Glycoproteins and their formations, including the two common types of glycoproteins and their site and modes of synthesis with associated diseass

1. Glycoproteins: Biological and
Clinical Significance
Presented By:
Dr. Nilesh Chandra

2. OBJECTIVES:
• Classes Of Glycoproteins
• Biosynthesis Of Different Classes Of
Glycoproteins
• Biological Roles Of Glycoproteins
• Glycoproteins And Diseases

3. Classes of Glycoproteins:
O-Linked
Glycoproteins
GalNAcSer(Thr)
linkage
GlcNAc-Ser[Thr]
linkage
N-Linked
Glycoproteins
Complex
Hybrid
High-
mannose
GPI-Linked
Glycoproteins
Other minor
groups

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

5. Dolichol-P-P-Oligosaccharide:
Structure of Dolichol (polyisoprenol)
Structure of dolichol-P-P-oligosaccharide
Dol-P-P-GlcNAc2Man9Glc3.

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

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

8. 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

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

10. Major Classes of N-Linked
Oligosaccharides:
• Complex
• Hybrid
• High-mannose
Share a common pentasaccharide, Man3GlcNAc2

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

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

13. 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.

14. Subclasses of O-glycosidic linkages :
1. The GalNAcSer(Thr) linkage -
(A)submaxillary mucins
(B)fetuin

15. Subclasses of O-glycosidic linkages
(contd.)
2. Gal-Gal-XylSer trisaccharide (link trisaccharide)-
Transfer of a Xyl residue to Ser from UDP-xylose
Two residues of Gal then added to the Xyl residue
Further chain growth on the terminal Gal
• O-glycosidic bond between xylose (Xyl) and Ser is
unique to proteoglycans

16. Subclasses of O-glycosidic linkages
(contd.)
3. Gal-hydroxylysine (Hyl) linkage-
– Hydroxylysine in collagen modified by the addition
of galactose or galactosyl-glucose through an O-
glycosidic linkage.
• Hydroxylysine - a glycosylation site that is
unique to collagen.

17. Subclasses of O-glycosidic linkages
(contd.)
4. GlcNAc-Ser[Thr] linkage –
– On many nuclear proteins and cytosolic proteins.
– a single GlcNAc attached to a serine or threonine
residue.
– formed by donation to Ser (or Thr) of a GalNAc
residue, employing UDP-GalNAc as its donor.

18. 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. Fetuine.g. Calnexin

19. GPI linkage
Acetylcholinesterase linkage to the plasma
membrane of the human red blood cell.

20. Biosynthesis of GPI anchors: Glypiation
• Begins in the endoplasmic reticulum.
• GPI anchor is assembled independently.
• The hydrophobic end cleaved off.
• Replaced by the GPI-anchor.
• GPI-linked proteins thought to be
preferentially located in lipid rafts.

21. Some GPI-Linked Proteins:
TABLE
Acetylcholinesterase (red cell membrane)
Alkaline phosphatase (intestinal, placental)
Decay-accelerating factor (red cell membrane)
5'-Nucleotidase (T lymphocytes, other cells)
Thy-1 antigen (brain, T lymphocytes)
Variable surface glycoprotein (Trypanosoma brucei)

22. Functions Served by Glycoproteins:
1. Structural molecule
2. Lubricant and protective agent
3. Transport molecule
4. Immunologic molecule
5. Hormone
6. Enzyme
7. Cell attachment-recognition site

23. Functions Served by Glycoproteins (contd.)
8. Interact with specific carbohydrates
9. Antifreeze
10.Receptor
11.Affect folding of certain proteins
12.Regulation of development
13.Hemostasis (and thrombosis)

24. Role of Oligosaccharide Chains of
Glycoproteins:
• Modulate physicochemical properties, eg,:
– Solubility
– Viscosity
– Charge
– Conformation
– Denaturation
– binding sites for various molecules, bacteria viruses
and some parasites
• Protect against proteolysis, from inside and
outside of cell

25. Schematic diagram of a mucin

26. Role of Oligosaccharide Chains of
Glycoproteins (contd.)
• Affect proteolytic processing of precursor proteins to
smaller products
• Are involved in biological activity, eg, of human
chorionic gonadotropin (hCG)
• Affect insertion into membranes, intracellular
migration, sorting and secretion
• Affect embryonic development and differentiation
• May affect sites of metastases selected by cancer
cells

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

28. 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

29. 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

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

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

32. 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.

33. Defects in CDG

34. 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.

35. 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–braindisease (MEB)

37. IEF pattern for transferrin (Type 2)

38. Some Diseases Involving Glycoproteins:
• Glycans Involved in the Binding of Micro-
organisms:
– Influenza virus A
– Human immunodeficiency virus type 1 (HIV-1)
– Helicobacter pylori
– Plasmodium falciparum
• Cancer: Increased branching of cell surface glycans
or presentation of selectin ligands may be important
in metastasis.

39. Some Diseases Involving Glycoproteins
(contd.)
Selectins in Inflammation & in Lymphocyte Homing:
• Three types of selectins:
Present on
– L-selectin : Polymorphonuclear leukocytes, Lymphocytes
– P-selectin : Endothelial cell, Platelets
– E-selectin : Endothelial cell
• All three bind sialyl-Lewisx, a structure present on
both glycoproteins and glycolipids

40. Schematic representation of the structure of sialyl-Lewisx

41. SUMMARY:
• Glycoproteins are of various types, based on their
linkages.
• Different types of linkages follow different
biosynthetic pathways.
• Glycoproteins play diverse roles in various important
functions of the body.
• A great many of the properties of the glycoproteins
are due to the oligosachharide component.

42. SUMMARY (contd.)
• Glycoproteins in Disease:
– Pathophysiology of :
• HEMPAS
• LAD II
• PNH
• I-Cell Disease
• CDGs
– Role in adhesion of micro-organisms
– Role in Chemotaxis and migration of leukocytes
– Cancer

43. 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.

44. Advanced Glycation End-Products:
Maillard reaction
• AGEs cause tissue damage in diabetes mellitus.

45. Some consequences of the formation of AGEs

46. 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