Extracellular Macromolecules Glycosaminoglycans; proteoglycans; glycoproteins; mucins Glycoprotein synthesis; plasma proteins Molecular immunology: innate.

Extracellular MacromoleculesExtracellular Macromolecules

Glycosaminoglycans; proteoglycans; Glycosaminoglycans; proteoglycans; glycoproteins; mucinsglycoproteins; mucins

Glycoprotein synthesis; plasma proteinsGlycoprotein synthesis; plasma proteins

Molecular immunology: Molecular immunology: innate immunity; inflammationinnate immunity; inflammation

Molecular immunology: Molecular immunology:

adaptive (acquired) immunityadaptive (acquired) immunity

Fibrous proteins: keratin, collagen and elastinFibrous proteins: keratin, collagen and elastin

Extracellular MacromoleculesExtracellular Macromolecules

1. Glycosaminoglycans Proteoglycans1. Glycosaminoglycans Proteoglycans GlycoproteinsGlycoproteins

MucinsMucins

Extracellular MacromoleculesExtracellular Macromolecules

macromoleculemacromolecule % carb.% carb.

glycosaminoglycansglycosaminoglycans** (GAGs) (GAGs) 100 100

proteoglycansproteoglycans** 90-9590-95

glycoproteinsglycoproteins 2-30 2-30

fibrous proteinsfibrous proteins 1-2 1-2

Examples of functions: Examples of functions:

mechanical supportmechanical support lubricationlubrication

cushioning cushioning adhesivesadhesives

cell spacerscell spacers selective filtersselective filters

* aka* aka mucopolysaccharides, mucoproteins, respectively mucopolysaccharides, mucoproteins, respectively11

Extracellular matrix in tissues Extracellular matrix in tissues ground substance + fibersground substance + fibersmacromolecules between cells macromolecules between cells

ground substance moleculesground substance molecules

GAGs/proteoglycans (mostly carbohydrate)GAGs/proteoglycans (mostly carbohydrate)–fibersfibers

fibrous proteins:fibrous proteins: structural structural

adhesive adhesive

•especially abundantespecially abundantin connective tissuein connective tissue

adhesionmolecules

Adapted from Hypercell

extra-cellularmatrix

basallamina

underlying cells

epithelial cells

2

GAG structureGAG structureexist as: exist as:

independent moleculesindependent moleculese.g.e.g., hyaluronate & heparin, hyaluronate & heparin

parts of larger structuresparts of larger structurese.g.e.g., in proteoglycans, in proteoglycans

heteropolysaccharidesheteropolysaccharides repeating structure: repeating structure:

disaccharide ( disaccharide (ABAB))nn ABABABABABAB…… where where AA is usually 1 uronic acid (hexose with is usually 1 uronic acid (hexose with C6C6 as COO as COO– – )) & & BB is 1 glycosamine (amino sugar) derivative is 1 glycosamine (amino sugar) derivative

unbranchedunbranched glycosidic linkageglycosidic linkage anomeric anomeric CC of 1 unit linked to hydroxyl of adjacent unit of 1 unit linked to hydroxyl of adjacent unit

A sugar

B sugar

33

GAG structure: repeating unitsGAG structure: repeating units GAGGAG A A sugarsugar B sugar B sugarhyaluronatehyaluronate glucuronateglucuronate N-acetyl glucosamineN-acetyl glucosamine

25*

44

GAG structure: repeating unitsGAG structure: repeating units GAGGAG A A sugarsugar B sugar B sugarhyaluronatehyaluronate glucuronateglucuronate N-acetyl glucosamineN-acetyl glucosamine

chondroitin sulfatechondroitin sulfate glucuronate glucuronate N-Ac galactosamine 4-SON-Ac galactosamine 4-SO44

dermatan sulfatedermatan sulfate iduronateiduronate " "

heparan sulfateheparan sulfate glucuronateglucuronate glucosamine N-SOglucosamine N-SO33, 6-SO, 6-SO44

heparin heparin iduronate 2-SOiduronate 2-SO44 " "

keratan sulfatekeratan sulfate galactosegalactose N-Ac glucosamine 6-SON-Ac glucosamine 6-SO44

