Inherited hematological disorders due to defects incoat protein (COP)II complex

Roberta Russo,1 Maria Rosaria Esposito,1 and Achille Iolascon1,2*

Many diseases attributed to trafficking defects are primary disorders of protein folding and assembly. However,an increasing number of disease states are directly attributable to defects in trafficking machinery. In this con-text, the cytoplasmic coat protein (COP)II complex plays a pivotal role: it mediates the anterograde transport ofcorrectly folded secretory cargo from the endoplasmic reticulum towards the Golgi apparatus. This reviewattempts to describe the involvement of COPII complex alteration in the pathogenesis of human genetic disor-ders; particularly, we will focus on two disorders, the Congenital Dyserythropoietic Anemia type II and theCombined Deficiency of Factor V and VIII. Am. J. Hematol. 00:000–000, 2012. VVC 2012 Wiley Periodicals, Inc.

IntroductionEukaryotic protein homeostasis, termed proteostasis, is

maintained by a network of pathways controlling proteinsynthesis, folding, trafficking, aggregation, disaggregation,and degradation [1]. In particular, the proteostasis networkis involved in maintaining the dynamic interplay betweenfolding and export of proteins [2]. Secretion is a fundamen-tal function of every cell. Current knowledge on proteinsecretion originates �30 years ago by the work of Paladeand colleagues [3]. This work first established the vesicletransport hypothesis, which states that the transfer of cargomolecules between organelles of secretory pathway ismediated by shuttling transport vesicles [4]. Accordingly tothis theory, newly synthesized secretory proteins passthrough a series of membrane-enclosed organelles includ-ing the endoplasmic reticulum (ER), the Golgi complex, andsecretory granules, before they reach the extracellularspace. Even proteins destined for residence at the plasmamembrane, endosomes, or lysosomes share the earlysteps of this pathway (i.e., the ER and the Golgi complex)with secreted proteins. The eukaryotic secretory pathway isresponsible for delivery of a wide number of proteins totheir specific cell location. Secretory proteins contain sort-ing elements that are recognized by the intracellular trans-port machinery at multiple steps of the way from synthesisto specific location [5]. Proper selection of appropriate pro-teins for incorporation into nascent vesicles is crucial forsecretory pathway homeostasis [6]. Coat protein complexesare major components of this machinery. Three well-char-acterized coat complexes, clathrin, and coat protein com-plexes I and II (COPI and COPII), have been described;they are multisubunit complexes that recognize specific pro-tein sorting signals and they selectively sort proteins intocarrier vesicles.This review describes fundamental principles as well as

contemporary aspects of protein intracellular trafficking pro-viding new insights that have emerged in the field of hema-tology diseases in recent years. Since protein intracellularpathway has been extensively covered by excellent reviews[4,6], it will not be covered in details in this review.In this review we will describe human genetic disorders

associated with defects in COPII machinery. In particular,we will focus on two COPII-related genetic disorders: theCongenital Dyserythropoietic Anemia type II (CDA II) andthe Combined Deficiency of Factor V and VIII (F5F8D).

Trafficking pathway ER–GolgiTransportation can be divided into four main phases: ves-

icle budding, the selection of proteins to transport, address-ing, and the fusion of the vesicle membrane with the target.

The vesicle bud by a membrane called the donor, whichalso allows the selective incorporation of cargo proteins inthe vesicle and retain residents in the donor compartment.These vesicles are directed to specific target compartmentswhere they can deliver their loads after fusion with themembrane [3]. The export of proteins from the ER hasbeen well defined in yeast, both S. cerevisiae [7] and P.pastoris [8], and in mammalian cells [9]. Figure 1 shows adiagrammatic representation of this pathway: nascent se-cretory proteins are translated and folded at the ER andthen packaged into COPII vesicles for anterograde trans-port to pre-Golgi and Golgi compartments. Forward trans-port of folded secretory proteins in COPII vesicles is alsobalanced by a retrograde transport pathway that relies onCOPI to recycle vesicle components and retrieve escapedER resident proteins (Fig. 1). While in S. cerevisiae vesiclebudding appears to occur stochastically across the entireER membrane, in P. pastoris and in mammalian cells, thisevent is highly organized, occurring at discrete sites calledtransitional ER (tER) or ER exit sites (ERES) [3,9–11].These sites are relatively immobile structures and face outtowards assemblies of ERGIC clusters (ER-Golgi intermedi-ate compartment) [12,13]. In the latter compartment, cargoproteins are separated from ER resident proteins and trans-ported to the cis-Golgi along microtubules. Conversely, ERresident proteins and cargo receptors, that display ER-re-trieval signals, are returned to the ER via COPI vesicles.Following cotranslational translocation, proteins destined tothe secretory pathway acquire their native conformationwithin the ER. A robust quality control system operates inthe ER to ensure that only properly folded proteins are

