Protein glycosylation and its associate disorders. Glycosylation is one of the post translational modifications important for the normal function of the protein such as cell adhesion, signalling etc.. defect in this process leads to fatal disorder such as cancer, PNH....
4. WHAT IS POST TRANSLATIONAL
MODIFICATION ? ? ?
Post translational modification
(PTM) is the chemical modification
of a protein after its translation.
4
5. Why PTM is necessary???
ďStability of protein
ďBiochemical activity (activity
regulation)
ďProtein targeting (protein
localization)
ďProtein signaling (protein - protein
interaction, cascade amplification)
5
7. Protein glycosylation :
⢠The attachment of sugar moieties to proteins to
form glycoprotein.
⢠Glycosylation is a critical function of the
biosynthetic-secretory pathway in the endoplasmic
reticulum (ER) and Golgi apparatus.
⢠Approximately half of all proteins typically
expressed in a cell undergo this modification.
⢠Secreted proteins, surface receptors and ligands
and organelle-resident proteins.
7
8. Function:
⢠In the ER, it is used to monitor the status of protein
folding.
⢠Facilitate their delivery to the correct destination.
⢠It act as ligands for receptors on the cell surface to
mediate cell attachment or stimulate signal transduction
pathways.
⢠It can affect protein-protein interactions by either
facilitating or preventing proteins from binding to
cognate interaction domains.
â˘Because they are hydrophilic, they can also alter the
solubility of a protein.
8
10. Glycoprotein differ from Proteoglycan:
Glycoprotein Proteoglycan
⢠Protein with
oligosaccharide chains
(glycans).
⢠Carbohydrates chain is
relatively short. The
chains are often branched
instead of linear
â˘N-linked or O-linked
saccharides.
⢠Found on cell surfaces
⢠Consists of a core protein
with glycosaminoglycan
(GAG) chain(s).
⢠The chains are long, linear
and are negatively
charged due to the
occurrence of sulfate and
uronic acid groups.
⢠Categorized depending
upon the nature of their
GAG chains.
⢠Found mainly in
connective tissues
10
11. Types of glycosylation:
N â Linked
O â Linked
Glypiation
C â Linked
Phosphoglycosylation
Glycan bind to the amino group of
asparagine in the ER
Monosaccharides bind to the hydroxyl
group of serine or threonine in the ER,
Golgi, cystosol and nucleus
Glycan core links a phospholipid and a
protein
Mannose binds to the indole ring of
tryptophan
Glycan binds to serine via
phosphodiester bond
11
16. N â linked glycosylation :
1. Synthesis of dolichol-linked
precursor oligosaccharide
(ER)
2. En bloc transfer of precursor
oligosaccharide to protein
(ER)
3. Processing of the
oligosaccharide (Golgi)
16
17. Dolichol phosphate
⢠It is an isoprenoid compound (90-100 carbons total)
made from dolichol by phosphorylation catalyzed by
a kinase that uses CTP as the energy and phosphate
source.
17
18. 18
Synthesis of precursor oligosaccharide and
en bloc transfer:
24. ⢠Complex oligosaccharides â contain multiple sugar types
⢠High-mannose oligosaccharides â multiple mannose
residues
⢠Hybrid â branches of both high mannose and complex
ďź common core stage become resistant to glycan removal
24
by endoglycosidase H (endo H)
25. 25
O â linked glycosylation :
⢠This type of glycosylation is essential in the
biosynthesis of mucins.
â˘O-glycosylation is also critical for the formation of
proteoglycan core proteins that are used to make
extracellular matrix components.
â˘Antibodies are often heavily O-glycosylated.
â˘O-glycosylation occurs post-translationally on serine
and threonine side chains in the Golgi apparatus.
26. 26
Continues. . .
⢠Involves glycosyltransferase acting in stepwise manner.
Each transferase is specifically for particular type of
linkage.
⢠The enzyme involved are located in various compartment
of golgi apparatus.
⢠Each glycosylation involves the appropriate nucleotide
sugar.
⢠Dolichol â p â p oligosaccharide is not involved and the
reaction is not inhibited by tunicamycin.
27. 27
Mechanism:
⢠Proteins trafficked into the Golgi are most often O-glycosylated
by N-acetylgalactosamine (GalNAc)
transferase.
⢠Transfers a single GalNAc residue to the β-OH group of
serine or threonine.