**opposite configuration in iduronate opposite configuration in iduronate glucuronate/iduronate: glucuronate/iduronate: epimers epimers at at C5C5 glucose/galactose: glucose/galactose: epimersepimers at at C4C4

25*

44

Hyaluronate (aka hyaluronan)mol wt: 106 – 107 (5000 – 50,000 monosaccharide units)very polar: 2 hydroxyls/unit 6 heteroatoms/unit

COO– every other unitbinds cations: Na+, Ca++

213 4 5 6

–

––

(glucuronate–N-acetyl glucosamine)3 (glcUA–glcNAc)3

A B A B A B

Display of HA in motion

55

Hyaluronate: structure & propertiesextended structure (charge repulsion)hydrophilic: binds 10 –100 × wt in H2Oadditional, loosely associated H2O, so that volume occupied ~1000 ×

weight

21 3 4

5 6

–

––

(glcUA–glcNAc)3 glcUAs in space-filling form (CPK)

Display of HA with glcUAs in CPK

66

Hyaluronatesolutions viscous, gel–like, compression-resistant

occurrence: EC matrix,esp. in developing tissue

healing wounds synovial fluid

functions: lubricantshock absorberflexible cementattachment sitepath for cell migration

made by fibroblastsdegraded by hyaluronidase

hyaluronidasebacterial hyaluronidase facilitates spread of infection

Alberts et al. Fig. 19-37

7

HeparinHeparin

mol wt ~ 10mol wt ~ 1044

~ 40 monosaccharide units~ 40 monosaccharide unitsmade & released from mast cells in lungs & livermade & released from mast cells in lungs & liver

88

heparin

cell

HeparinHeparin

mol wt ~ 10mol wt ~ 1044

~ 40 monosaccharide units~ 40 monosaccharide unitsmade & released from mast cells in lungs & livermade & released from mast cells in lungs & liveralso associated with luminal surface of endotheliumalso associated with luminal surface of endotheliumanticoagulantanticoagulant

forms complex with antithrombin IIIforms complex with antithrombin III this complex binds to thrombin, inactivating itthis complex binds to thrombin, inactivating it as a result, clot growth is limitedas a result, clot growth is limited fast-acting, making it therapeutically usefulfast-acting, making it therapeutically useful

88

heparin

cell

Extracellular MacromoleculesExtracellular Macromolecules

macromoleculemacromolecule % carb.% carb.

glycosaminoglycans* (GAGs)glycosaminoglycans* (GAGs) 100 100

proteoglycans*proteoglycans* 90-9590-95

glycoproteinsglycoproteins 2-30 2-30

fibrous proteinsfibrous proteins 1-2 1-2

Examples of functions: Examples of functions:

mechanical supportmechanical support lubricationlubrication

cushioning cushioning adhesivesadhesives

cell spacerscell spacers selective filtersselective filters

* aka* aka mucopolysaccharides, mucoproteins, respectively mucopolysaccharides, mucoproteins, respectively

Proteoglycans (PGs)Proteoglycans (PGs)composed of as many as 200 GAG chains covalently bonded to a core protein via serine side composed of as many as 200 GAG chains covalently bonded to a core protein via serine side

chains chains molecular weight range: 10molecular weight range: 1055 – 10 – 1077 GAG chains:GAG chains: chondroitin sulfate, heparan sulfate, chondroitin sulfate, heparan sulfate, dermatan sulfate, dermatan sulfate,

keratan sulfatekeratan sulfateExamplesExamplesdecorindecorin

many connective tissuesmany connective tissues binds type I collagen, TGF-binds type I collagen, TGF-ββ

perlecanperlecan basal laminaebasal laminae structural & filtering functionstructural & filtering function

aggrecanaggrecansyndecan (syndecan (slide 13slide 13)) GAG chains

coreprotein

99

PG in basal lamina of renal glomerulus

network offibrousproteins &perlecanPG forms filter

adapted from Alberts et al., 3 ed., Fig. 19-56

type IV collagen

laminin

perlecan

entactin

1010

Proteoglycans: aggrecanProteoglycans: aggrecan~100 GAG chains/molecule~100 GAG chains/molecule~100 monosaccharides/GAG chain~100 monosaccharides/GAG chaineach "bristle" = 1 GAG chaineach "bristle" = 1 GAG chaineach GAG chain is either each GAG chain is either chondroitin sulfatechondroitin sulfate

