glycans: potential diagnostic and prognostic application •one of the causes of micro‐and...
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Glycans:potential diagnostic and prognostic application
Professor Jerka DumićDepartment of Biochemistry and Molecular Biology
Faculty of Pharmacy and BiochemistryUniversity of Zagreb
Glycoconjugates
glycoconjugates classification
structural and functional characteristics
biosynthesis of glycoconjugates
diversity of glycan functions
physiological receptors of glycans
examples glycoconjugates roles in heath, disease and therapy
The terms
glycan – mono‐, oligo‐ or polysaccharide; free or covalently attached to another molecule glycoconjugate
glycosylation – a process of enzymatic addition of glycan to aglycon (protein or lipid)
the most abundant posttranslational protein modification
saccharides are directed molecules – non‐reducing and reducing end
glycobiology – structure, biosynthesis, biology and evolution of saccharides and proteins which recognize glycans
the term introduced in 1988 (Rademacher, Parekh, and Dwek ; 1988 Annu Rev Biochem)
non‐reducing end
reducing end
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GLYCATION• non‐enzymatic adding of carbohydrates
• randomly chosen protein amino‐groups
• advanced glycation end products (AGEs)
• hyperglycemia (increased glucose concentration)
• one of the causes of micro‐ and macrovascular complications in diabetes mellitus type 1 and 2
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
Repertoar of monosaccharides foundin glycoconjugates is limited
HOCH 2 O
HO
OH
HO OH O
OHOH
OH
OH
H3C O
OHHO O H
HO O
OHOH
C CO
OHNCO
CH3
HO
COH
HOH2 C
HOCH2 O
OHHO
OH
HOHOCH2 O
OHHO
OH
OH
HOCH2 O
HO
OHNH
COH 3C
OHHO CH2 O
HO
O H
HO
NHC
OH3 C
N-acetyl--D-galactosamine(GalNAc)
-D-glucose(Glc)
-D-galactose(Gal)
N-acetyl--D-glucosamine(GlcNAc)
-D-mannose(Man)
-L-fucose(Fuc)
-D-xylose(Xyl)
N-acetylneuraminic acid (Neu5Ac, Sia)
D-glucuronic acid L-iduronic acid D-arabinose D- and L-ramnoseD- galacturonic acid N-glycolylneuraminic acid (Neu5Gc)
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Potential diversity of glycan structures is enormous
1. monosaccharide sequence
2. glycosidic bond position
3. anomeric configuration
( or ) of glycosidic linkage
4. number of branching points
5. position of branching point
HOCH2 O
HONH
COH3C
OH
O
HOCH2 O
OHHO
OO
O
OH
OH
OH
CH2
3
6
1
pentamere ABCDE number of isomeresoligosaccharides 2 144 640oligopeptides 120
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
majority of animalnih glycolipids
cerebrosides – only one monosaccharide
do not contain phosphate, so usually are not charged• galactocerebrosides – in the membranes of neural cells• glucocerebrosides – in the membranes of other cells
• sulphatides or sulphogalactocerebrosides contain sulphate ‐ negatively charged
globosides – ceramide + simple oligosaccharide (Lactosyilceramide)
gangliosides – ceramide + more complex oligosaccharide
galacto‐cerebroside
Glycolipids – sfingoglycolipids (glycosfingolipids)
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Glycolipids – glycosfingolipids ‐ gangliosides
Gangliosides – ceramide + more complex oligosaccharide contain sialic acid – negatively charged more than 60 types – GM1, GM2, GM3 cell‐cell recognition, hormone receptors mostly in brain (6% of lipids in brain)
serotype determinants Immune reactivity
Bacterial lipopolisaharides
specificchain
core
lipid A
