Glycoproteins
Charlotte V. Bañes, MD
Department of Biochemistry
Davao Medical School Foundation
Introduction
• Glycobiology
– Study of the role of sugars in health and disease
• Glycome
– The entire complement of sugars
• Glycomics
– Study of glycomes, including genetic, physiologic, pathologic and
other aspects
Introduction
• Glycoproteins
– Proteins that contain oligosaccharide
chains
• Glycosylation
– Most frequent post-translational
modification of proteins
– Enzymatic attachment of sugars
• Glycation
– Non-enzymatic attachment of sugar to
proteins
– Have pathologic consequences (diabetes)
Importance
• Examples of glycoproteins
– Almost all human plasma proteins (except
albumin)
– Blood group substances
– Some hormones
• Alterations in the structure of glycoproteins of
cancer cells lead to metastasis
Functions of Glycoproteins
Function Glycoproteins
Structural molecule Collagen
Lubricant and protective agent Mucin
Transport molecule Transferrin, ceruloplasmin
Immunologic molecule Immunoglobulins, histocompatibility antigens
Hormone Chorionic gonadotropin, TSH
Enzyme Various (eg alkaline phosphatase)
Cell attachment recognition site Various proteins involved in cell-cell, virus-
cell, bacterium-cell, hormone-cell interactions
Receptor Various proteins involved in hormone and drug
action
Hemostasis Glycoprteins found on the surface membranes
of platelets
Functions of Glycoproteins
Principal sugars found in Glycoproteins
Sugar Type Structure
Galactose (Gal) Hexose
Glucose (Glc) Hexose
Mannose (Man) Hexose
N-Acetylneuraminic acid Sialic acid (nine C
(NeuAc) atoms)
Principal sugars found in Glycoproteins
Sugar Type Structure
Fructose (Fuc) Doexyhexose
N-Acetylgalactosamine Aminohexose HO
(GalNAc)
N-Acetylglucosamine Aminohexose
(GlcNAc)
Xylose (Xyl) Pentose
Principal sugars found in Glycoproteins
• NeuAc
– Found at the termini
– Attached to subterminal Gal or Gal-Nac
• Sulfate
– Attached to Gal, Gal-NAc, Glc-Nac
• Sources
– All except glucose are synthesized from glucose
– Diet
Nucleotide Sugars
“Sugars participate in biochemical reactions in the form
of nucleotide sugars.”
• UDP-Glc
– First nucleotide sugar reported
Nucleotide
Glycoproteins and their corresponding Nucleotide Sugars
Sugar Nucleotide sugar
Galactose (Gal) UDP-Gal
Glucose (Glc) UDP-Glc
Mannose (Man) GDP-Man
N-Acetylneuraminic acid (NeuAc) CMP-NeuAc
Fructose (Fuc) GDP-Fuc
N-Acetylgalactosamine (GalNAc) UDP-GalNAc
N-Acetylglucosamine (GlcNAc) UDP-GlcNAc
Xylose (Xyl) UDP-Xyl
Nucleotide Sugars - Sythesis
• Occurs in the CYTOSOL
• Requires
– Nucleoside triphosphate
– Phosphorylated sugar
• Enzymes
– Phosphorylase
– Epimerase
Nucleotide Sugars - Sythesis
Formation of UDP-Gal:
UDP-Glc pyrophosphorylase
UTP + Glc-1-P UDP-Glc + pyrophosphate
UDP-Glc epimerase
UDP-Glc UDP-Gal
Formation of UDP-Glucose
Nucleotide Sugars
• Glycosylation occurs within the lumen of
Golgi apparatus
• Nucleotide sugars are transpoted from the
cytosol across the Golgi membrane through
carrier systems
Nucleotide Sugars
• Antiport system
– Influx of 1 molecule of nucleotide sugar is
balanced by the efflux of 1 molecule of the
corresponding nucleotide
– Transport nucleotide sugars across the Golgi
membrane
Antiport System
UMP
PPi
Golgi
apparatus UDP
-Glc
Nucleotide Sugars
galactosyl transferase
UDP-Gal + protein protein-Gal + UDP
nucleoside diphosphate phosphatase
UDP UMP + Pi
Major Classes of Glycoproteins
Major Classes of Glycoproteins
• O-linked
– Contains O-glycosidic linkage
– Involves –OH of serine or threonine and a sugar (GalNAc)