Conflict of interest: Nothing to report

*Correspondence to: Achille Iolascon, CEINGE – Biotecnologie Avanzate,Via Gaetano Salvatore 486, 80145 Naples, Italy. E-mail: achille.iolascon@unina.it

1CEINGE Biotecnologie Avanzate, University Federico II of Naples, Naples,Italy; 2Department of Biochemistry and Medical Biotechnologies, UniversityFederico II of Naples, Naples, Italy

Contract grant sponsor: Italian Ministero dell’Universita e della Ricerca;Contract grant number: MUR-PS 35-126/Ind.

Contract grant sponsor: Regione Campania; Contract grant numbers:DGRC2362/07, LSHM-CT-2006-037296.

Contract grant sponsor: Italian Telethon Foundation; Contract grantnumber: GGP 09044.

Received for publication 6 April 2012; Revised 28 May 2012; Accepted 7 June2012

Am. J. Hematol. 00:000–000, 2012.

Published online in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/ajh.23292

Critical Review

VVC 2012 Wiley Periodicals, Inc.

American Journal of Hematology 1 http://wileyonlinelibrary.com/cgi-bin/jhome/35105

allowed into transport vesicles [14]. Nascent cargo isretained and/or not recognized by the export machineryuntil the cargo is fully folded and assembled [14,15].

COPII complex assembly and evolutionMost of the sequential assembly of COPII machinery has

been well defined in yeast. COPII recruitment is initiated bythe activation of the small GTPase Sar1 by its ER-localizedguanine exchange factor (GEF), Sec12. Sar1 initiates vesi-cle formation on ER membranes through the exchange ofGDP for GTP, which induces tight membrane association ofSar1 and the subsequent recruitment of the heterodimericcomplex comprising of Sec23/24 COPII components.Sec23 is a GTPase activating protein (GAP) that stimulatesthe enzymatic activity of Sar1, whereas Sec24 is the adap-tor protein that captures specific cargo into the nascentvesicle. The Sar1-GTP/Sec23/Sec24 ‘‘pre-budding’’ com-plex in turn recruits the Sec13/Sec31 heterotetramer, whichforms the outer layer of the COPII coat, a flexible coatcage that can accommodate various sizes of vesicles, andlikely functions to cross-link adjacent prebudding complexesand complete the vesicle biogenesis process [16,17] (Fig.2). Two elegant studies [7,18] have identified many of themolecular steps in the anterograde protein trafficking; how-ever, they remained confined to unicellular yeast or mam-malian cells in culture as model systems. Less is knownabout the behavior of the secretory pathway within the con-text of the entire multicellular organism and how its mal-function might influence development and organ homeosta-sis. The increase in size of eukaryotic cell was accompa-nied by an increase in complexity of the molecularmachinery of these transport routes, i.e., number and typesof coat proteins, regulatory GTPases, fusion and recruit-ment factors [19]. One possible way in which evolutionmodifies function is to develop protein orthologues and iso-forms, i.e., by gene duplication and/or by alternative mRNAsplicing. Mammalian orthologues have been identified foreach of the five core COPII components and, in somecases, multiple isoforms of these proteins exist, eachencoded by a different gene. These are generally indicatedby an alphabetical suffix. Two mammalian isoforms of Sar1and Sec23, and four mammalian isoforms of Sec24 havebeen reported [20–23]. Only one form of mammalianSec13 has been described [24]. To date, only two mamma-

lian proteins corresponding to the Sec31p yeast protein,Sec31A [25,26] and Sec31B [27], have been identified.

