gene.aFurther information on approved treatments and clinical trials can be found on the Web. Useful addresses for registered clinical trials in the UnitedStates and Europe are: https://www.clinicaltrials.gov/; https://eudract.ema.europa.eu/.bApproved by the European Medicines Agency but not by the US Food and Drug Administration.cApproved by the US Food and Drug Administration but not by the European Medicines Agency.

Now, after the first phase of ERT development, there are sufficient clinical data to documentthe successes of this approach. However, important limitations have emerged, and it is necessaryto develop strategies to improve its efficacy.

Significant variability in clinical benefit has been observed among different patients. After aninitial period of improvement, the disease may again progress in some patients. In MPS I, II,

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and VI, the correction of pathology is insufficient in tissues such as bone, cartilage, and heart(30), which are major pathological sites. In Pompe disease, skeletal muscle pathology remainspartly refractory to ERT, particularly in patients with advanced stages of the disease (31, 32). Thebenefits and cost effectiveness of ERT have been carefully evaluated in a multicenter study in theUnited Kingdom (33).

A major drawback of ERT has been the limited bioavailability of intravenously injected re-combinant enzymes. Recombinant enzymes are large molecules that do not freely diffuse acrossmembranes and are unable to reach therapeutic concentrations in some target tissues, particularlythe brain. A major challenge in the coming years will be to discover a means that allows recom-binant enzymes to cross the blood–brain barrier (BBB). Given that approximately two-thirds ofLSDs cause neurological symptoms and progressive neurodegeneration, the major therapeuticgoals in treating many LSDs are to obtain corrective enzyme levels in the brain and correct thepathology in the central nervous system (34, 35).

Strategies to improve the delivery of enzymes to the central nervous system are currentlyundergoing preclinical and clinical evaluation. For example, β-glucuronidase, which is deficientin MPS VII, has been chemically modified to increase its plasma half-life and facilitate its trafficthrough the BBB (36, 37). Other approaches, based on the use of so-called Trojan horses, relyon chimeric enzymes fused with peptides to allow penetration of the BBB and brain deliverythrough specific pathways (such as the apolipoprotein and receptor pathways). These approacheshave been evaluated in preclinical studies for α-iduronidase (38, 39), iduronate-2-sulfatase (40),arylsulfatase A (41), and tripeptidyl peptidase I (42). Sorrentino et al. (43) showed that a chimericheparan sulfamidase, produced by liver cells after adeno-associated virus (AAV) 2/8 gene delivery,could cross the BBB via transcytosis and correct the enzyme deficiency in a mouse model of MPSIIIA. The chimeric enzyme was engineered by adding the signal peptide from the highly secretediduronate-2-sulfatase and the BBB-binding domain of apolipoprotein B.

In addition to these approaches, preclinical studies are evaluating the use of invasive proceduresto deliver recombinant enzymes directly into the cerebrospinal fluid as treatment for severallysosomal disorders. The objectives of these approaches are to alleviate spinal cord compressionand to improve the neurological or cognitive outcome of patients. Intrathecal administrationand lumbar or cisterna magna puncture have been studied in animal models of various typesof mucopolysaccharidoses (44–47). Intrathecal ERT has been translated into human therapy formucopolysaccharidoses types I and VI (48). Devices for continuous intrathecal infusion have beendeveloped and tested in preclinical studies. Some clinical trials of intrathecal administration ofERT are ongoing, and others have been completed (Table 1).

Attempts have also been made to improve the efficiency of ERT targeting muscle in Pompedisease by enriching the recombinant enzyme with M6P moieties (49, 50) or by producing achimeric enzyme fused with a peptide derived from insulin-like growth factor 2 (51, 52). Enhancedlysosomal transport of recombinant enzymes has also been obtained in animal models of Pompeand Fabry diseases by coupling the enzymes with polymer nanocarriers coated with an antibodythat is specific to intercellular adhesion molecule-1 (ICAM-1) (53, 54).

An alternative strategy to improve muscle targeting by recombinant human GAA is based onthe enhancing effect of β2-agonists on the expression of the MPR at the plasma membrane ofmyofibers (55, 56). The use of β-2-agonists as adjunctive therapy to ERT is being evaluated in aclinical trial (Table 1).

