What is Glycoprotein ?: Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. This process is known as glycosylation. The carbohydrate is attached to the protein during the following modifications: Co-translational modification & Post-translational modification. In proteins that have segments extending extracellularly, the extracellular segments are often glycosylated.

1. Glycoproteins
Mr.Tapeshwar Yadav
Final Year, PG
Mamata Medical College-
Khammam,A.P,INDIA
06/01/12

2. What is Glycoprotein ?:
 Glycoproteins are proteins that contain
oligosaccharide chains (glycans) covalently
attached to polypeptide side-chains.
This process is known as glycosylation.
The carbohydrate is attached to the protein
during the following modifications: Co-
translational modification & Post-translational
modification.
 In proteins that have segments extending
extracellularly, the extracellular segments are
often glycosylated.

4. Glycoproteins and Proteoglycans
Glycoproteins
Proteins conjugated to
saccharides lacking a
serial repeat unit
Proteoglycans
Proteins conjugated to
polysaccharides with
serial repeat units
Protein >> carbohydrate
Carbohydrate >> protein

5. 3 major classes:-
1)N-linkage (N -acetylglucosamine to asparagine)
2)O-linkage (N -acetylgalactosamine to serine)
3)Glycosyl-phosphatidyl-inositol (GPI) linkage
 N-glycosidic linkage (ie, N-linked), involving the amide nitrogen
of asparagine and N -acetylglucosamine (GlcNAc-Asn)
 O-glycosidic linkage (ie, O-linked), involving the hydroxyl side
chain of serine or threonine and a sugar such as N -
acetylgalactosamine (GalNAc-Ser[Thr])
 linked to the carboxyl terminal amino acid of a protein via a
phosphoryl-ethanolamine moiety joined to an
oligosaccharide (glycan), linked via glucosamine to
phosphatidylinositol (PI).
CLASSIFICATION OF GLYCOPROTEINS:-

6. 1)N-linked Glycans:
Important N-linked glycans
• are found in ovalbumin and the immunoglobulins.
• Part of the recognition of immunoglobulins is due to the
sequence of the oligosaccharide chains of the glycans.
• A very important further use of N-linked
oligosaccharides is in intracellular targeting in eukaryotic
organisms.
• Proteins destined for certain organelles or for excretion
from the cell are marked specifically by oligosaccharides
during posttranslational processing to ensure they arrive
at their proper destinations.

7. N-linked Glycoproteins

8. 2)O-Linked Glycans:
• Mucins, which are found extensively in salivary
secretions, contain many short O-linked glycans.
• Increase the viscosity of the fluids in which they
are dissolved.
• Function is intracellular targeting and molecular
and cellular identification.
• Example: blood group antigens.
• Antarctic fish contain a glycoprotein that serves
as an "antifreeze", preventing the freezing of
body fluids, even in extremely cold water.

9. O-linked Glycoproteins

11. Glycopeptide bonds
Type I
Type II Type III
N-Glycosyl linkage to Asn
O-Glycosyl linkage to Ser (Thr) O-Glycosyl linkage to 5-HOLys
O
H
OH
HN
H
H
HNH
OH
CH2OH
H
C CH2 CH
O
COOH
NH2
C CH3
O
OH
OH
O
H
H
HNH
OH
CH2OH
H
CH2 CH COOH
NH2
C CH3
O
OH
OH
O
H
H
OHH
OH
CH2OH
H
CH
CH2
CH2
CH2
NH2
CHH2N COOH
Glc
NAc
Asn
Glc
NAc
Ser HOLysGlc

13. Principal Sugars Found in Human Glycoproteins
Sugar Type Abbrevi-
ation
Nucleotide
Sugar
Comments
Galactose
Hexose Gal UDP-Gal Often found subterminal to NeuAc in N-linked
glycoproteins. Also found in the core
trisaccharide of proteoglycans.
Glucose Hexose Glc UDP-Glc Present during the biosynthesis of N-linked
glycoproteins but not usually present in
mature glycoproteins. Present in some clotting
factors.
Mannose Hexose Man GDP-Man Common sugar in N-linked glycoproteins.
N-Acetyl
neuramin
ic acid
Sialic acid
(nine C
atoms)
NeuAc CMP-
NeuAc
Often the terminal sugar in both N- and O-
linked glycoproteins. Other types of sialic acid
are also found, but NeuAc is the major species
found in humans. Acetyl groups may also occur
as O-acetyl species as well as N-acetyl.