outer membrane of Gram‐bacteria (E. coli, Salmonella typhimurium)
selective passage of nutritients and toxic substances
some of them are toxic for humans
ABO system of blood groups
30 systems of blood groups krvnih grupa; antigenes of erytrocytes
Karl Landsteiner, 1900 (Nobel prize, 1930)
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ABO system of blood groups
on erythrocytes, ali on other cells as well (endothelial, epithelial cells)
most of them are glycoproteins, and smaller part are glycolipids
Fuc
Gal
GalNAc
Varki, Essentials ofGlycobiology, 2nd edition
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
Glycoproteins glycans linked to proteins through N‐ or O‐glycosidic linkage
N‐glycosidic linkage O‐glycosidic linkage
in N‐linked oligosaccharides N‐acetylglucosamine on the reducing end is linked to Asn in the sequence Asn‐X‐Ser/Thr of the protein through C1 carbone
O‐glycosidic linkage is formed between C1 carbon at the reducing end of monosaccharide or oligosaccharide and hidroxyaminoacid, Ser or Thr (on the reducing end often N‐acetylgalactosamine is present)
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Types of N‐linked glycans glycans linked to proteins by N‐glycosidic linkage
N‐glycosidic linkage
3 types of N‐linked glycans contain common core –
oligomannose type complex type hybride type
Man GlcNac Gal Fuc Neu5Ac (Sia))
Man1‐6(Man1‐3) Man1‐4GlcNac1‐4GlcNac‐Asn‐X‐Ser/Thr
Varki, Essentials of Glycobiology, 2nd edition
Types of O‐linked glycans
glycans linked to proteins by O‐glycosidic linkage
O‐glycosidic linkage diverse structures
several core types
the most frequent contain GalNAcSer/Thr
mucins
Mucins
glycoproteins Mr > 200 kDa
glycan part (mostly O‐linked) 50‐90%
highly hydratized (contain Sia)
protective physical barier at the epithelial surfaces
solubile and membrane
mucosal tissue of gastro‐intestinal, respiratory and reproductive tract
structure of saliva mucin
O‐linked glycans N‐linked glycans
repeating sequences (rich in Ser, Thr)
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Mucins changed expression and/or changed glycosylation in different types
of tumors laboratory diagnostics and novel therapeutical approaches?
mucins changed glycans
basalmembrane
blood vessel
secretion ofmucins in circulation
healthy tissue tumor tissue
Varki, Essentials of Glycobiology, 2nd edition
MUCINS
laboratory diagnosticsMUC1 (CA 15‐3, CA27.29 ) – breast carcinoma
CA 19‐9 (sialyl‐lacto‐N‐fucopentose II) –pancreas cacrcinoma
CA50 – (sijalil‐lakto‐N‐tetraoza) – lung carcinoma
CA242 – adenocarcinoma of pancreas, colorectal carcinoma
normal MUC1 tumor MUC1
Glycosylation of MUC1 mucin is significantlyreduced in tumor cells
O‐linked glycans extend functional protein domains above membrane surface
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Examples of glycoproteins from different organisms
GLYCOPROTEIN source Mr % sugar
MEMBRANE & VIRUS GLYCOPROTEINSglycophorin hum. erythrocytes 31 000 60hemaglutinin influenza virus 210 000 25rodopsin bovine retina 40 000 7
ENZYMESalkalne phosphatase murine liver 130 000 18carboxypeptidase Y yeast 51 000 17
HORMONES & CYTOKINESchorionic gonadothropine hum. urin 38 000 31erythropoetin hum. urin 34 000 29interferon hum. leukocytes 26 000 20
SERUM GLYCOPROTEINSimunoglobuline hum. serum 150 000 10thyroglobuline bovine thyreoidea 670 000 8prothrombine hum. serum 72 000 8
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
Proteoglycans
Proteoglycans vs. glycoproteins?