Another limitation of ERT is the patient’s immune response to recombinant enzymes, mainlyin patients who are cross-reactive material–negative (57). Increased antibody titers have been asso-ciated with severe immune reactions and a reduced clinical efficacy for ERT. Immune modulationhas been used to prevent these problems in some LSDs, such as Pompe disease (58, 59).

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Novel manufacturing procedures are being developed, and these are expected to lower the highcosts of ERT. Treating a single LSD patient may cost as much as several hundred thousand dollarsper year. Such an expense may limit patients’ access to ERT and heavily impact the budgets ofnational health systems (33). Recombinant β-glucocerebrosidase has been manufactured in plantsor plant-cell expression systems, and in 2012 it was approved for clinical use to treat Gaucherdisease (60). Other plant-based production processes have been either reported in the literatureor patented (61–63).

Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation uses hematopoietic stem cells derived from a healthydonor as a therapeutic agent. These cells have a dual effect: they can both repopulate specific tissuesand secrete functional lysosomal hydrolases in the extracellular space and into blood circulation.The secreted normal enzyme can be recaptured by the recipient cells and cross-correct the enzymedefect in these cells. This approach is effective only for some LSDs; it has been shown to beineffective for several others. The best results are obtained when the procedure is performed in arestricted therapeutic window, early in the course of the disease, and in specific subsets of patients.Thus, guidelines have been developed to determine which patients are eligible and to promotetimely intervention (64, 65).

Pharmacological Chaperone Therapy

Pharmacological chaperone therapy is based on the concept that loss-of-function diseases areoften caused by missense mutations that disrupt the three-dimensional conformation of mutantproteins. Misfolded proteins may be recognized by the quality control systems of the endoplasmicreticulum (ER) and degraded, retained in the ER, or abnormally glycosylated and mistrafficked(66). Thus, in these protein misfolding diseases the loss of function is not due to the loss of catalyticactivity but rather is the result of degradation or inappropriate trafficking of the aberrant protein.Small-molecule ligands (pharmacological chaperones) can interact with mutant proteins, favortheir native conformation, enhance their stability, and allow for correct trafficking. As a result,the enzymatic activity of the mutant protein is partially rescued. Chaperone therapy has beenproposed as a feasible strategy for treating some LSDs (66–68) and is presently under evaluationin phase I/II clinical trials (Table 1).

An important evolution in pharmacological chaperone therapy was the demonstration thatsome chaperones not only are able to rescue the endogenous, mutant, misfolded proteins, butcan also enhance the physical stability, and possibly the efficacy, of the wild-type recombinantenzymes that are commonly used for ERT. This effect was demonstrated in preclinical in vitroand in vivo studies for Pompe, Fabry, and Gaucher diseases (69–73). Some trials evaluating thepotential of the combination of ERT and chaperones are in progress, and others have alreadybeen completed (74) (Table 1).

Proteostasis Regulators

Other small-molecule-based approaches have been proposed to rescue the mutated enzymes inLSDs. Chaperones are ligands that specifically interact with mutant proteins (and are thus able torescue a single protein), but proteostasis regulators can adjust the capacity for proteostasis, a com-plex network that controls protein synthesis, folding, trafficking, aggregation, and degradation.Regulation of proteostasis has been proposed as a strategy to treat two distinct LSDs caused by adeficiency of β-hexosaminidase A, Gaucher disease and GM2 gangliosidosis (Tay–Sachs disease)

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(75). Regulation of proteostasis has also been achieved through the overexpression of the TFEBgene in Gaucher disease fibroblasts (76). Downregulation of the ER-resident protein FKBP10also resulted in enhanced proteostasis of β-glucocerebrosidase (77).

Substrate Reduction Therapy

Substrate reduction therapy inhibits specific steps in the biosynthetic pathways of substrates torestore the equilibrium between the synthesis of substrates and their degradation by lysosomalenzymes (78, 79). This task is generally accomplished by using small-molecule enzyme inhibitorsthat are involved in substrate biosynthesis. A substrate-reducing agent, miglustat (Actelion Phar-maceuticals, San Francisco, CA), has already been approved for clinical use to treat type 1 Gaucherdisease (80) and Niemann–Pick disease type C (81). A novel substrate inhibitor, eliglustat tartrate(Genzyme, a Sanofi Company, Cambridge, MA), has been introduced recently and evaluated in aphase II clinical trial (82) for treating Gaucher disease. The flavonoid genistein has been proposedas a treatment for MPS (83). A phase III clinical trial using high-dose oral genistein aglycone is inprogress (Table 1).