COPII-related human genetic disordersPresently, mutations in three components of the COPII

core trafficking machinery have been assigned to humangenetic disorders: they are the Chylomicron Retention Dis-ease (CMRD), the Cranio-lenticulo-sutural dysplasia (CLSD),and the Congenital Dyserythropoietic Anemia type II (CDAII). We propose a classification of these diseases on the ba-sis of the major clinical findings. However, this classificationis only for a didactic purpose. So, we classified CMRD andCLSD as non-hematological disorders; conversely, CDA IIwill be discussed in the same section of the Combined Defi-ciency of Factor V and VIII (F5F8D), which is due to defectsof F5/F8 selective cargo receptor (Table I).

Non-hematological disordersChylomicron Retention Disease (CMRD) (OMIM

246700), or Anderson’s disease, is a fat malabsorption dis-order, in which enterocytes fail to secrete chylomicrons inlymph after a fat meal. This defect is usually diagnosed ininfants presenting with failure to thrive, chronic diarrhea,low plasma vitamin E levels, hypocholesterolemia and hy-pobetalipoproteinemia with selective absence of apoB48 inthe post prandial state [28]. The low plasma lipid levels andlow fat-soluble vitamin levels commonly cause mild periph-eral neuropathy with diminished or absent deep tendonreflexes and definite or borderline mental retardation. How-ever, neurological signs may develop more frequently laterin untreated individuals [29,30]. A typical histological findingin the intestinal biopsy has been noted, with a distinctivewhite stippling, resembling hoar frosting, covering the mu-cosal surface of the small intestine. The enterocytes con-tain accumulations of large lipid droplets free in the cyto-plasm as well as membrane-bound lipoprotein-sized struc-tures [30–32]. In 1987, in vitro studies of small intestinalexplants from CMRD patients showed a normal apoB-48protein synthesis, but impaired chylomicron synthesis inview of the altered glycosylation. The authors postulated adefect in the formation and secretion of chylomicronsresulting from a defect in glycosylation [33]; this observa-tion was the prelude to the identification of the causativegene, 16 years later. Indeed, after the exclusion of APOBas causative gene [34], in 2003 Jones et al. demonstratedthat the CMRD is caused by missense substitutions inSAR1B, one of two paralogous Sar1 proteins in humans[35] (Fig. 2). SAR1B mutations lead to an impaired chylomi-cron trafficking between ER and Golgi, with a subsequentaccumulation of prechylomicron transport vesicles in thecytoplasm of the enterocytes [29]. CMRD is a very rare

Figure 2. Model for COPII vesicle assembly. In yeast, COPII-coated vesiclesform by the sequential binding of Sar1-GTP, the inner complex proteins Sec23-Sec24 and the outer complex components Sec13-Sec31 on the endoplasmic retic-ulum (ER). The transport of both integral membrane cargo and soluble secretorycargo is shown. [Color figure can be viewed in the online issue, which is availableat wileyonlinelibrary.com.]

Figure 1. Schematic representation of ER-Golgi transport. After translation, foldednascent proteins ( ) are exported from the ER in COPII anterograde transportvesicles ( ). In mammalian cells, COPII vesicles generate a structure known asERGIC. The ERGIC is a site for concentrating retrograde cargo into COPI vesicles( ), which bud from pre-Golgi and Golgi compartments to recycle vesicle compo-nents and retrieve resident proteins ( ) that have escaped the ER. [Color figurecan be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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recessively inherited disease with less than 50 cases hav-ing been reported in the literature. The diagnosis is oftendelayed because symptoms are nonspecific, but recentlyclinical guidelines for the diagnosis, follow-up, and treat-ment have been proposed [36].The Boyadjiev-Jabs syndrome or Cranio-lenticulo-sutural