14. Fucose Deoxy
hexose
Fuc GDP-
Fuc
May be external in both N- and O-linked
glycoproteins or internal, linked to the
GlcNAc residue attached to Asn in N-linked
species. Can also occur internally attached
to the OH of Ser (eg, in t-PA and certain
clotting factors).
N-
Acetylgalactosamine
Amino
hexose
GalNAc UDP-
GalNAc
Present in both N- and O-linked
glycoproteins.
N-
Acetylglucosamine
Amino
hexose
GlcNAc UDP-
GlcNAc
The sugar attached to the polypeptide chain
via Asn in N-linked glycoproteins; also found
at other sites in the oligosaccharides of
these proteins. Many nuclear proteins have
GlcNAc attached to the OH of Ser or Thr as a
single sugar.
Xylose Pentos
e
Xyl UDP-
Xyl
Xyl is attached to the OH of Ser in many
proteogly-cans. Xyl in turn is attached to two
Gal residues, forming a link trisaccharide. Xyl
is also found in t-PA and certain clotting
factors.

15. Functions Served by Glycoproteins
Function Glycoproteins
Structural molecule Collagens
Lubricant and protective agent Mucins
Transport molecule Transferrin, ceruloplasmin
Immunologic molecule Immunoglobulins, histocompatibility
antigens
Hormone Chorionic gonadotropin, thyroid-
stimulating hormone (TSH)
Enzyme Various, eg, alkaline phosphatase
Cell attachment-recognition site Various proteins involved in cell-cell (eg,
sperm-oocyte), virus-cell, bacterium-cell,
and hormone-cell interactions

16. Antifreeze Certain plasma proteins of cold-water fish
Interact with specific carbohydrates Lectins, selectins (cell adhesion lectins),
antibodies
Receptor Various proteins involved in hormone and
drug action
Affect folding of certain proteins Calnexin, calreticulin
Regulation of development Notch and its analogs, key proteins in
development
Hemostasis (and thrombosis) Specific glycoproteins on the surface
membranes of platelets

17. Functions
1)Structural:
Glycoproteins are found throughout matrices. They
act as receptors on cell surfaces that bring other cells and
proteins (collagen) together giving strength and support to
a matrix.
 Proteoglycan-linking glycoproteins cross links proteoglycan
molecules and is involved in the formation of the ordered
structure within cartilage tissue.
 In nerve tissue glycoproteins are abundant in gray matter
and appear to be associated with synaptosomes, axons,
and microsomes.
 In certain bacteria the slime layer that surrounds the
outermost components of cell walls are made up of
glycoproteins of high molecular weight.

18. 2)Protection:
High molecular weight polymers called mucins are found
on internal epithelial surfaces.
 They form a highly viscous gel that protects epithelium form
chemical, physical, and microbial disturbances. Examples of
mucin sites are the human digestive tract, urinary tract, and
respiratory tracts.
 Mucins are also found on the outer body surfaces of fish to
protect the skin.
 Not only does mucin serve the function of protection, but it
also acts as a lubricant.
 Human lacrimal glands produce a glycoprotein which protects
the corneal epithelium from desiccation and foreign particles.
 Human sweat glands secrete glycoproteins which protect the
skin from the other excretory products that could harm the
skin.

19. 3)Reproduction:
 Glycoproteins found on the surface of spermatozoa
appear to increase a sperm cell's attraction for the egg by
altering the electrophoretic mobility of the plasma
membrane.
 Actual binding of the sperm cell to the egg is mediated by
linked glycoproteins serving as receptors on the surface
of each the two membranes.
 The zona pellucida is an envelope made of glycoprotein
that surrounds the egg and prevents polyspermy from
occurring after the first sperm cell has penetrated the
egg's plasma membrane.
 Hen ovalbumin is a glycoprotein found in egg white that
serves as a food storage unit for the embryo.