or or keratan sulfatekeratan sulfateGAG chains linked to GAG chains linked to serser side chains of core protein side chains of core protein

corecoreproteinprotein

GAG chainsGAG chains1111

An aggregate of aggrecans & hyaluronanmajor GAG–PG

in cartilagelink proteins bind

noncovalentlywith bound H2O,

disperses shocks,compressive force

~ cell sizeadhesion proteins

link to collagen & cells

degraded by chondroitin sulfatase, etc

ç 1μm è

hyalur-onan

link proteins

keratansulfate chondroitin

sulfate

Alberts et al. Fig. 19-41

core protein

12

Proteoglycans: Proteoglycans: syndecansyndecan

GAG chainsGAG chains

corecoreproteinprotein

cell-surface PGcell-surface PGcore protein domainscore protein domains intracellularintracellular transmembranetransmembrane extracellularextracellular

5 GAGs attached5 GAGs attachedfunctionsfunctions interactionsinteractions

cell-cellcell-cellcell-matrixcell-matrix

growth factor receptorgrowth factor receptorLehninger et al. Fig. 9-22

outside

inside

1313

GAG synthesis & breakdownGAG synthesis & breakdown

synthesissynthesis activated precursors:activated precursors: UDP–monosaccharide UDP–monosaccharide

derivativesderivativese.ge.g., UDP–glucuronate., UDP–glucuronate

residues added one at a time in Golgi complexresidues added one at a time in Golgi complex sulfate moietiessulfate moieties

donor: PAPS (active sulfate)donor: PAPS (active sulfate)degradationdegradation

lysosomeslysosomes specific glycosidases & sulfatasesspecific glycosidases & sulfatases mucopolysaccharidosesmucopolysaccharidoses

genetic disordersgenetic disordersaccumulation of GAG due to absence of a specific glycosidase accumulation of GAG due to absence of a specific glycosidase

or sulfataseor sulfatase

–UDP

1414

adenine––

––

Extracellular MacromoleculesExtracellular Macromolecules

macromoleculemacromolecule % carb.% carb.

glycosaminoglycans (GAGs)glycosaminoglycans (GAGs) 100 100

proteoglycansproteoglycans 90-9590-95

glycoproteins*glycoproteins* 2-30 2-30

fibrous proteinsfibrous proteins 1-2 1-2

* polypeptide with 1 or more oligosaccharide side chains* polypeptide with 1 or more oligosaccharide side chains

1515

Glycoproteins: functions of glyco moietiesGlycoproteins: functions of glyco moietiesincrease protein’s solubility & hydrophilicity (increase protein’s solubility & hydrophilicity (sl 19sl 19))stabilize protein againststabilize protein against

denaturationdenaturation proteolysisproteolysis

markersmarkers direct protein's destinationdirect protein's destination

organelleorganelleplasma membraneplasma membraneexport (secretion)export (secretion)

indicate protein's lifetime (indicate protein's lifetime (sl 21sl 21)) part of the protein's receptor recognition site (part of the protein's receptor recognition site (sl 23sl 23))

signal factors such as hormones, cytokinessignal factors such as hormones, cytokinescell-cell adhesion proteinscell-cell adhesion proteins

Glycosylation:Glycosylation: one kind of one kind of post-translational modificationpost-translational modificationothers:others: phosphorylation phosphorylation

carboxylation carboxylation

1616

Glycoprotein structureGlycoprotein structurepolypeptide with 1 or more oligosaccharide side chainspolypeptide with 1 or more oligosaccharide side chainsoligosaccharide linked to polypeptide in two ways:oligosaccharide linked to polypeptide in two ways:typetype linked to side chain of linked to side chain of organelle where sugarsorganelle where sugars

are added to proteinare added to protein

O-linkedO-linked serine (serine (serser), threonine (), threonine (thrthr), ), Golgi complex lumenGolgi complex lumen (O-glycoside)(O-glycoside) hydroxylysine (in collagen)hydroxylysine (in collagen)

