glycoproteins N and O glycosylation synthesis and functions

Glycoproteins

1. Glycoproteins (N & O Glycosylation ).

2. OBJECTIVES: Glycoproteins Biological Roles Of Glycoproteins Glycosylation (N & O linkage) Diseases

3. Glycoproteins They are proteins that contain oligosaccharide ( glycan ) chains covalently attached to their polypeptide backbones. Eight sugars predominate in human glycoproteins Glucose, Galactose , Mannose, N-Acetyl neuraminic acid, Fucose , N- Acetylgalactosamine , N- Acetylglucosamine , Xylose .

4. Functions Served by Glycoproteins:

5. Classes of Glycoproteins:

6. O- glycosidic linkage- hydroxyl side chain of serine or threonine and a sugar such as Nacetylgalactosamine ( GalNAc -Ser[ Thr ]) N- glycosidic linkage- amide nitrogen of asparagine and N- acetylglucosamine ( GlcNAcAsn ) Glycosylphosphatidylinositol -anchored (GPI-anchored, or GPI-linked)- carboxyl terminal amino acid of a protein via a phosphoryl -ethanolamine moiety joined to an oligosaccharide ( glycan ), which in turn is linked via glucosamine to phosphatidylinositol

7. O-linkage ( N -acetylgalactosamine to serine)

8. O-linkage The GalNAcSer ( Thr ) linkage is the predominant linkage. This type of linkage is found in mucins Proteoglycans contain a Gal-Gal- Xyl -Ser trisaccharide Collagens contain a Gal- hydroxylysine ( Hyl ) linkage. Many nuclear proteins ( eg , certain transcription factors) and cytosolic proteins contain side chains consisting of a single GlcNAc attached to a serine or threo -nine residue ( GlcNAc -Ser[ Thr ]).

9. Carrier systems ( permeases , transporters) are necessary to transport nucleotide sugars across the Golgi membrane. Systems transporting UDP-Gal, GDP-Man, and CMP- NeuAc into the cisternae of the Golgi apparatus

10. antiport systems ; influx of one molecule of nucleotide sugar is balanced by the efflux of one molecule of the corresponding nucleotide ( eg,UMP , GMP, or CMP)

11. Biosynthesis of O-Linked Glycoproteins: Stepwise donation of sugars from nucleotide sugars such as: UDP- GalNAc UDPGal CMP- NeuAc Enzymes involved: membrane-bound glycoprotein glycosyltransferases . Enzymes located in various subcompartments of Golgi apparatus.

12. Schematic diagram of a mucin

13. N-linkage ( N -acetylglucosamine to asparagine)

14. The boxed area encloses the pentasaccharide core common to all N-linked glycoproteins

15. Dolichol -P-P-Oligosaccharide: Structure of Dolichol ( polyisoprenol ) Structure of dolichol -P-P-oligosaccharide Dol-P-P-GlcNAc 2 Man 9 Glc 3 .

16. Biosynthesis of DolicholP -P-oligosaccharide: Dolichol Dolichol phosphate ( Dol -P) Dol -P + UDP- GlcNac Dol -P-P- GlcNac +UMP dolichol kinase ATP ADP

17. Biosynthesis of DolicholP -P-oligosaccharide (contd.)

18. 1= oligosaccharide: protein transferase 2= - glucosidase I 3= - glucosidase II 4= endoplasmic reticulum 1,2-mannosidase 5=Golgi apparatus - mannosidase I I= N - acetylglucosaminylphosphotransferase II= N -acetylglucosamine-1-phosphodiester α - N - acetylglucosaminidase

19. 6= N- acetylglucosaminyltransferase I 7= Golgi apparatus - mannosidase II 8= N - acetylglucosaminyltransferase II 9= fucosyltransferase 10= galactosyltransferase 11= sialyltransferase .