Gene Therapy

Gene therapy is directed toward increasing or restoring defective enzyme activity in patients’ cellsand tissues by delivering a wild-type copy of the defective gene. LSDs appear to be excellentcandidates for gene therapy (84). These disorders are monogenic and, in principle, it is possible tocure the disease by correcting the gene defect. In addition, small increases in enzymatic activity (aslittle as <10%) may be sufficient to produce clinical benefit and phenotypic correction of LSDs.

Although this approach is generally based on delivering the therapeutic gene to patients usingviral vectors as carriers, a broad range of different strategies may be exploited, depending on thetissues that need to be targeted and on the characteristics of the protein or enzyme that must bereplaced.

A first approach is to correct the genetic information in all of the patient’s cells and tissues bysystemically delivering the therapeutic gene under the control of a ubiquitous promoter in orderto allow synthesis of the wild-type protein in situ. However, because most lysosomal hydrolases aresecreted and can be recaptured by neighboring or distant cells via the MPR pathway, an alternativestrategy may be based on transducing specific cells or organs that may act as protein factories andthus cross-correct other tissues. This approach has advantages when compared with ERT becausefactory cells provide stable and sustained amounts of the normal enzyme in the blood circulation.

Transduction of specific tissues or cells can be achieved in various ways. An in vivo strategymay use systemic delivery and a tissue-specific promoter or direct injection into a target organ.Alternatively, an ex vivo strategy has also been developed that engineers a patient’s bone marrowcells and then injects the engineered cells back into the patient.

Depending on the delivery strategy being used, different viral vectors and promoters thatcontrol the expression of the gene of interest can be used as therapeutic agents. Currently, AAVsare the vector of choice for in vivo gene therapy, and lentiviruses are the vectors of choice for exvivo gene therapy.

Preclinical data have been obtained from small- and large-animal models of LSDs for all ofthese strategies and viral vectors. Some viral vectors have been designated as orphan drugs and areto be tested in clinical trials (Table 1). For some diseases—including MPS II, MPS IIIA, MPS VI,Pompe disease, neuronal ceroid lipofuscinoses, Gaucher disease, and Fabry disease—clinical trialsare already in progress (Table 1). The ex vivo approach has been successfully used in a clinical

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trial to treat patients affected by metachromatic leukodystrophy (MLD), a severe and progressiveneurodegenerative LSD caused by arylsulfatase A (ARSA) deficiency. A lentivirus vector has beenused to transfer a functional ARSA gene into hematopoietic stem cells from three patients withpresymptomatic, late-infantile MLD (85). After reinfusion of the gene-corrected hematopoieticcells, all patients showed correction of enzyme levels and arrest of neurodegeneration, indicatingthe clinical efficacy of this approach.

ADVANCES IN UNDERSTANDING PATHOPHYSIOLOGY AND ITSIMPACT ON THE DEVELOPMENT OF TREATMENTS

In recent years novel areas of therapeutic intervention have been identified in addition to thosetypically directed towards correcting the enzyme defect and reducing storage. Some of the exam-ples below clearly show that as the lysosomal biology is revealed, new therapeutic strategies canbe devised, in most cases based on the manipulation of specific molecular pathways.

Manipulation of the autophagic pathway has been reported as a potential target for therapyin Pompe disease (86). Genetic suppression of autophagy in the animal model of Pompe diseaseresulted in a substantial reduction in glycogen accumulation in skeletal muscle and in improvedefficacy of ERT.