• N-linked
– Contains N-glycosidic linkage
– Involves the amide nitrogen of asparagine and Glc-NAc
• Glycosylphosphatidylinositol – anchored (GPI-linked)
– Linked to the –COOH terminal amino acid of a protein via a
phosphoryl-ethanolamine moiety joined to an oligosaccharide which in
turn is linked via glucosamine to phosphatidylinositol
• oligosaccharide chains can vary in number
• oligosaccharides can have different types of
linkages
– Eg: glycophorin contain both O- and N- linked
oligosaccharides
O-glycosidic linkages
• Subclasses:
– GalNAc-Ser (Thr)
• Predominant
• Found in mucin
– Gal-Gal-Xyl-Ser
• proteoglycans
– Gal-hydroxylysine
• collagens
– GlcNAc-Ser
• Nuclear and cytosolic proteins
Mucins
• Major characteristics
– High content of O-linked oligosaccharides (>50%)
– Repeating amino acid sequences (rich in serine,
threonine, proline)
– High sugar content water-holding capacity;
resistant to proteolysis
– Extended structure high viscosity
Mucins
• Function:
– Secretory
• Found in mucus present in secretions of GI, respiratory,
and reproductive tracts
• Lubrication
• Protective barrier on epithelial cells
– Membrane bound
• Cell-cell interactions
• May contain or mask certain surface antigens
Mucins
O-linked Glycoproteins - Synthesis
• Polypeptide chains are encoded by mRNA, translated
in ribosomes
• Glycosylation occurs in the Golgi apparatus
• Enzymes used in glycosylation are found in the Golgi
apparatus (glycoprotein glycosyl transferases)
• Glycosylation occurs by stepwise donation of sugars
from nucleotide sugars
N-linked Glycoproteins
• Asn-Glc-Nac linkage
• Most readily accessible glycoprotein
• Found in membrane bound and circulating
glycoproteins
N-linked Glycoproteins
• Three major classes:
– Complex
– Hybrid
– High -mannose
• Common pentasaccharide
– Man3GlcNAc2
– Share an initial common mechanism of
biosynthesis
Major Classes of N-linked Glycoproteins
• Complex
– Contain terminal NeuAc residues
– underlying N-acetyllactosamine (Gal and GlcNAc
residues)
• Mannose-rich
– Have 2 to 6 additional mannose residues
• Hybrid
– Contain features of both complex and mannose rich
classes
Major Classes of N-linked Glycoproteins
Biosynthesis of N-linked Glycoproteins
• Steps:
1. Assembly and transfer of Dolichol-P-P-
oligosaccharide common petasaccharide core
structure
2. Processing of oligosaccharide chain different
classes
Assembly of Dolichol-P-P-oligosaccharide
• Dolichol
– Polyisoprenol of eukaryotes
– Participate in the synthesis of N-linked
glycoproteins and GPI anchors
– Longest occuring hydrocarbon made up of a single
repeating unit
– Composed of 17-20 repeating unit
Assembly of Dolichol-P-P-oligosaccharide
• Phosphorylation of dolichol
Dol
ATP
dolichol kinase
ADP
Dol P
Assembly of Dolichol-P-P-oligosaccharide
Dol P
UDP-GlcNAc
GlcNAc-P transferase
UMP
Dol P P Glc NAc
Assembly of Dolichol-P-P-oligosaccharide
Dol P
UDP-GlcNAc
GlcNAc-P transferase
UMP
Dol P P Glc NAc
UDP-GlcNAc
GlcNAc-P transferase
UMP
Dol P P Glc NAc Glc NAc
Assembly of Dolichol-P-P-oligosaccharide
Dol P P Glc NAc Glc NAc
GDP-M
GDP
Dol P P Glc NAc Glc NAc Man
Assembly of Dolichol-P-P-oligosaccharide
Dol P P Glc NAc Glc NAc
GDP-M
GDP
Dol P P Glc NAc Glc NAc Man
4(GDP-M)
4(GDP)
Man
Dol P P Glc NAc Glc NAc Man
Man Man Man
Assembly of Dolichol-P-P-oligosaccharide
Man
Dol P P Glc NAc Man
2 Man Man Man
4(Dol-P-M)
4(Dol-P)
Man Man
Man
Man Man
Dol P P Glc NAc Man
2 Man Man Man
Assembly of Dolichol-P-P-oligosaccharide
Man Man
Man
Man Man
Dol P P Glc NAc Man
2 Man Man Man