Glycoprotein = glycans + proteinProteoglycan = glycosaminoglycans + protein
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Repeatedly linked huge number of specific disaccharides yeild glycosaminoglycans (GAG)
‐ = highly hydratized gelovs (1000 x bigger volumen) protection of tissue drying
they are all linked on proteins except hialuronate (proteoglycans)
PGs bild basic substance of extracellular matrix in which proteins are merged
conective tissues ‐bones, tetives, cartilage, skin, blood vessel walls…
repeating of disaccharides (50‐1000; hialuronic acid 25 000) ‐ GlcNAc or GalNac and uronic acid) (glucuronic or iduronic asid)
sulphate group (except hialuronate)
Voet and Voet, Biochemistry
Proteoglycans the main components of
conective tissue –macromolecules on cell surface and in extracellular matrix
structure:
core protein (transmembrane or extracellular)
long, linear chains of GAGs are covalently linked to the protein (trough Ser)
• turned into extracellular space
• sulphatized highly hydratized
• the biggest part of Mr
• biological activity
Syndecan structure
Heparin vs. heparan‐sulphatea) Heparan sulphate (HS)
‐ produced by almost all cells in the body‐ anticoagulant
b) Heparin‐ produced by mastocytes from conective tissue‐ in pharmacy as anticoagulant‐ in organism? – protection from bacteria?
weak sulphatation
highly sulphatated domian
core protein (Ser‐Gly)n
core protein
(GlcNSO3‐IdoA,2S),variabile O‐sulphatation on C6
and rare on C3 of aminosugar
(GlcNAc‐GlcA)2
(GlcNSO3 ,6S ‐ IdoA,2S)
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Extracellular matrix network extracellular proteoglycans, fibrilar matrix proteini (colagen, elastin,
fibronectin) and cell membrane proteins
anchoring of the cells in the matrix
directioning of cell migration during tissue development
Informationtransfer
Classification of proteoglycans
depending on homology, function and localizationInterstitial PGs‐decorin, biglycan‐maintaince of ECM structure
Agrecan family‐agrecan, versican‐cind to hialuronan‐in cartilage
PGs of secretory granules‐serglicin‐secretory granules in cytoplasm‐enable storage of + molecules (e.g. proteases) in granules
Cell membrane PGs ‐syndecan (transmembrane domain), glypican (bound onto GPI anchor)‐bind ligands participate in cellular signaling)
Varki, Essentials ofGlycobiology, 2nd edition
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
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Glycosylphosphatidylinositol (GPI) anchors
for “anchoring” of membrane proteins
core:Man(12)Man(14)Man(14) GlcNH2 (14)PI
modifications:• different oligosaccharides on mannose• fatty acids on inositol
ER lumen
cytosol COOH COOH
NH2
NH2
NH2NH2
C‐terminal part of cleaved peptide
protein anchored by GPI anchor
GPI
PP P P
Adding of proteins to GPI anchor occurs in ER
Common classes of glycoconjugates
Sia
Modified from Varki A. 1997. FASEB J. 11: 248–255; Fuster M. and Esko J.D. 2005. Nat. Rev. Can. 7: 526–542.
Regulatory modification ‐binding of N‐acetylglucosamine (O‐GlcNAc)
one molecule of GlcNAc linked by O‐glycosidic linkaged on Ser or Thr
in cytoplasm or nucleus
in all eukariotes
function ? – regulation cell signaling and transcription
Cell lacking GlcNAc‐transferase are not viabile
c‐myc, p53, Hsp, RNA polymerase...
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Central dogma of molecular biology
DNA RNA Protein Enzyme
Carbohydrate
LipidOrganism
Cell Glycoconjugate
“glycan structure” is characteristic for each cell, tissue and organism there’s no template for glycan synthesis glycan structures depend on expression of enzymes, specificity of
substrate and the availability of other molecules required for glycansynthesis
Synthesis of main types ofanimal glycoconjugatas (glycosylation)
protein
dolichol
oligosaccharide
sugar
GPI anchor
ceramide
Varki, Essentials ofGlycobiology, 2nd edition
Glycosylation starts in ER...
• oligosaccharide is transfered from dolichol onto propteinby membrane enzyme oligosaccharyl‐transferase
Cooper, Stanica:molekularni pristup
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...and continous in GA
in the process of glycan biosynthesis >500 gene products are involved (glycosyltransferases and glycosidases, transporters....)