20. 4)Adhesion:
 Glycoproteins serve to adhere cells to cells and cells to
substratum. Cell-cell adhesion is the basis for the
development of functional tissues in the body.
 In different domains of the body, different glycoproteins act to
unite cells. For example, nerve cells recognize and bind to one
another via the glycoprotein N-CAM (nerve cell adhesion
molecule).
 N-CAM is also found on muscle cells indicating a role in the
formation of myoneural junctions.
 Substrates with the appropriate receptor will bind to the cell
related to that receptor. For example, a substrate containing
the glycoprotein fibronectin will be recognized and adhered to
by fibroblasts.
 The fibroblasts will then secrete adhesion molecules and
continue to spread, producing a pericellular matrix.

21. 5)Hormones:
 There are many glycoproteins that function as hormones such as
human chorionic gonadotropin (HCG) which is present in human
pregnancy urine.
 Another example is erythropoietin which regulates erythrocyte
production.
6)Enzymes:
 Glycoprotein enzymes are of three types. These are
oxidoreductases, transferases, and hydrolases.

22. 7)Carriers:
Glycoproteins can bind to certain molecules
and serve as vehicles of transport. They can
bind to vitamins, hormones, cations, and
other substances.
8)Inhibitors:
Many glycoproteins in blood plasma have
shown antiproteolytic activity.
Example:glycoprotein a1-antichymotrypsin
inhibits chymotrypsin.

23. 9)Defense:
In beetles pygidial glands secrete a glycoprotein
disinfecting paste that covers the body and
hardens.
This shell provides protection against attack by
bacteria and fungi.
10)Freezing-point depression:
Glycoproteins were found in the sera of antarctic
fishes to decrease the freezing point due to their
apparent interaction with water.
11)Vision:
 In bovine visual pigment a glycoprotein forms
the outer membranes of retinal rods.

24. 12)Immunological:
 The interaction of blood group substances with
antibodies is determined by the glycoproteins on
erythrocytes.
 Many immunoglobulins are actually glycoproteins .
 Soluble immune mediators such as helper, suppressor,
and activator cell have been shown to bind to
glycoproteins found on the surface of their target cells.
 B and T-cells contain surface glycoproteins that attract
bacteria to these sites and bind them.
 It can direct phagocytosis. Because the HIV virus
recognizes the receptor protein CD4, it binds to helper
T cells which contain it.

25. Some Functions of the Oligosaccharide Chains of
Glycoproteins
• Modulate physicochemical properties, eg, solubility,
viscosity, charge, conformation, denaturation, and binding
sites for various molecules, bacteria viruses and some
parasites.
• Protect against proteolysis, from inside and outside of cell.
• Affect proteolytic processing of precursor proteins to
smaller products.
• Involved in biologic 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.

26. Some Important Methods Used to
Study Glycoproteins
METHOD USE
Periodic acid–Schiff reagent Detects glycoproteins as pink bands after
electrophoretic separation.
Incubation of cultured cells with a
radioactive sugar
Leads to detection of glycoproteins as
radioactive bands after electrophoretic
separation.
Treatment with appropriate endo- or
exoglycosidase or phospholipases
Resultant shifts in electrophoretic
migration help distinguish among proteins
with N-glycan, O-glycan, or GPI linkages
and also between high mannose and
complex N-glycans.
Sepharose-lectin column chromatography To purify glycoproteins or glycopeptides
that bind the particular lectin used.
Compositional analysis following acid
hydrolysis
Identifies sugars that the glycoprotein
contains and their stoichiometry.

27. Mass spectrometry Provides information on molecular mass,
composition, sequence, and sometimes
branching of a glycan chain.
NMR spectroscopy To identify specific sugars, their sequence,
linkages, and the anomeric nature of
glycosidic linkages.
Methylation (linkage) analysis To determine linkages between sugars.
Amino acid or cDNA sequencing Determination of amino acid sequence.
Contd…

28. Clinical Significances of Glycoproteins
Glycoproteins on cell surfaces are important
for communication between cells, for
maintaining cell structure and for self-
recognition by the immune system.
The alteration of cell-surface glycoproteins
can, therefore, produce profound
physiological effects, of which several are
listed below.

