essentials of glycobiology lecture 19 april 29th. 2004 ajit varki lectins in er-golgi “quality...

Essentials of Glycobiology

Lecture 19

April 29th. 2004

Ajit Varki

Lectins in ER-Golgi “Quality Control”

R & L-type Lectins(Excluding Plant Lectins)

Current Classification of LectinsFamilies with known protein sequence homologies

Calnexin group (e.g., Calnexin, Calcireticulin, Calmegin) *”L-type” lectins (e.g., (ERGIC-53 and VIP-36 in ER-Golgi pathway, Plant Lectins *"P-type" lectins (Mannose-6-Phosphate Receptors) *"C-type" lectins (e.g., Selectins, Collectins etc.) * Galectins (formerly "S-type" lectins) *"I-type" lectins (includes Siglec family)

*”R-type" lectins (e.g., GalNAc-SO4 receptors, Plant Lectins) “Eglectins” (Frog Egg lectins) Eel Agglutinins (Fucolectins) Hyaluronan-binding proteins Ficolins Pentraxins

Sequence homologies not known (Examples)CD11b/CD18 (beta3-integrin, CR3) Complement Factor HTNF, Interleukins & Cytokines Ameoba lectin TachylectinsAnnexins

Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

Model of Calnexin (CNX), Calreticulin (CRT) and ERp57 interacting with a nascent chain of a glycoprotein in the ER.

• Phosphorylation of CNX cytosolic tail increases ribosome association.

• CNX, CRT and ERp57 interact co- and post-translationally with glycoproteins after two glucoses are rapidly trimmed

• Glycans are recognized by a lectin activity of CNX and CRT.

•CNX and CRT cooperate with ERp57 - allowing this thiol oxidoreductase to interact optimally with cysteines in the glycoprotein substrate and promote proper formation of disulfide bonds.

The Calnexin-Calreticulin cycle

Schematic representation of Calnexin and Calreticulin

(a) Calnexin is a type 1 membrane protein of 570 residues, Calreticulin is a soluble, lumenal protein of 400 residues

(b) A cartoon of the CRT P-domain NMR structure is shown

Evolution of the Calnexin-related Lectins Dodd R.B. and Drickamer,K. Glycobiology 11:71-79, 2001

Calmegin

• Loss of this ER resident chaperone leads to production of sterile sperm

• Calmegin -/- sperm defective in migrating into the oviducts and in binding to egg plasma membrane.

• Despite impaired adhesion, calmegin -/- sperm can fertilize eggs when zona pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term.

Calmegin

• Calmegin binds to sperm membrane proteins, Fertilin alpha and beta, during spermatogenesis. Calnexin not able to bind

• In Calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm.

• This explains why Calmegin and Fertilin beta knockout mouse lines share a common infertile phenotype, and also reveal the importance of maturation of sperm membrane proteins in ER

Initial Processing of N-linked Glycans

ER-mannosidase I may act as a “Timer” for Protein Degradation

• When trimming by ER-mannosidase I is prevented by inhibitors or genetic manipulation, Endoplasmic Reticulum-Associated Degradation (ERAD) of glycoproteins stops.

• ER mannosidase removes single alpha-1,2-linked Man. Resulting Man8 appears to serve as part of signal for degradation

• Removal of Man not sufficient, because most proteins that have folded normally are mannose-trimmed before leaving the ER.

• Since the mannosidase is slow-acting, it may provide a “timer function” protecting against premature degradation of the most recently synthesized glycoproteins.

ER Mannosidase(s) may act as “Timer(s)” for Protein Degradation

Recognition by EDEM?( ER-degradation enhancing mannosidase-like protein)

Sec 61





Amphoterin

*Have defined Carbohydrate Recognition Domains (CRDs)

Evolutionary relationships of Animal and Plant L-type LectinsDodd R.B. and Drickamer,K. Glycobiology 11:71-79, 2001

Intermediate Compartment

Rough EndoplasmicReticulum

SecretoryGranule Early

EndosomeENDOSOME

Lysosome

N-GLYCOSYLATIONTRANSLATION

LateEndosome

ENDOSOME

"UNCOVERING"

Golgi Stacks

Trans Golgi Network

Lectins in the ER-Golgi Pathway

ERGIC-53

VIP-36

Calnexin/Calcireticulin

ERGIC-53 (MR60)(rat p58)

• Originally identified as protein reacting with antibodies against ER-Golgi Intermediate Compartment (ERGIC)

• MR60 indepedently identified as a mannose-binding protein.