3(Dol-P-Glc)
3(Dol-P)
Man Man
Man
Man Man
Dol P P Glc NAc Man
2 Man Man Man Glc Glc Glc
Assembly of Dolichol-P-P-oligosaccharide
• 1st 7 sugars
– donated by nucleotide sugars
– UDP-GlcNAc (2), GDP-M (5)
• Last 7 sugars
– donated by Dol-P- sugars
Man Man
Man
Man Man
Dol P P Glc NAc Man
2 Man Man Man Glc Glc Glc
Formation of N-glycosidic bond
• Catalyzed by oligosaccharide: protein
transferase
– Recognize Dol-P-P-(GlcNAc)2-R
– Strong preference to Dol-P-P-(GlcNAc)2-M9-Glc3
– Forms an N-glycosidic link between Dol-P-P-
(GlcNAc)2-M9-Glc3 and Asn residue of Asn-X-
Ser/Thr tripeptide
• X- any amino acid except proline, aspartic acid,
glutamic acid
Formation of N-glycosidic bond
Asn-X-Ser/Thr + Dol-P-P-(GlcNAc)2-M9-Glc3
(GlcNAc)2-M9-Glc3 + Dol-P-P
Asn-X-Ser/Thr Dol-P + Pi
Processing of Oligosaccharide Chain
• Reaction 1
– oligosaccharide: protein transferase
– Formation of N-glycosidic bond
Processing of Oligosaccharide Chain
• Reaction 2 and 3
– Removal of terminal Glc residue by glucosidase I
– Removal of the next 2 Glc residue by glucosidase
II
Processing of Oligosaccharide Chain
Man Man
Man
Man Man
Asn X Ser/ Glc NAc Man
Thr
2 Man Man Man Glc Glc Glc
Processing of Oligosaccharide Chain
Man Man
Man
Man Man
Asn X Ser/ Glc NAc Man
Thr
2 Man Man Man Glc Glc
Processing of Oligosaccharide Chain
Man Man
Man
Man Man
Asn X Ser/ Glc NAc Man
Thr
2 Man Man Man Glc
Processing of Oligosaccharide Chain
Man Man
Man
Man Man
Asn X Ser/ Glc NAc Man
Thr
2 Man Man Man
Processing of Oligosaccharide Chain
• Processing of complex chains
– Reaction 4 and 5:
• Removal of 4 external Man residues
– Reaction 6
• GlcNAc residue is added to the Man residue of the Man α1-arm
• GlcNAc transferase 1
– Reaction 7
• Reduction of mannose residues to the core number of three
• Mannosidase (Golgi α1-mannosidase II)
Processing of Oligosaccharide Chain
• Processing of complex chains
– Reaction 8
• 2nd GlcNAc is added to the peripheral Man residue
• GlcNAc transferase II
– Reaction 9, 10, 11
• Addition of Fuc, Gal, NeuAc residues
• Catalyzed by fucosyl, galactosyl, sialyl transferase
Processing of Oligosaccharide Chain
Sites of Glycosylation
• Endoplasmic Reticulum
– Assembly of Dol-P-P-oligossacharide
– Addition of oligosaccharides to protein
– Removal of Glc and some Man residues
• Golgi Apparatus
– Reaction 5 (Golgi α-mannosidase I)
– Reaction 6 (GlcNAc transferase I)
– Reactions 9,10, 11 (fucosyl, galactosyl, sialyl
transferases)
Inhibitors of Glycosylation
• Tunicamycin
– Inhibits GlcNAc-P transferase
– Inhibits addition of GlcNAc to dol-P (1st step in the
synthesis of dol-P-P-oligo)
• Doexynojirimycin
– Inhibits of glucosidase I and II
• Swainsonine
• Inhibits mannosidase II
• GalNAc –benzyl
– Inhibits extension of O-linked chains beyond GalNAc
CLINICAL IMPORTANCE
Glycoproteins and Fertilization
• Oocyte’s zona pellucida
contains 3 glycoproteins
(ZP3) which contains an O-
linked glycoprotein
– Functions as a receptor for the
sperm
• Sperm contains galactosyl
transferase which interacts
with ZP3 initiates
acrosomal reaction
Glycoproteins and Fertilization
• PH3 – glycoprotein in sperms facilitates binding of the PM of
sperm and PM of oocyte
• Drugs or antibodies that inhibits functions of PH3 and ZP3
may act as contraceptive agents
Glycoproteins and Inflammation
• Selectins
– glycoproteins that participate in
intercellular adhesions
• Adhesion of neutrophils to
endothelial cells is mediated
by selectins and other adhesion
molecules found in neutrophils
and in the endothelial surfaces.
