GlycoproteinsMr.Tapeshwar Yadav

Final Year, PGMamata Medical College-

Khammam,A.P,INDIA

06/01/12

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.

Glycoproteins and Proteoglycans

GlycoproteinsProteins conjugated to saccharides lacking a serial repeat unit

ProteoglycansProteins conjugated to polysaccharides with serial repeat units

Protein >> carbohydrate

Carbohydrate >> protein

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:-

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.

N-linked Glycoproteins

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.

O-linked Glycoproteins

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

OH

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

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 neuraminic 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.

Fucose Deoxyhexose

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

Aminohexose

GalNAc UDP-GalNAc

Present in both N- and O-linked glycoproteins.

N-Acetylglucosamine

Aminohexose

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 Pentose

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.

Functions Served by GlycoproteinsFunction 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

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

Functions1)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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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…

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.

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.

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-

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).

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).

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).

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.

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

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.

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.

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.

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.

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.

References

1) Harper's Illustrated Biochemistry, 28the2) Lippincott’s Illustrated Biochemistry, 4the3) Textbook of Biochemistry (Vasudevan), 6the 4) www.google.com

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