dysplasia (CLSD) (OMIM 607812) is an autosomal reces-sive disorder characterized by late-closing fontanels, suturalcataracts, facial dysmorphisms, and skeletal defects. CLSDwas originally mapped to chromosome 14q13-q21 in fivemales and one female from a large consanguineous SaudiArabian family of Bedouin descent [37]. Subsequently, ithas been showed that this disorder arises from a missensesubstitution (F382L) in SEC23A, a gene encoding one oftwo paralogous proteins of the inner layer of COPII vesicle[38] (Fig. 2). Cell-free vesicle budding assays show that theF382L-SEC23A protein retains many aspects of wild-typefunction, including Sec24 binding, membrane binding andintrinsic GAP activity. However, the mutant protein showsreduced recruitment of the Sec13-Sec31 outer coat com-plex, especially when paired with SAR1B, indicating distinctaffinities of the two human Sar1 paralogs for the SEC13-SEC31A complex [39].Studies on Sec23a-deficient embryos of zebrafish sug-

gested that disrupted ER export of the secretory proteinsrequired for normal morphogenesis, such as collagen typeII, accounts for CLSD [40]. This alteration leads mutantchondrocytes to accumulate large amount of matrix pro-teins into extended ER compartments and proteasomes,unable to transport them to the Golgi for posttranslationalmodifications and eventually to the extracellular matrix.Lang et al. hypothesize that chondrocytes sense the ab-sence of extracellular matrix maintaining abnormally hightranscription of collagen genes. This excessive level of tran-scription might further exacerbate accumulation of unfoldedproteins in the ER and trigger the response of the ER qual-ity control system [40]. Similarly to those observed inSec23a-deficient zebrafish embryos, nonsense mutations inSec23 were also found to have serious developmental con-sequences in worms [41].

Hematological disordersCongenital dyserythropoietic anemia type II. Congeni-

tal Dyserythropoietic Anemia type II (CDA II) (OMIM224100) is an autosomal recessive disorder characterizedby ineffective erythropoiesis: this is suspected if there aresymptoms and signs of increased hemoglobin turnover,such as mild jaundice due to indirect hyperbilirubinemiaand low or absent haptoglobin, but reticulocytosis does notcorrespond to the degree of anemia. The main clinical find-ings of CDA II are moderate to severe normocytic or micro-cytic anemia, chronic or intermittent jaundice, splenomegaly[42]. CDA II is associated with a well-defined morphological

phenotype: peripheral blood smears show distinct anisopoi-kilocytosis with basophilic stippling and a few (occasionallybinucleated) mature erythroblasts. Bone marrow shows 5–10 times more erythroblasts than normal (erythroid hyper-plasia) [43,44]. Early erythroblasts are relatively normal, butmore than 10% of all erythroid cells are binucleated withequal size of two nuclei or multinucleated [45]. In addition,upon electron microscopy, vesicles of ER appear to be run-ning beneath the plasma membrane of CDA II erythroblas-tic cells [46]. Similarly to others COPII-related human disor-ders [35,37], CDA II shows a number of abnormalitiesaffecting glycosylation and/or levels of erythrocyte glyco-conjugates. The most useful for diagnosis is the hypoglyco-sylation of the erythrocyte anion exchanger 1 (AE1 or band3), which represents a key for the diagnosis [47] and sug-gests a defect in vesicles trafficking. As already stated, itseems that the altered glycosylation lead the accumulationof large amount of unfolded proteins in the ER and triggersthe response of the ER quality control system, the unfoldedprotein response (UPR) system [40]. The reduced glycosy-lation is associated with a decreased activity of AE1 in redblood cells from CDA II patients; furthermore the CDA IIerythrocytes were found to contain higher amounts of ag-gregate AE1 than control erythrocytes [48]. AggregatedAE1 has been reported to bind naturally occurring antibod-ies, possibly mediating the phagocytic removal of red bloodcells [49]. These results suggested that the hemolysisfound in CDA II patients may be ascribed to clustering ofthe AE1, leading to IgG binding and phagocytosis. All typesof CDA share a high incidence of splenomegaly, cholelithia-sis and iron overload [42], which in turn leads to secondaryhaemochromatosis: this represents the most importantlong-term complication encountered by patients after thefirst years of life. Of note, iron overload is not dependenton (albeit enhanced by) transfusions. Indeed, even inpatients with mild or moderate anemia, ferritin levels shouldbe checked at least annually, because iron overload mayapproach risk levels at any age. Haemochromatosis canlead to organ damage if not recognized and properlytreated [50,51]. Splenomegaly becomes apparent in 75% ofall patients in the first 3 decades of life [50]. The efficiencyof splenectomy is still not established well; however,reduced hypoglycosylation of band 3 seems to have a rele-vant role in spleen removal of red blood cells. The mainbenefit of splenectomy is abrogation of transfusion require-ments and increase of the hemoglobin concentration insevere cases [51].The prevalence of CDAs in Europe has been recently