Glycoproteins and Diseases
• Cancer
– Increased branching of cell
surface glycans or presentation of
selectin ligands metastasis
– At the primary site, cancer cells
secrete high levels of small
integrin-binding ligand, N-linked
glycoproteins
– Cancer cells with enhanced
adhesive and migratory
capabilities can detach from the
primary tumour mass and degrade
the basement membrane to invade
the stroma
Glycoproteins and Diseases
• Congenital disorders of glycosylation
– Autosomal recessive disorder
– Affect the CNS psychomotor retardation, and other features
– Type1: mutations in genes encoding phosphomannomutase2 (PMM2)
• Causes CDG 1a
• Involved in the synthesis of dol-P-P-oligosaccharide
• Oral mannose may have benefits in treatment
– Type2: mutations in genes encoding GlcNAc transferase 2
• Causes CDG2a
• Involved in processing N-glycan chains
Glycoproteins and Diseases
• Heredetary erythroblastic multinuclearity with
a positive acidified lysis test (HEMPAS)
– Abnormalities in certain enzymes (mannosidase II)
involved in the biosysthesis of N-glycans
– Affects RBC
Glycoproteins and Diseases
Paroxysmal Nocturnal Hemoglobinuria
Mutations in phosphatidylinositol glycan class A
(PIG-A) gene of certain hematopoietic cells
Defective synthesis of the enzyme that links
glucosamine to PI (GlcNH2-PI linkage) of GPI
anchors
Decreased amounts in the RBC membrane of GPI
anchored proteins (decay accelerating factor), CD59
Components of the complement system (MAC) not
opposed by DAF/ CD59 lysis of RBC
Glycoproteins and Diseases
• Leukocyte adhesion deficiency, type II
– Mutations affecting a Golgi located GDP-fucose
transporter
– Leads to absence of fucosylated ligands for
selectins
– Decreased neutrophil rolling
– Patients suffer from life threatening, recurrent
bacterial infections; psychomotor and mental
retardation
Glycans bind viruses and bacteria
Glycans bind viruses and bacteria
• Glycans bind specifically to a variety of
molecules like proteins or other glycans
• Bind viruses and many bacteria
Glycans bind viruses and bacteria
• Influenza A virus
– Binds to cell surface receptors
molecules containing NeuAc
via hemagglutinin
– Neuraminidase – release of
newly synthesized virus from
the infected host cell to infect
other cells
– Inhibitors of neuraminidase
(zanamivir,
oseltamivir)prevents spread of
virus to other cells
Glycans bind viruses and bacteria
• HIV-1
– Cause of AIDS
– Attaches to cells via
gp120 (surface
glycoproteins)
– gp41 used to fuse with
the host cell membrane
Glycans bind viruses and bacteria
• Helicobacter pylori
– Major cause of peptic ulcer
– Binds to two glycans in the
gastric epithelium
– Establishes a stable
attachment, secretes
ammonia and other subtances
leading to ulceration
‘Never give yourself a chance to fail’