N-linkedN-linked asparagine (asparagine (asnasn)) rough ER lumenrough ER lumen (N-glycoside)(N-glycoside)

1717

Glyco moiety structureoligosaccharide chain extends away from protein surfaceoligosaccharide chain extends away from protein surfaceunits mostly hexoses in pyranose (6-atom ring) formunits mostly hexoses in pyranose (6-atom ring) formbranchedbranchedglycosidic links varied: glycosidic links varied:

αα or or ββ1,2; 1,3; 1,41,2; 1,3; 1,4

terminal terminal sugarsugaroften often sialatesialate

asn

2

7

2

7

Stryer 4ed., p. 4631818

asn

Mucins: salivary glycoproteinsMucins: salivary glycoproteins

mol wt ~ 10mol wt ~ 1066

~800 short ~800 short (disaccharide) (disaccharide) side chainsside chains

terminal sugar is terminal sugar is sialatesialate anionic sugaranionic sugar at end of glyco chains at end of glyco chains

of many glycoproteinsof many glycoproteinsvery hydrophilic, very hydrophilic,

extended structureextended structure

galNAcsialate

–

2

~ ~

1919

Mucins: modification & aggregationMucins: modification & aggregation

sialidase sialidase (neuraminidase)(neuraminidase) catalyzes hydrolysiscatalyzes hydrolysis

of sialates from mucins of sialates from mucins secreted by oral bacteriasecreted by oral bacteria

products:products: less hydrophilic, less hydrophilic,

less Hless H22O-soluble, O-soluble, more folded, more folded, more aggregatedmore aggregated

part of the enamel pellicle part of the enamel pellicle & dental plaque matrix& dental plaque matrix

sialidasex H2O galNAc

sialate

~ ~

~~

x

~~

2020

Role of glyco moiety in controlling protein lifetimeRole of glyco moiety in controlling protein lifetime

many blood proteins have glyco chains with terminal many blood proteins have glyco chains with terminal sialatesialateendothelial surface endothelial surface sialidasessialidases slowly remove sialates from these circulating proteins slowly remove sialates from these circulating proteinsrate of sialate removal depends on protein's structurerate of sialate removal depends on protein's structure

now-exposed now-exposed gal–glcNAcgal–glcNAc…… residues bind to residues bind to asialoglycoprotein receptorasialoglycoprotein receptor on liver cell on liver cell surfacesurface

protein is then endocytosed & broken downprotein is then endocytosed & broken down

sialoglycoprotein: sia–gal–glcNAc–[core sugars]–protein

asialoglycoprotein: gal–glcNAc–[core sugars]–protein

2121

Blood group typesBlood group types

A: have A: have –– enzyme to add enzyme to add galNAcgalNAc to core sugars to core sugars–– antibody to type B antigen antibody to type B antigen

B: have B: have –– enzyme to add enzyme to add galgal to core sugars to core sugars–– antibody to type A antigen antibody to type A antigen

O: have O: have –– neither enzyme neither enzymeAB: have AB: have –– both enzymes (either both enzymes (either galNAcgalNAc or or galgal

added to core sugars)added to core sugars)

Type O cell surface: gal–glcNAc–gal–glc–protein†

Type A cell surface: galNAc–gal–glcNAc–gal–glc–protein†

Type B cell surface: gal–gal–glcNAc–gal–glc–protein†

* 6-deoxygalactose†or lipid

both antibodies

neither antibody

|fucose

|fucose

|fucose*

2222

core sugarscore sugars

Glyco moiety-binding proteins: lectinsGlyco moiety-binding proteins: lectinscontain sites that bind specific glyco structurescontain sites that bind specific glyco structurese.g., e.g., asialoglycoprotein receptorasialoglycoprotein receptor described on described on sl 21sl 21important in intercell adhesion (i.e., lectins are CAMs: important in intercell adhesion (i.e., lectins are CAMs: cell cell

adhesion moleculesadhesion molecules))selectins:selectins:

plasmaplasmamembranemembranelectins thatlectins thatmediatemediatecell-cellcell-cellrecognitionrecognition& adhesion& adhesion

Lehninger et al. Fig. 7-372323