Stimulation of lysosomal exocytosis has also been identified as a strategy for treating LSDs.Lysosomes are able to secrete their contents through a Ca2+-dependent process. Medina et al.(87) showed that this process is modulated by TFEB, and that overexpression and activation ofTFEB result in enhanced clearance of substrates in cells in two LSDs: MSD and Pompe disease.Overexpression of TFEB reduced glycogen load and lysosomal size, improved autophagosomeprocessing, and alleviated excessive accumulation of autophagic vacuoles in cultured myoblastsfrom a Pompe disease murine model (88). In vivo TFEB gene delivery mediated by AAV2/1 ad-ministered by intramuscular injection resulted in nearly complete glycogen clearance and restoredmuscle architecture. TFEB-induced cellular clearance of stored substrates through enhanced exo-cytosis appears to be particularly attractive because it may be obtained in all LSDs, irrespective ofthe metabolic defect.

Cyclodextrin, a cholesterol-sequestering agent, has been shown to mobilize cholesterol fromlate endosomes and lysosomes, bypassing the functions of the genes NPC1 and NPC2, which areinvolved in Niemann–Pick disease type C. Clinical trials using cyclodextrin to treat this disorderare in progress (Table 1). In the same disorder, other studies have shown that depletion oflysosomal Ca2+ stores is another potential target for therapeutic intervention and that curcumincould compensate for this calcium defect in lysosomes (12). The combined use of curcumin andthe nonsteroidal anti-inflammatory drug ibuprofen led to improved neuroprotection in an NPC1-defective mouse model (89).

Heat-shock protein 70 (Hsp70) has been shown to enhance lysosomal stability by modulatingthe sphingolipid composition of membranes. In cells from patients with Niemann–Pick diseasetype A, acid sphingomyelinase deficiency could be partially restored by treatment with Hsp70,indicating that this may be an additional strategy for treating LSDs (90, 91).

CONCLUSIONS

In spite of the remarkable progress that has been made in treating LSDs, these disorders stillrepresent a large and growing unmet medical need. Future research must confront importantchallenges. Relatively short-term tasks are to translate into clinical use the strategies that arecurrently under development and to optimize existing therapies. In particular, it will be important

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to improve the bioavailability and targeting of therapeutic agents, to reduce the impact of therapieson patients’ quality of life, and to lower the costs of therapies.

Researchers also need to explore the efficacy of therapeutic protocols based on the combinationof different approaches, to address all the manifestations of multisystem disorders such as LSDs,and to consider the development of personalized therapies. Finally, a major and exciting challengewill be to improve the understanding of the pathophysiology of LSDs to identify new targets fortherapy.

DISCLOSURE STATEMENT

G.A. received travel support for meeting attendance from BioMarin Pharmaceuticals, Inc.; Acte-lion Pharmaceuticals Ltd.; Genzyme, a Sanofi Company; and Shire Pharmaceuticals Ltd. G.A. alsoreceived consultancy fees from Actelion Pharmaceuticals Ltd.; Genzyme, a Sanofi Company; andShire Pharmaceuticals Ltd. A.B. received funding from the European Research Council, Fon-dazione Telethon, Team Sanfilippo Foundation, San Filippo Children’s Research Foundation,Center for Orphan Disease Research and Therapies, Beyond Batten Disease Foundation, andHuffington Foundation.

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Annual Review ofMedicine

Volume 66, 2015Contents

A Tale of Two Tumors: Treating Pancreatic and ExtrapancreaticNeuroendocrine TumorsDaniel M. Halperin, Matthew H. Kulke, and James C. Yao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Metformin in Cancer Treatment and PreventionDaniel R. Morales and Andrew D. Morri � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �17

Neoadjuvant Therapy for Breast CancerDimitrios Zardavas and Martine Piccart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �31

Neuroblastoma: Molecular Pathogenesis and TherapyChrystal U. Louis and Jason M. Shohet � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �49

Pharmacogenetics of Cancer DrugsDaniel L. Hertz and James Rae � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Recent Therapeutic Advances in the Treatment of Colorectal CancerKristen K. Ciombor, Christina Wu, and Richard M. Goldberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � �83

Regulation of Tumor Metastasis by Myeloid-Derived Suppressor CellsThomas Condamine, Indu Ramachandran, Je-In Youn, and Dmitry I. Gabrilovich � � � � � �97

Targeting HER2 for the Treatment of Breast CancerMothaffar F. Rimawi, Rachel Schiff, and C. Kent Osborne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

The DNA Damage Response: Implications for Tumor Responses toRadiation and ChemotherapyMichael Goldstein and Michael B. Kastan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 129