20. Differences between O- & N- linked Glycoproteins: N-Linked Glycoproteins Synthesis:- Cotranslationally En-bloc transfer of Oligosachharide chain on the protein and further modification. Enzymes not membrane bound. Dolichol P-P-Oligosaccharide involved. Inhibited by Tunicamycin O-Linked Glycoproteins Synthesis:- Post- translationally Oligosaccharide chain synthesized on the protein. Enzymes membrane bound. Dolichol not involved. Not inhibited by Tunicamycin e.g. Mucin e.g. Calnexin

21. Some Diseases Due to Glycoproteins: Disease HEMPAS  Leukocyte adhesion deficiency, type II Paroxysmal nocturnal hemoglobinuria I-cell disease Congenital disorders of glycosylation

22. I-Cell Disease: Summary of the causation of I-cell disease

23. Congenital Disorder of Glycosylation Previously called carbohydrate-deficient glycoprotein syndrome. Glycosylation of a variety of tissue proteins and/or lipids is deficient or defective. CDG-I mainly in ER & related to steps prior processing of glycan chains and transfer of oligosac chain to protein. CDG-II includes all defects localized in the processing of N- glycans on the glycosylated protein. These are situated mainly in the Golgi compartment. Often cause serious, sometimes fatal, malfunction of several different organ systems (especially the nervous system, muscles, and intestines) in affected infants.

24. Diseases due to O-linked Glycoproteins: Inborn errors of O- glycan metabolism can lead to severe autosomal recessive multisystem syndrome with neurologic involvement, some defects, for example, those in persons with the Bombay blood group do not produce a clinical phenotype.

25. Defects of O glycosylation Name Defect Clinical features Defects in mucin -type o-glycan biosynthesis (familial tumoral calcinosis (FTC) UDP- GalNAc transferase 3 ( ppGalNAc T3) abnormal cleavage and secretion of the phosphaturic factor FGF23 . massive calcium deposits in skin and subcutaneous tissues and unresponsiveness to parathyroid hormone Defects in o- galactosyl glycan biosynthesisLysyl hydroxylase-1 deficiency Ehlers– Danlos syndrome type Via Deficiency or mutation in Lysyl hydroxylase-1 neonatal kyphoscoliosis , generalized joint laxity, skin fragility, and severe muscle hypotonia at birth Defects in o- mannosyl glycan biosynthesis Walker–Warburg syndrome (WWS) and limb-girdle muscular dystrophy type 2K Muscle–eye–brain disease (MEB)

26. HEMPAS: Hereditary Erythroblastic Multinuclearity With A Positive Acidified Lysis Test . Also known as Congenital Dyserythropoietic Anemia Type II. Claimed to be due to defects in alpha– mannosidase II Characterized by Ineffective erythropoiesis Hemolysis & erythroblast morphological abnormalities Hypoglycosylation of some red blood cell (RBC) membrane proteins. Treatment consists of frequent blood transfusions and chelation therapy

27. Leukocyte adhesion deficiency (LAD) II: A congenital disorder of glycosylation Mutations affecting the activity of a Golgi-located GDP- fucose transporter Marked decrease in neutrophil rolling Subjects suffer Life-threatening, recurrent bacterial infections Psychomotor and mental retardation The condition appears to respond to oral fucose

28. Paroxysmal Nocturnal Hemoglobinuria : Scheme of causation of paroxysmal nocturnal hemoglobinuria

29. SUMMARY Glycoproteins Functions Served by Glycoproteins Classes of Glycoproteins N and O linkage Glycoproteins in Disease: I-Cell Disease CDGs others

30. References: Harper's Illustrated Biochemistry, Twenty-Eighth Edition. Lehninger’s Principles of Biochemistry, Fifth Edition. Stryer’s Biochemistry, Sixth Edition. Dennis JW, Nabi IR, Demetriou M. Metabolism, cell surface organization, and disease. Cell. 2009 Dec 24;139(7):1229-41. Review. Vagin O, Kraut JA, Sachs G. Role of N-glycosylation in trafficking of apical membrane proteins in epithelia. Am J Physiol Renal Physiol. 2009 Mar;296(3):F459-69. Epub 2008 Oct 29. Luther KB, Haltiwanger RS. Role of unusual O- glycans in intercellular signaling. Int J Biochem Cell Biol. 2009 May;41(5):1011-24. Epub 2008 Oct 8. Review.

31. O- mannosyl glycan O- Mannosyl glycans are a less common type of protein modification present on a limited number of glycoproteins in the brain, nerves and skeletal ms. Eg α- Dystroglycan It is a laminin receptor 2 subunits α- DG interacts with laminin-2 in the basal lamina and β- DG binds to dystrophin induces Ach receptor clustering at neuromuscular junctions