Cooper, Cell: molecular approach
Main characteristics of N‐ and O‐glycosylation
N‐glycosylation
it starts in ER, continues in GA
big oligosaccharide Glc3Man9(GlcNac)2 built on lipid carrier – dolichol is trensferred onto protein
it occurs co‐translationaly– as soon as Asn apears in ER
glycosyltransferases andglycosidases are involved
O‐glycosylation
it occurs in GA (in ER only the first sugar is added onto protein)
monosaccharides are added one by one
It occurs post‐translationaly on particular Ser i Thr
only glycosyltransferases are involved, but not glycosidases
Fine details of N‐glycans strongly affect effector funcions of IgG
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Single IgG molecule is produced as a mixture of over 100 glycoforms
Erythropoietin synthesized in kidney induces production of erythrocytes 165 aa, 3 N‐glycosylation sites, 1 O‐glycosylation site
glycans account 40% of molecular mass, N‐linked glycans – heavely sialylated
in circulation ‐ desilalylation
in vivo EPO activity and its half‐life in circulation are directly related to appropriate N‐glycosylation (in vivo activity of deglycosylated EPO is less than 10% of glycosylated EPO because incompletely glycosylated forms are rapidly cleared by filtration in the kidney and through the action of asialoglycoprotein receptors on hepatocytes and macrophages
• Amgen Inc. (Thousand Oaks, CA) regurarly throws out as much as 80% of produced erythropoietin because the attached sugars are incorrect
Isoelectric focusing of transferrin
useful analysis in estimation of liver function
screning for Congenital disorders of Glycosylation
asialo-Tf
Ctrl CDG - I CDG- II
+
-
1 2 3
pentasialo-Tf
tetrasialo-Tf
trisialo-Tf
disialo-Tf
monosialo-Tf
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Problems in glycome solving
complex, non‐linear structures
there’s no template for their synthesis
“assembly‐line” system, which includes hundreds of gene products
numerous glycosylation sites on which different glycans are attached
specific cell, tissue, organ glycosylation pattern, dependent on the state and the activity of the system
there’s no why to synthetise lager amount of glycans in vitro
it is not possible to change specific glycan/glycoconjugate
lack of specific, sensitive and user‐ friendly techniques for glycananalysis
Which quantities do we analyze?
glucose 1 mol 1 mmol 1 μmol180 g 0.18 g 0.18 mg
1 mol1 mmol 10‐3
1 μmol 10‐6
1 nmol 10‐9
1 pmol 10‐12
1 fmol 10‐15
1 amol 10‐18
1 amol 1 mm1 fmol 1 m1 pmol 1 km1 nmol 1 000 km – Zagreb ‐ Napoli
1 μmol 1 000 000 km – 25x Earth's circumference at the Equator1 mmol 1 000 000 000 km – 7x distance from Earth to Sun1 mol 1 000 000 000 000 km – 2.5 light years
Diversity of glycan functions
modulation of physical and chemical characteristics (solubility, viscosity, charge, conformation, denaturation)
funtional effects
interactions receptor‐ligand
regulatory effects
intracellular directioning of proteins into organeles
directioning of cells into tissues
fertilisation (interactions sperm‐egg)
embrional development and tissue differentiation
control of immune system
cancerogenesis and metastasis
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During evolution glycan structurs got novel functions
prokariotes eukariotesmonocellular multicellular
structures on the cellsurface
modulation of protein
activities
intercellularsignaling
protein folding
‐(glyco)proteins that specifically recognize and bind carbohydrates
‐ “interpreters” of molecularinformation stored in oligosaccharide structures ofglycoconjugates
‐ minimal polypeptide sequence responsible for binding of crbohydrates ‐ carbohydrate recognition domain, CRD