Pathogenesis of Macrophage Activation Syndrome and Potential forCytokine-Directed TherapiesGrant S. Schulert and Alexei A. Grom � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 145

Cardiovascular Disease in Adult Survivors of Childhood CancerSteven E. Lipshultz, Vivian I. Franco, Tracie L. Miller, Steven D. Colan,

and Stephen E. Sallan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Advances in Nanoparticle Imaging Technology forVascular PathologiesAnanth Annapragada � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 177

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Vasopressin Receptor Antagonists, Heart Failure, and PolycysticKidney DiseaseVicente E. Torres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 195

ADAMTS13 and von Willebrand Factor in ThromboticThrombocytopenic PurpuraX. Long Zheng � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 211

Current Therapies for ANCA-Associated VasculitisLindsay Lally and Robert Spiera � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

Changing Practice of Anticoagulation: Will Target-SpecificAnticoagulants Replace Warfarin?Gowthami M. Arepally and Thomas L. Ortel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241

The Mechanisms and Therapeutic Potential of SGLT2 Inhibitors inDiabetes MellitusVolker Vallon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 255

Extranuclear Steroid Receptors Are Essential for SteroidHormone ActionsEllis R. Levin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Impact of the Obesity Epidemic on CancerPamela J. Goodwin and Vuk Stambolic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 281

Obesity and Cancer: Local and Systemic MechanismsNeil M. Iyengar, Clifford A. Hudis, and Andrew J. Dannenberg � � � � � � � � � � � � � � � � � � � � � � � � 297

The JAK-STAT Pathway: Impact on Human Disease and TherapeuticInterventionJohn J. O’Shea, Daniella M. Schwartz, Alejandro V. Villarino,

Massimo Gadina, Iain B. McInnes, and Arian Laurence � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 311

Management of Postmenopausal OsteoporosisPanagiota Andreopoulou and Richard S. Bockman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

The Gut Microbial Endocrine Organ: Bacterially Derived SignalsDriving Cardiometabolic DiseasesJ. Mark Brown and Stanley L. Hazen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 343

H7N9: Preparing for the Unexpected in InfluenzaDaniel B. Jernigan and Nancy J. Cox � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

Treatment of Recurrent and Severe Clostridium Difficile InfectionJ.J. Keller and E.J. Kuijper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 373

Emerging Technologies for Point-of-Care Managementof HIV InfectionHadi Shafiee, ShuQi Wang, Fatih Inci, Mehlika Toy, Timothy J. Henrich,

Daniel R. Kuritzkes, and Utkan Demirci � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

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Understanding HIV Latency: The Road to an HIV CureMatthew S. Dahabieh, Emilie Battivelli, and Eric Verdin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Lessons from the RV144 Thai Phase III HIV-1 Vaccine Trial and theSearch for Correlates of ProtectionJerome H. Kim, Jean-Louis Excler, and Nelson L. Michael � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 423

T Cell–Mediated Hypersensitivity Reactions to DrugsRebecca Pavlos, Simon Mallal, David Ostrov, Soren Buus, Imir Metushi,

Bjoern Peters, and Elizabeth Phillips � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 439

Synthetic Lethality and Cancer Therapy: Lessons Learned from theDevelopment of PARP InhibitorsChristopher J. Lord, Andrew N.J. Tutt, and Alan Ashworth � � � � � � � � � � � � � � � � � � � � � � � � � � � � 455

Lysosomal Storage Diseases: From Pathophysiology to TherapyGiancarlo Parenti, Generoso Andria, and Andrea Ballabio � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 471

From De Novo Mutations to Personalized Therapeutic Interventionsin AutismWilliam M. Brandler and Jonathan Sebat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 487

Ketamine and Rapid-Acting Antidepressants: A Window into a NewNeurobiology for Mood Disorder TherapeuticsChadi G. Abdallah, Gerard Sanacora, Ronald S. Duman, and John H. Krystal � � � � � � � � 509

Indexes

Cumulative Index of Contributing Authors, Volumes 62–66 � � � � � � � � � � � � � � � � � � � � � � � � � � � 525

Cumulative Index of Article Titles, Volumes 62–66 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 529

Errata

An online log of corrections to Annual Review of Medicine articles may be found athttp://www.annualreviews.org/errata/med

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