⢠Some proteins are O-glycosylated with GlcNAc, fucose,
xylose, galactose or mannose, depending on the cell and
species.
⢠Sugar nucleotides are used as monosaccharide donors for
O-glycosylation.
29. Glypiation :
The covalent attachment of a glycosylphosphatidylinositol
(GPI) anchor is a common post-translational modification
that localizes proteins to cell membranes.
29
GPI anchors consist of :
⢠Phosphoethanolamine linker that binds to the C-terminus
of target proteins
â˘Glycan core structure
⢠Phospholipid tail that anchors the structure in
membrane
32. 32
Post-Glycosylation Modifications
⢠Sulfation at Man and GlcNAc residues in the production
of glycosaminoglycans (GAGs), which are components of
proteoglycans in the extracellular matrix.
â˘Acetylation of sialic acid to facilitate protein-protein
interactions.
⢠Phosphorylation, such as with Man residues on precursor
lysosomal proteins (mannose 6-phosphate) to ensure
trafficking to lysosomes by binding to mannose 6-
phosphate receptor (M6PR) in the Golgi.
34. 34
Fertilization
⢠Zona pellucida: ZP 1-3
⢠ZP 3 O linked
glycoprotein act as
receptor for sperm.
⢠Sperm surface contain
galactosy transferase.
⢠Induce the acrosomal
reaction.
35. 35
Erythropoietin is a vital hormone :
⢠Secreated by kidney and
stimulate production of
RBC.
⢠165 amino acid, N
glycosylated to 3 asp
residue & O glycosylated on
serine residue.
â˘Mature EPO is 40%
carbohydrate by weight and
enhance stability.
â˘Unglycosylated protein has
only about 10% of bioactivity.
36. 36
Blood groups are based on protein
glycosylation pattern :
ď Common
oligosaccharide : O
antigen.
ď A antigen contain extra
N â acetylgalactosamine
by type A transferase.
ď B antigen contain
galactose by type B
transferase.
38. 38
Some factors affecting the glycoprotein
processing enzymes :
Factor Comment
Previous enzyme Certain glycosyltransferase act only on oligosaccharide chain
if it has been acted upon by another processing enzyme.
Development The cellular profile of processing enzyme may change during
development of their gene turned on or off.
Intracellular
location
For eg, if an enzyme inserted in to the membrane of ER it may
never encounter golgi located processing enzymes.
Protein
conformation
Differences in conformation of different protein may facilitate
or hinder access of processing enzyme to identical
oligosaccharide chain.
Species Same cells eg: fibroblast from different species may exhibit
different pattern of processing enzymes.
Cancer Cancer cell may exhibit processing enzymes different from
those of corresponding normal cell.
40. Abnormalities in biosynthesis of glycoprotein
40
Disease
Cancer
Congenital disorders of glycosylation
Congenital dyserythropoetic anemia
type II
Leukocyte adhesion deficiency type II
Paroxysmal nocturnal hemoglobinuria
(PNH)
I â cell disease
41. 41
Cancer :
⢠Loss of normal topology
and polarisation of
epthelial tissue in cancer
result in mucins
secretion in to
bloodstream.
⢠Tumor cells invading the
tissues and bloodstream
also present such
mucins on their cell
surface.
43. 43
Congenital disorders of glycosylation :
â˘Autosomal recessive disorder.
â˘Generally affect the central nervous system.
â˘Result in psychomotor retardation.
⢠Type I disorder â mutation in gene coding for
phosphomannomutase 2 ( involved in synthesis of
dolichol P P oligosaccharide)
⢠Type II disorder â mutation in gene coding for GlcNAc
transferase 2 ( involved in process of N â glycan
chain.
â˘Atleast 15 distinct disorders have been recognised.
44. Congenital Dyserythropoietic
Anemia Type II :
â˘Maturation of N-glycans from oligomannose to complex-type
44
structures - essential role in cell adhesion and
recognition events in metazoan organisms.
⢠ι-glucosidases and ι-mannosidases in the endoplasmic
reticulum (ER) and Golgi complex to result in the
Man3GlcNAc2 core structure necessary for conversion to
complex-type N-glycans
45. 45
Leukocyte adhesion deficiency type II
⢠Rare autosomal recessive disorder characterized
by immunodeficiency resulting in recurrent infections.
⢠Recurrent bacterial infections, defects
in neutrophil adhesion.