29. 1. The ABO blood group antigens are the
carbohydrate moieties of glycolipids on the
surface of cells as well as the carbohydrate
portion of serum glycoproteins.
When the ABO carbohydrates are associated with
protein in the form of glycoproteins they are
found in the serum and are referred to as the
secreted forms.
Some individuals produce the glycoprotein forms
of the ABO antigens while others do not.
 This property distinguishes secretors from non-
secretors, a property that has forensic
importance such as in cases of rape.

30. 2. The truncation of erythrocyte surface
glycoproteins leads to cell clumping, as in
congenital dyserythropoietic anemia type II. Also
referred to as HEMPAS (hereditary erythroblastic
multinuclearity with positive acidified-serum
test).
3. Several viruses, bacteria and parasites have
exploited the presence of cell-surface
carbohydrates, principally associated with protein
(glycoproteins), using them as portals of entry
into the cell. Ex-

31. i) The malarial parasite Plasmodium vivax, binds
to the erythrocyte chemokine receptor known
as the Duffy blood group antigen (also known
as the erythrocyte receptor for interleukin-8)
to infect erythrocytes.
ii)Rabies virus binds to cells through interactions
with neural cell adhesion molecule (N-CAM).

32. 4. Some glycoproteins are tethered to the membrane by a
lipid linkage:
 The protein is attached to the carbohydrate through
phosphatidylethanolamine (PE) linkage, and the carbohydrate is
in turn attached to the membrane via linkage to
phosphatidylinositol (PI), which anchors the structure within the
membrane.
 The linkage is called a glycosylphosphotidylinositol (GPI) anchor.
 Decay-accelerating factor(DAF) prevents erythrocyte lysis by
complement. When this factor is lost from the erythrocyte
surface, abnormal hemolysis occurs, with the end result of
hemoglobin accumulation in the urine.
 Other important GPI linked proteins are the enzymes
acetylcholinesterase, intestinal and placental alkaline
phosphatase and 5'-nucleotidase, the cell adhesion molecule N-
CAM (neural cell adhesion molecule) and the T-cell markers Thy-
1 and LFA-3 (lymphocyte function associated antigen-3).

33. 5. Defects in the proper targeting of
glycoproteins to the lysosomes can also lead
to clinical complications:
 Deficiencies in the enzyme responsible for the
transfer of GlcNAc-1-P to Man residues (GlcNAc
phosphotransferase) in lysosomal enzymes leads to
the formation of dense inclusion bodies formation in
the fibroblasts.
 Two disorders related to deficiencies in the targeting
of lysosomal enzymes are termed I-cell disease
(mucolipidosis II) and pseudo-Hurler polydystrophy
(mucolipidosis III, also called mucolipidosis-HI).

34.  I-Cell disease:-
 Deficiency of GlcNAc phosphotransferase,
 Resulting in abnormal targeting of certain lysosomal enzymes.
 characterized by:
o severe psychomotor retardation,
o skeletal abnormalities,
o coarse facial features,
o painful restricted joint movement, and
o early mortality.
 Pseudo-Hurler polydystrophy is less severe; it progresses
more slowly, and afflicted individuals live to adulthood.

35. Congenital Disorders of Glycosylation (CDG)
 Autosomal recessive disorders
 Multisystem disorders that have probably not been recognized in
the past
 Generally affect the central nervous system, resulting in
psychomotor retardation and other features
 Type I disorders are due to mutations in genes encoding enzymes
(eg, phosphomannomutase-2 [PMM-2], causing CDG Ia) involved in
the synthesis of dolichol-P-P-oligosaccharide
 Type II disorders are due to mutations in genes encoding enzymes
(eg, GlcNAc transferase-2, causing CDG IIa) involved in the
processing of N-glycan chains
 At least 15 distinct disorders have been recognized
 Isoelectric focusing of transferrin is a useful biochemical test for
assisting in the diagnosis of these conditions; truncation of the
oligosaccharide chains of this protein alters its iso-electric focusing
pattern.
 Oral mannose has proved of benefit in the treatment of CDG Ia