assessed [52]. After this evaluation, CDA II seems to bethe most frequent form of CDAs. The geographic distribu-tion of affected patients suggests a higher frequency of thegene in Italy and in the Mediterranean countries as

TABLE I. Human Diseases Associated to Proteins Defects of the COP II Complex

Disease GeneChromosomeLocalization Protein function Inheritance Clinical phenotype Prevalence

Cranio-lenticulo-suturaldysplasia (CLSD)

SEC23A 14q21.1 GAP AR Delay in closure of fontanels, suturalcataracts, facial dysmorphism,skeletal efects

Unknown

Congenital DyserythropoieticAnemia type II (CDA II)

SEC23B 20p11.2 GAP AR Anemia, jaundice, low reticulocytecount, splenomegaly,hemochromatosis

0.7/100,0000a

Chylomicron RetentionDisease (CMRD)

SAR1B 5q31.1 GTPase AR Malabsorption of fat,hypobetalipoproteinemia

Unknown

Combined Deficiency ofFactor V and VIII (F5F8D)

LMAN1 18q21.3 Transmembrane lectin,Soluble protein

AR Bleeding symptoms, epistaxis,menorrhagia, and excessivebleeding during or after trauma

1/100,000–1/100,00,00MCFD2 2p21

aPrevalence referring to the European population [Ref. 59].

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compared with central and northern Europe. The combinedprevalence of CDA I and CDA II (based on all casesreported in the last 42 years) varies widely among Euro-pean regions, with minimal values of 0.04 cases/million inScandinavia and the highest value in Italy (2.49/million).The regional distribution of the Italian patients demon-strated a clustering in Southern Italy [52]. CDA II (367cases) is relatively more frequent than CDA I (122 cases),with an overall ratio of �3.0.The CDA II locus was originally mapped to 20q11 [53].

However, in a refined contig build (build 36.3), the markerswith the highest CDA II lod scores overlap the minimalhomozygosity region on the short arm of chromosome 20[54]. With the assumption that the cis, median and trans N-glycan Golgi processing of erythroblast glycoproteins wasimpaired, the SEC23B gene became a likely candidate forCDA II [55,56]. SEC23B encodes the second paralogousprotein of the inner layer of COPII vesicle (Fig. 2). To date,59 different causative mutations in SEC23B gene have beendescribed, localized along the entire coding sequence of thegene [55–60]. The vast majority of patients had two muta-tions (in the homozygous or compound heterozygous state),in accordance with the pattern of autosomal recessive inheri-tance. Homozygosity for nonsense mutations in SEC23Bgene must be lethal since no patient with such a genotypehas ever been observed. The association of one missensemutations and one nonsense mutation tends to produce amore severe presentation than two missense mutations.Although most of the mutations in SEC23B gene are theresults of sporadic and independent events, four mutationsaccounted for more than 50% of mutated alleles, which is aguide for a rational molecular diagnosis [57,58]. A clusteriza-tion of these mutations seems to be relevant in SouthernItaly, where a founder effect has been observed [61],accordingly to the regional distribution of the Italian patients.If mutations in SEC23B gene explain the impaired proc-

