glycans: potential diagnostic and prognostic …...11/6/2014 1 glycans: potential diagnostic and...

11/6/2014

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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

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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

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

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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

O HHO O H

HO O

OHOH

C CO

OHNCO

CH3

HO

COH

HOH2C

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


Sia


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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)

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)

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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


Sia


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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)

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 typeMan GlcNac Gal Fuc Neu5Ac (Sia))

Man1‐6(Man1‐3) Man1‐4GlcNac1‐4GlcNac‐Asn‐X‐Ser/Thr

Varki, Essentials of Glycobiology, 2nd edition

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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

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O‐linked glycans extend functional protein domains above membrane surface

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

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Sia


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

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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)

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

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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)



Sia


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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


Sia


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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...

Central dogma of molecular biologyDNA 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

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Synthesis of main types ofanimal glycoconjugatas (glycosylation)

protein

dolichol

oligosaccharide

sugar

GPI anchor

ceramide


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

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Fine details of N‐glycans strongly affect effector funcions of IgG

Single IgG molecule is produced as a mixture of over 100 glycoforms

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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

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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

During evolution glycan structurs got novel functions

prokariotes eukariotesmonocellular multicellular

structures on the cellsurface

modulation of protein

activities

intercellularsignaling

protein folding

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‐(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

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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)

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

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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

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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

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)

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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

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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

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

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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)

glycans: potential diagnostic and prognostic …...11/6/2014 1 glycans: potential diagnostic and...

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