1. Essentials of Glycobiology Lecture 19 April 29th. 2004 Ajit Varki Lectins in ER-Golgi “Quality Control” R & L-type Lectins (Excluding Plant Lectins)

2. Current Classification of Lectins Families 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 H TNF, Interleukins & Cytokines Ameoba lectin Tachylectins Annexins Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

3. 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.

4. The Calnexin-Calreticulin cycle

5. 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

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

7. 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.

8. 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

9. Initial Processing of N-linked Glycans

10. 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.

11. ER Mannosidase(s) may act as “Timer(s)” for Protein Degradation Recognition by EDEM? ( ER-degradation enhancing mannosidase-like protein) Sec 61

12. Current Classification of Lectins Families 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 H TNF, Interleukins & Cytokines Ameoba lectin Tachylectins Annexins Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

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

14. Lectins in the ER-Golgi Pathway ERGIC-53 VIP-36 Calnexin/Calcireticulin

15. 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

16. 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.

17. 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.

18. 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

19. Predicted protein products Normal ERGIC-53: ERGIC-53 Mutations 510 aa 402 aa 7 aa 10 aa 0 aa 140 aa 30 aa 202 aa 213 aa 302 aa 239 aa 304 aa 274 aa 370 aa 383 aa 405 aa 456 aa 507 aa 383 aa KKFF mRNA Protein D.Ginsburg

20. Lectins in the ER-Golgi Pathway ERGIC-53 VIP-36 Calnexin/Calcireticulin

21. 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.

22. 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

23. Current Classification of Lectins Families 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 H TNF, Interleukins & Cytokines Ameoba lectin Tachylectins Annexins Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

24. Domain Organization of R-Type CRDs in Nature Dodd R.B. and Drickamer,K. Glycobiology 11:71-79, 2001

25. J. Baenziger

27. 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

28. 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.