‐ Interactions between CRD and binding determinants are non‐covalent, low‐affinity (Kd 10‐6), but numerous thus increasing
specificity
Lectins – physiological receptors of glycans
Feinberg i sur. Science 294:2163, 2001
61Man2 1GlcNAc1Man
3 1Man2 1GlcNAcα β
βα
1
4 5
3
2
cian – CRD ICAM-3 DCyellow – part of Man9 structure
Membranes of all cells are virtually covered with complex carbohydrates
>80% of membrane and >50% animal proteins proteins are glycosylated > 1% genes encoding enzymes involved in glycosylation
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Cell directing into the tissues (immune response)
selectins at the site of inflammation interact with membrane glycoproteins and trigger the extravasation of leukocytes
Immune responese
Mannose binding lectin/protein (MBL, MBP) is important player of innate immune responese
Glycoproteins at the surfaces of lower organisms contain mostly highly mannosylated structures which can be recognized by MBL ‐ induction of complement reaction which can kill pathogenesi (ane‐anti‐body). Children with non‐functional MBL suffer of fungal infecctions... In adults is not essential, except in immunodefficient persons (HIV, immunosupressive therapy)
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LECTINS
‐ physiological receptors of glycoconjugates
interaction of galectin‐3 CRD and βGal14βGlcNAc12αMan13(βGal14βGlcNAc12αMan16)βMan14GlcNAc
‐ “interpreters” of biological informations stored in oligosaccharide structures of glycans
– cell‐cell interactions
– infection
– immune reactions
– metastasis formation
Uropathogenic E. coli P‐type
Glycan structures on the membrane glycoproteins of endothelial cells of
urinary tract
na vrhovima pila P-tipa UPEC su adhezini – lektini koji
Gram‐, asporogenic, aerobe (anaerobe) bacteria
causes 90% of uncomplicated infections of urinary tract
adhesins – P‐type and Type 1 fimbriae (pila) = lectins that binds α‐Gal‐α(1,4)‐Gal and mannose
carriers of such glycans are more succesible to the infections with UPEC (99%)
pigeon ovalbumin contains numerous glycans with α‐Gal‐α(1,4)‐Gal –possible therapeutic application?
flagelae
pili supramolecular structures consisting ofa short, flexibile tip fibrillum attached tothe distal end of a thicker rod structure
nucleoid ribosomes
cytosolplasma membrane periplasma cell wall outer membrane
Combating infection – possible approach
CELL
SURFACE GLYCOPROTEIN
BACTERIA
LECTIN
CARBOHYDRATE = DRUG
LECTIN= DRUG
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Adhesion protects invading microorganism from elimination by ntural clensing mechanims
Inhibitors of adhesion prevent infection
(adapted from the Ph.D. thesis of Dina Zafriri, Tel Aviv University, 1988).
Microbial adherence and anti‐adhesive therapy
Inflammation
• involves numerous cell‐cel and cell‐matrix interaction
• usually a beneficial protective reaction to tissue injury
• underlines many pathophysiological conditions
TARGETING INFLAMMATION COMPONENTS
COMMON THERAPEUTIC APROACH
Galectin‐3
• ‐galactoside binding lectin
• monomer, Mr 26200‐30300, 249 aa
– rich Gly/Pro N‐terminal domain
– C‐terminal carbohydrate binding domain
• preferentially binds poly‐LacNAc chains
• LGALS3 gene (chromosome 14, locus q21‐q22, 17 kb, 6 exons, 5 introns)
• present in almost all cellular compartments depending on cell type and in extracellular space
lactose
N-terminal domain
(Hughes, 2001)
β1,6
n
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Gal-3
Gal-3
Gal-3Gal-3 Gal-3 Gal-3
Gal-3Bcl-2
PI3KK-Ras Akt
AktP
GTP
Caspase-8
Caspase-3
Caspase-9
Cytochrome c
Raf-1
MEK
ERK
Gal-3
JNK
Ask-1
Gal-3
Synexin
Gal-3Chrp
Cytokeratans
? in vivo
?