⢠Resulting from a general deficiency of fucose possibily
due to defect in transformation of GDP â mannose in to
GDP â fucose.
46. 46
Paroxysmal nocturnal hemoglobinuria
(PNH)
Acquired mutations in the PIG â A gene of certain hematopoietic cells
Defective synthesis of the GlcNH2 â PI linkage of GPI anchors
Decreased amounts in the RBC membrane of GPI anchored proteins,
with decay accelerating factor and CD59 being special importance
Certain components of the complement system are not opposed by
DAF and CD59, resulting in complement mediated lysis of red cell.
48. 48
I â cell disease
⢠Part of the lysosomal storage disease
⢠Defective phosphotransferase (an enzyme of
the Golgi apparatus)
⢠The proteins are instead excreted outside the cell --
the default pathway for proteins moving through
the Golgi apparatus.
⢠Lysosomes cannot function without these proteins
⢠A buildup of these substances occurs within
lysosomes because they cannot be degraded,
resulting in the characteristic "I cells," or "inclusion
cells."
51. 51
Analysis Of Glycoprotein :
ďMass Spectrometry.
ďGlycoprotein Gel Stain.
ďX Ray Crystallography.
ďNMR Spectroscopy.
52. Inhibitors of enzyme involved in N â Glycosylation
52
Inhibitor Site of action
Tunicamycin Inhibits GlcNAc-P transferase, the enzyme
catalysing addition of GlcNAc to dolichol-P.
Deoxynojirimycin Inhibitor of glucosidases I and II
Swainsonine Inhibitor of mannosidase II
53. 53
Summary :
⢠Glycosylation is one of the most important posttranslational
modification of proteins.
⢠There are various types of carbohydrateâprotein glycosidic
linkage and a great variety of structures of proteinâlinked
oligosaccharides (glycans).
⢠At least certain of the oligosaccharide chain of glycoprotein
encode biological information.
⢠Glycosidases hydrolyze specific linkages in oligosaccharides and
used to explore both the structural and functions of
glycoprotein.
⢠Lectins are carbohydrate binding proteins involved in cell
adhesion and many other biological process.
54. 54
Continues . . .
⢠The major class of glycoproteis are O â linked, N â linked and GPI
linked.
⢠The endoplasmic reticulum and golgi apparatus plays a major
role in glycosylation reactions involved in the biosynthesis of
glycoproteins.
⢠The oligosaccharide chain of O â linked glycoproteins are
synthesized in stepwise addition of sugars donated by nucleotide
sugars.
⢠The synthesis of N â linked glycoproteins involves specific
dolichol â p â p oligosaccharide and various enzymes. It can
synthesis complex, hybrid or high mannose type.
55. 55
Continues . . .
⢠Alterations of glycosylation in many diseases are used for
diagnostic and prognostic purposes.
⢠Geneticallyâbased defects in protein glycosylation are the
reason of severe diseases that is a direct evidence for the
importance of glycosylation.
⢠Various glycosylation pathways are studied as potential
targets for therapeutic purposes.
56. 56
References :
⢠Cox. MM, Nelson. DL; Lehninger Principle of Biochemistry; 5th
edition.
⢠Berg JM, Tymoczko JL, Stryer L; Biochemistry; 7th edition.
⢠Murray RK, Bender DA, Botham KM, Kennelly PJ et al; Harper
Illustrated Biochemistry; 29th edition.
⢠Schwarz F, Aebi M; Mechanisms and principles of N-linked
protein glycosylation; Current Opinion in Structural Biology 2011,
21:576â582.
⢠Spiro R. G. Protein glycosylation: Nature, distribution, enzymatic
formation, and disease implications of glycopeptide bonds.
Glycobiology. 12, 43R-56R.
⢠Varki A, Cummings RD, Esko JD, et al., Essentials of Glycobiology.
2nd edition.
⢠Chapman NR, Christopher LR; The role of carbohydrate in sperm-
ZP3 adhesion; Molecular Human Reproduction vol.2 no.10.
All N-linked glycoproteins have the same precursor glycan structure
Trimming and adding sugars to diversify the glycans on individual glycoproteins.
This maturation pathway to generate diverse oligosaccharides is highly ordered, such that each step is dependent upon the previous step.
The Golgi segregates specific enzymes into different cisternae to facilitate this step-wise process