essing of CDA II erythroblast glycoproteins, it is not clearhow these impede the ultimate cell division along erythroiddifferentiation, generating binucleated erythroblasts. Inter-estingly, SEC23B was identified as a component of mid-body, a transient ‘‘organelle-like’’ structure remnant of celldivision just prior to abscission [16]. It remains to be estab-lished, whether SEC23B plays an active role in erythrocytemidbody assembly or deconstruction or whether glycosyla-tion impairment indirectly affects cytokinesis.CDA II and CLSD are caused by mutations in the two

human paralogs of the same COPII component. So, howSEC23A e SEC23B lead to specific phenotypes? In CLSD,primary calvarial osteoblasts showed a very low level ofSEC23B, suggesting these cells are specifically affectedbecause of insufficient SEC23B protein available to providenormal SEC23 function. Conversely, unaffected tissues inCLSD patients might retain normal function through expres-sion of sufficient SEC23B to complement the loss ofSEC23A function [39]. Similarly, in CDA II, it was showedan increased SEC23B expression during in vitro erythroiddifferentiation, 5–7 fold over SEC23A expression, whereasno gross RNA expression difference between the paralogsin primary dermal fibroblasts was observed [56]. However,studies on zebrafish morphants showed that both Sec23genes carry specific but partially redundant roles in cranio-facial cartilage maturation [40,56]: in addition, sec23b mor-phants show a typical erythroid phenotype, with a signifi-cant increase in immature, binucleated erythrocytes [56].Combined deficiency of factor V and VIII. An alterna-

tive mechanism to protein secretion COPII-mediatedinvolves selective packaging of secreted proteins with thehelp of specific cargo receptors (Fig. 3). A good example ofthis is the resulting defects in secretion of blood clotting

factors in patients with F5F8D (OMIM 227300). This is anautosomal, recessive bleeding disorder that is distinct fromthe co-inheritance of deficiency of both FV and FVIII. It ischaracterized by a mild-to-moderate bleeding tendencymanifested during or after trauma, tooth extraction, surgery,labor, and abortions. Menorrhagia is common, but hematu-ria, gastrointestinal, and intramuscular bleeding are rare.Affected individuals are characterized by concomitantly lowlevels of both FV and FVIII, usually between 5 and 30% ofnormal values, with normal platelet count, prolonged pro-thrombin time (PT), and partial thromboplastin time tests(PTT) [62,63]. Congenital F5F8D is estimated to beextremely rare, affecting males and females in equal num-bers. In the general population, increased frequency isassociated with consanguineous marriage. Indeed, thehighest reported occurrence of F5F8D is among MiddleEastern Jews and non-Jewish Iranians (1:100,000), wherecustomary consanguineous marriages are frequent [63].However, this disorder may be significantly under-diag-nosed because of the often mild bleeding symptoms, ormisdiagnosed as single factor deficiencies in many coun-tries with limited hematology/genetics infrastructure [64].The majority (70%) of F5F8D patients have mutations in

LMAN1 [65], with the remaining subset of patients resultingfrom mutations in MCFD2 [66]. To date, at least 51 muta-tions affecting the LMAN1 and MCFD2 proteins have beendescribed, 70% of which are located in the LMAN1 gene[67]. Almost all LMAN1 mutations reported to date are nullmutations; in contrast, in the MCFD2 gene have been iden-tified mostly missense mutations [68]. Although patientswith LMAN1 mutations and patients with MCFD2 mutationsare considered clinically indistinguishable, a genotype-phe-notype correlation became evident with the comparison ofa large number of patients with known mutations in eitherof the genes. Indeed, patients with MCFD2 mutations tendto have FV and FVIII levels at a lower range than patientswith LMAN1 mutations [64].LMAN1 (also known as ERGIC-53) is a mannose-selec-

tive lectin recycling from the ER to the ERGIC. MCFD2(multiple coagulation factor deficiency 2) is a small, solubleprotein with two Ca21-binding motifs. Both proteins form astable Ca21-dependent complex with 1:1 stoichiometrythat specifically aids in the transport of glycosylated FV andFVIII from the ER to the Golgi compartment. The intracellu-lar localization of this complex at the ERGIC suggests aspecific cargo receptor function for FV and FVIII in the earlysecretory pathway [69,70]. Cargo receptors are transmem-brane proteins, required for the efficient ER to Golgi trans-port of many soluble secretory proteins. They also containER exit signals on the cytoplasmic side that are recognizedby the SEC24 component of COPII [71]. To date, mostcargo receptors are identified in yeast [72]. However, the

