Glycoproteins(N & O Glycosylation ).

Presented By: Dr. Kapil Dev

OBJECTIVES:

• Glycoproteins

• Biological Roles Of Glycoproteins

• Glycosylation (N & O linkage)

• Diseases

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 .

Functions Served by Glycoproteins:

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

• 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

O-linkage (N -acetylgalactosamine to serine)

O-linkage

1. The GalNAcSer(Thr) linkage is the predominant linkage. This type of linkage is found in mucins

2. Proteoglycans contain a Gal-Gal-Xyl-Ser trisaccharide

3. Collagens contain a Gal-hydroxylysine (Hyl) linkage. 4. 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]).

• 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

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)

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.

Schematic diagram of a mucin

N-linkage(N -acetylglucosamine to asparagine)

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

Dolichol-P-P-Oligosaccharide:

Structure of Dolichol (polyisoprenol)

Structure of dolichol-P-P-oligosaccharide

Dol-P-P-GlcNAc2Man9Glc3.

Biosynthesis of DolicholP-P-oligosaccharide:

• Dolichol Dolichol phosphate (Dol-P)

• Dol-P + UDP-GlcNac Dol-P-P-GlcNac +UMP

dolichol kinase

ATP ADP

Biosynthesis of DolicholP-P-oligosaccharide (contd.)

1= oligosaccharide: protein transferase2= -glucosidase I3= -glucosidase II 4= endoplasmic reticulum 1,2-mannosidase 5=Golgi apparatus -mannosidase II= N-acetylglucosaminylphosphotransferase II= N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase

6= N-acetylglucosaminyltransferase I7= Golgi apparatus -mannosidase II8= N-acetylglucosaminyltransferase II 9= fucosyltransferase10= galactosyltransferase11= sialyltransferase.

Differences between O- & N- linked Glycoproteins:

N-Linked GlycoproteinsSynthesis:-

• 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 GlycoproteinsSynthesis:-

• Post- translationally• Oligosaccharide chain

synthesized on the protein.

• Enzymes membrane bound.

• Dolichol not involved.• Not inhibited by Tunicamycin

e.g. Mucin e.g. Calnexin

Some Diseases Due to Glycoproteins:

DiseaseHEMPAS

Leukocyte adhesion deficiency, type II

Paroxysmal nocturnal hemoglobinuria

I-cell disease

Congenital disorders of glycosylation

I-Cell Disease:

Summary of the causation of I-cell disease

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.

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.

Defects of O glycosylationName 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 deficiencyEhlers–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)

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

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

Paroxysmal Nocturnal Hemoglobinuria:

Scheme of causation of paroxysmal nocturnal hemoglobinuria

SUMMARY

• Glycoproteins • Functions Served by Glycoproteins • Classes of Glycoproteins• N and O linkage• Glycoproteins in Disease:

• I-Cell Disease• CDGs• others

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.

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