Alix/AIP1
Gal-3CBP70
Gal-3pre-mRNA processingGemin4
CD95Growth factor receptor
proliferationGal-3
Gal-3Cyclin E
Cyclin A
p27KIP1
transport
?
p21WAF1/CIP1
Cyclin D
enhancement/stabilization of TF binding
tion
Gal-3Nucling
CREB Sp1
Gal-3
Tcf-4-catenin
Gal-3
Tcf-4-catenin
Gal-3
regulation of Wnt signaling
TTF-1
Axin
Apoptosis
Nucleus
Extracellularspace
Cytosol
c-myc
(Dumic et al., 2006)
Gal-3
Gal-3
Gal-3
TR EGFR TCR
CD7CD29
Gal-3
Gal-3 Gal-3
Gal-3Gal-3
CD66a CD66b FcR
IgE
Gal-3Gal-3
Gal-3
Lamp 1/2
Gal-3
Gal-3
CEANCA-160(CD66a)
Gal-3
CD11b/C4.4ACD18
Gal-3 Gal-3
CD98
Gal-3
NG2
Apoptosis
Regulation of cell adhesion
Activation of neutrophils
Angiogenesis
(31 integrin)
CD49c
Cross-linking
Lattice formation
Receptor life-timeregulation
Activation of mast cells
Cross-linking
Lattice formation
Regulation of TCR signaling/T-cell activation
?
Ca2+ influx
?
?
Cytochrome c
Caspase-3
Neural adhesion molecules(MAG, N-CAM, L1)
Gal-3
Gal-3
Laminin 1, 5, 10
Fibronectin
Gal-3
VitronectinGal-3
LPS
Gal-3
AGE
Gal-3
Mucin-1
Gal-3
Mac-2BP
Gal-3
-subunit
Haptoglobin(cancer-associated
glycoform)
Circulation
Extracellular space
Cytosol
Galectin-3 ND –non-glycan interaction
Galectin-3 CRD –glycan interaction
Gal-3
Gal-3
Collagen IV
Gal-3
Tenascin-C, -R
Gal-3
Hensin
Gal-3
Elastin
(Dumic et al., 2006)
Galectin‐3 in inflammation and fibrosis...
upregulated
– in humans in: ̶ in mice in:
• strong lamina propria fibroblast‐stimulating factor
• elevated in obesity and negatively correlates with glycated hemoglobin in type 2 diabetes
• prognostic marker in patients with chronic heart failure
• diagnostic marker for thyroid cancer
– hepatic fibrosis
– renal fibrosis
– cardiac fibrosis
– liver cirrhosis
– idiopathic lung fibrosis
– chronic pancreatitis
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PRO‐INFLAMMATORY
ANTI‐INFLAMMATORY
Gal‐3
Macrophages
LPS/IFN‐γ
Classical activation
IL‐4/IL‐13
Alternative activation
TLR4
IL‐4 RIL‐13 R1
iNOS
IL‐6
TNF‐
NO
M1
Arginase
MR
FIZZ1
IL‐10
IL‐12
• tissue destruction
• pathogen phagocytosis
• inflammation
• tissue repair
• cellular debris phagocytosis
• fibroblast proliferation
M2
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GLYCANS ARE INVOLVED IN MANY MEDICAL PROBLEMS
• tumor development and metastasis
• some storage diseases and mucopolysaccharidoses
• congenital disorders of glycosylation (CDGs)
• microbial infections
• allergies, autoimmune diseases and other immune disorders
• rejection of xenotransplants
• disruption of homeostasis
• individual (non) response to therapy
– and many others
Glycosylation changes are related to many diseases
potential diagnostic and/or prognostic markers
• Alzheimer disease – liqur acetylcholine esterase
• diabetes mellitus – nuclear and cytosolic proteins ( O‐GlcNAc)
• rheumatoide arthritis, JRA – serum IgG
• ulcerative colitis – colon mucose ( O‐Ac Sia)
• cystic fibrosis – secretory proteins ( Sia); mucosal glycoproteins ( Fuc & sulphatation)
• IgA nephropathy – serum IgA ( sialylation and galactosylation of O‐glycans)