• Subsequent cloning showed that they were identical and represented the human homologue of rat p58 protein.

• Type I transmembrane, nonglycosylated protein with a lumenal domain, a transmembrane domain, and a short cytoplasmic domain.

• In cells, present as dimers and hexamers

• Lumenal domain has N-terminal carbohydrate recognition domain (CRD) and a membrane-proximal helical domain

Overall structure of Rat p58 (ERGIC) Strands related to Plant leguminous lectins are in red and

dark blue. Arrow in B indicates ligand binding site.

ERGIC-53 (MR60)(rat p58)

• Cytoplasmic tail contains KKFF sequence at its C terminus that is essential for both ER exit and Golgi retrieval

• Cycles between the ER and Golgi compartment

• Blocking export of ERGIC-53 from ER impaired but did not block export of the lysosomal enzyme cathepsin C

• Glycan structure binding to ERGIC-53 in the ER appears to be Man9, i.e., the N-glycan from which three terminal glucose residues have been trimmed

• Presumed to act as a lectin-like receptor involved in facilitating export from the ER of a subset of secretory glycoproteins.

Combined Factors V and VIII Deficiency

• Autosomal recessive

• >> 100 families worldwide

• ?Most frequent in Sephardic and Middle Eastern Jews

• Coordinate reduction in Factor V and Factor VIII

• Antigen and activity of each ~5-30%

• Not due to mutations in Factor V or Factor VIII genes

• Bleeding more severe than single factor deficiency

Predicted protein productsNormalERGIC-53:

ERGIC-53Mutations

510 aa

402 aa

7 aa10 aa

0 aa

140 aa30 aa

202 aa213 aa

302 aa

239 aa

304 aa

274 aa

370 aa383 aa

405 aa456 aa507 aa

383 aa

KKFF

mRNAProtein

D.Ginsburg

Intermediate Compartment

Rough EndoplasmicReticulum

SecretoryGranule Early

EndosomeENDOSOME

Lysosome

N-GLYCOSYLATIONTRANSLATION

LateEndosome

ENDOSOME

"UNCOVERING"

Golgi Stacks

Trans Golgi Network

Lectins in the ER-Golgi Pathway

ERGIC-53

VIP-36

Calnexin/Calcireticulin

VIP36

• 36 kDa vesicular-integral membrane protein originally isolated from MDCK epithelial cells as component of glycolipid-enriched detergent-insoluble complexes containing apical marker proteins

• Localised to Golgi and the early secretory pathway

• Luminal domain shows homology to leguminous plant lectins and to ERGIC-53.

• Recognizes high-mannose type glycans containing alpha1-2 Man residues.

• Binding of Vip36 to mannose independent of Calcium, optimal at pH 6.

VIP36

• Glycoproteins recognized by VIP36 secreted ~2-fold more efficiently from apical than from basolateral membrane.

• Upon overproduction of VIP36 apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins changed from ~ 2 to ~ 4, and secretion of VIP36-recognized glycoproteins was greatly stimulated

• Mutant version of VIP36, with no lectin activity, has no effect on the distribution of glycoproteins to apical and basolateral membranes





Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

Domain Organization of R-Type CRDs in Nature

Dodd R.B. and Drickamer,K. Glycobiology 11:71-79, 2001

J. Baenziger

Role of Glycosylation in Regulating The Ovarian Cycle

Circulatory half-life of the glycoprotein hormone lutropin (LH) is precisely regulated by a GalNAc-4-sulfate receptor expressed in hepatic endothelial cells. Rapid clearance from circulation contributes to episodic rise and fall of LH levels that is essential for maximal stimulation of the G protein-coupled LH receptor.

J. Baenziger

Mi et alJ Clin Invest. 2002, 2:269-76.•Complete genetic ablation of the Man/GalNAc-4-

SO4 receptor results in death in utero.

•Heterozygous female mice clear LH from the circulation more slowly and have smaller litters due to a reduction in the rate of implantation.

•Reduction fully correctable by exogenous progesterone and estrogen, indicating that rate of LH clearance is critical for the production of sufficient progesterone and estrogen to support implantation.

•Thus, Man/GalNAc-4-SO4 receptor regulates endocrinological status of female and is also essential for an early event in embryonic development.

essentials of glycobiology lecture 19 april 29th. 2004 ajit varki lectins in er-golgi “quality...

Documents

er slide

rtype lectins

calmegin calmegin

ctype lectins

stype lectins

ptype lectins mannose

calnexinrelated lectins

er mannosidase