Figure 3. Representation of LMAN1-MCFD2 complex. LMAN1 binds to theSEC24 component of COPII vesicle via C-terminal domain, on the cytoplasmicside. MCFD2 interacts with LMAN1 in the ER lumen in a 1:1 stechiometry.The dot-shaped structure (in black) on FVIII/FV represents glycosylation site onB-domain of both factors. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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LMAN1-MCFD2 protein complex is the only well-character-ized cargo receptor in mammalian cells [70]. The majorityof LMAN1 is localized to the ERGIC at steady state, it alsocycles between the ER and ERGIC in live cells. This isachieved through signal peptides, ER exit and ER retrievalmotifs, located at the C-terminal of the protein that interactwith COPII and COPI respectively. Conversely, MCFD2lacks the ER exit and retrieval signals; thus, it requiresLMAN1 binding for proper intracellular localization [66,67].Given the ubiquitous expression pattern of LMAN1 and

MCFD2 and the presence of their orthologues in lowereukaryotes without a blood clotting system, additional cargoproteins that are targeted by this complex probably alsoexist. Accordingly, cathepsin C, cathepsin Z and 1-antitryp-sin have also been reported as potential cargo proteins de-pendent on LMAN1 for efficient secretion [73,74]. However,LMAN1-deficient mice, with reduced FV and FVIII plasmalevels to �50% of wild-type mice, show no differences towild type mice in the levels of cathepsin C and cathepsin Zin liver lysates or a1-antitrypsin levels in plasma [75]. Whatare the unique features of FV and FVIII that enable them toboth interact with the LMAN1-MCFD2 complex? FV andFVIII are two plasma glycoproteins, which share a function-ally dispensable domain (B-domain) that is heavily glycosyl-ated. B-domain deleted FVIII exhibits markedly reducedbinding to the LMAN1-MCFD2 complex [69], suggesting aninteraction between the lectin LMAN1 and sugar sidechains of the heavily glycosylated B domains of FV andFVIII. However, unglycosylated FVIII can still interact withthe LMAN1-MCFD2 complex [69], suggesting that protein–protein interactions are also involved [76].

Conclusions and HypothesesIn the proteostasis network, nascent proteins are shuttled

through a series of membrane-enclosed organelles bytransport vesicles into the extracellular space. In this reviewwe focused on four human genetic disorders due to altera-tion in COPII trafficking machinery. Although these disor-ders are caused by alterations in ubiquitous proteinsinvolved in the same pathway, they are characterized byvery different clinical manifestations. Thus, an outstandingquestion is why alterations in these proteins give tissue-specific as opposed to generalized clinical manifestations.At present we don’t know the exact pathogenetic mecha-nisms; we could only make some hypotheses. The first isthat the repertoire of COPII paralogs available for coat po-lymerization should dictate the nature of the vesicles bud-ding from the ER. It has been assumed that the differential,tissue-specific expression of COPII paralogs, as well asdistinctive affinities between COPII subunit paralogs, wouldconfer different properties to vesicles according to therequirements of ER export [11,22,39,71]. This become par-ticularly evident in the pathogenesis of CDA II and CLSD,in which mutations in the two paralogs of SEC23, A and B,result in very different phenotypes. Another explanation forthe selective tissue vulnerability could lie in the highdemand of special tissue-specific cargoes (for example,band 3 in red blood cells), which might require high levelsand full function of a particular trafficking component to becorrectly transported [77]. However, further studies areneeded in order to define the role of each COPII paralogsto understand the pathogenesis of the genetic disordersrelated to alteration of this complex.

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