36. CDG-Ia:
most commonly occurring CDG, with
appearance in individuals of European.
 children are ataxic and have skeletal abnormalities
consisting of long limbs and short torsos.
 Due to defective synthesis of coagulation factors by the
liver (primarily factor XI, antithrombin III, protein C and
protein S), patients have severe coagulation defects.
 Hepatomegaly with consequent liver dysfunction.
 CDG-Ia results from mutations in
phosphomannomutase 2 (PMM2) the enzyme that is
required to convert Man-6-P to Man-1-P used in the
generation of GDP-Man.
 Over 60 mutations in PMM2 have been identified that
either decrease enzyme activity or stability.

37. CDG-IIc:
more commonly referred to as leukocyte adhesion
deficiency syndrome II (LAD II).
 LAD II belongs to the class of disorders referred to as primary
immunodeficiency syndromes as the symptoms of the disease
manifest due to defects in leukocyte function.
 Symptoms:
o unique facial features,
o recurrent infections,
o persistent leukocytosis,
o defective neutrophil chemotaxis and
o severe growth and mental retardation.
 The genetic defect resulting in LAD II is in the pathway of fucose
utilization leading to loss of fucosylated glycans on the cell surface.

38. Summary
 Glycoproteins are widely distributed proteins—with diverse
functions—that contain one or more covalently linked carbohydrate
chains.
 The carbohydrate components of a glycoprotein range from 1% to
more than 85% of its weight and may be simple or very complex in
structure. Eight sugars are mainly found in the sugar chains of
human glycoproteins: xylose, fucose, galactose, glucose, mannose,
N-acetylgalactosamine, N-acetylglucosamine and N-
acetylneuraminic acid.
 At least certain of the oligosaccharide chains of glycoproteins
encode biologic information; they are also important to
glycoproteins in modulating their solubility and viscosity, in
protecting them against proteolysis, and in their biologic actions.
 The structures of oligosaccharide chains can be elucidated by gas-
liquid chromatography, mass spectrometry, and high-resolution
NMR spectrometry.
 Glycosidases hydrolyze specific linkages in oligosaccharides and are
used to explore both the structures and functions of glycoproteins.

39. Contd…
 Lectins are carbohydrate-binding proteins involved in cell adhesion
and many other biologic processes.
 The major classes of glycoproteins are O-linked (involving an OH of
serine or threonine), N-linked (involving the N of the amide group
of asparagine), and glycosylphosphatidylinositol (GPI)-linked.
 Mucins are a class of O-linked glycoproteins that are distributed on
the surfaces of epithelial cells of the respiratory, gastrointestinal,
and reproductive tracts.
 The endoplasmic reticulum and Golgi apparatus play a major role in
glycosylation reactions involved in the biosynthesis of glycoproteins.
 The oligosaccharide chains of O-linked glycoproteins are
synthesized by the stepwise addition of sugars donated by
nucleotide sugars in reactions catalyzed by individual specific
glycoprotein glycosyltransferases.

40. Contd…
 In contrast, the synthesis of N-linked glycoproteins involves a
specific dolichol-P-P-oligosaccharide and various glycotransferases
and glycosidases. Depending on the enzymes and precursor
proteins in a tissue, it can synthesize complex, hybrid, or high-
mannose types of N-linked oligosacccharides.
 Glycoproteins are implicated in many biologic processes. For
instance, they have been found to play key roles in fertilization and
inflammation.
 A number of diseases involving abnormalities in the synthesis and
degradation of glycoproteins have been recognized. Glycoproteins
are also involved in many other diseases, including influenza, AIDS,
rheumatoid arthritis, cystic fibrosis and peptic ulcer.
 Developments in the new field of glycomics are likely to provide
much new information on the roles of sugars in health and disease
and also indicate targets for drug and other types of therapies.

42. References
1) Harper's Illustrated Biochemistry, 28the
2) Lippincott’s Illustrated Biochemistry, 4the
3) Textbook of Biochemistry (Vasudevan), 6the
4) www.google.com

43. “To be good & to do good that is the whole of
religion”