Revisiting some O Glycans Table 1 Different types of O linked glycans in humans Type of O linked glycan Structure and peptide linkage Mucin type R GalNAc1 Ser Thr GAG Secreted plasma membrane R GlcA1 3Gal1 3Gal1 4Xyl1 Ser O linked GlcNAc Proteoglycans GlcNAc1 Ser Thr O linked Gal Glycoprotein Nuclear and cytoplasmic Glc1 2Gal1 O Lys Collagens Dystroglycan O linked Man NeuAc2 3Gal1 4GlcNAc1 2Man1 Ser Thr O linked Glc Xyl1 3Xyl1 3Glc1 Ser EGF protein domains O linked Fuc NeuAc2 6Gal1 4GlcNAc1 3Fuc1 Ser Thr EGF protein domains Glc1 3Fuc1 Ser Thr TSR repeats Wopereis et al 2006 Clinical Chemistry 52 574 600 Table 2 Diversity of mucin type O linked glycans Core Structure Human tissue 1 Gal1 3GalNAc 2 Gal1 3 GlcNAc1 6 GalNAc All blood cells 3 GlcNAc1 3GalNAc Colon and saliva 4 GlcNAc1 3 GlcNAc1 6 GalNAc 5 GalNAc1 3GalNAc Meconium 6 GlcNAc1 6GalNAc Ovarian tissue 7 GlcNAc1 6GalNAc 8 Gal1 3GalNAc Most cells and secreted proteins Mucin secreting cell types Bronchia Wopereis et al 2006 Clinical Chemistry 52 574 600 1 Glycosaminoglycans and Proteoglycans Glycosaminoglycans anionic polysaccharides made up of repeating disaccharide units one of the sugars is an amino sugar N acetyl glucosamine GlcNAc or N acetyl galactosamine GalNAc at least one of the sugars is negatively charged due to a carboxyl group or a sulfate Wopereis et al 2006 Clinical Chemistry 52 574 600 Examples hyaluronic acid hyaluronan D GlcA 1 3 D GlcNAc 1 4 n H OH H OOC H H O O HO O O O HO H H H OH NHCOCH3 H H H 2 heparan sulfate heparin D GlcA 1 4 D GlcNAc 1 4 n H H OH OOC H O H O O o HO H H HO OH H NHCOCH3 H H o H or L IdoA 2 OSO3 1 4 D GlcNSO3 6 OSO3 1 4 n H OSO3 H H H H O O O HO OOC HO H OSO3 H NNSO3O H H H O chondroitin sulfate CS D GlcA 1 3 D GalNAc 4 SO3 1 4 n H OSO3 OH COO H H O O H O O O HO H H OH H NHCOCH3 H H H dermatan sulfate DS L IdoU 1 3 D GalNAc 4 SO3 1 4 n H OSO3 OH H H O HO H O OOC O O H OH H O H H H NHCOCH3 H 3 keratan sulfate D Gal 1 4 D GlcNAc 6 SO3 1 3 n OH OH H OSO3 H O H H O O O H O HO OH H H H NHCOCH3 H H Most GAGs are synthesized onto proteins and exit as glyconjugates known as proteoglycans The only GAG known to be synthesized independent of a protein is hyaluronic acid HA hyaluronan HA is synthesized at the plasma membrane while proteoglycans are synthesized in the ER Golgi 4 HA is an essential polysaccharide in animals and is found in pathogenic bacteria and viruses HA is a structural element in the vitreous humor of eye synovial fluid and skin of vertebrates HA interacts with proteins e g CD44 RHAMM fibrinogen influences angiogenesis cancer cell motility wound healing and cell adhesion HA is a polymer made up of the repeating disaccharide GlcA 1 3 GlcNAc 1 4 n n can exceed 30 000 i e Mr 107 HA is synthesized by HA synthase an integral membrane Mn or Mg requiring protein that has both 1 3GlcAT and 1 4GlcNAcT activity HA synthases range from 419588 amino acids 49 69 kD The vertebrate enzymes DG42 HAS1 HAS2 HAS3 the streptococcal HasA and the Paramecium bursaria chloreall virus A98R share several regions of sequence similarity 5 Expression of an active recombinant HAS from Streptococci pyogenes in E coli showed that a single purified gene product synthesized high Mr HA when incubated in vitro with UDP GlcA and UDP GlcNAc DeAngelis Weigel P H 1994 Biochemistry 33 9033 9039 Group A Streptococci has an HA synthesis operon has composed of 3 genes hasA hyaluronate synthase hasB UDP Glc dehydrogenase hasC UDP Glc pyrophosphorylase Fig 4 Photomicrographs of recombinant E coli Panel A India ink staining 1 000 magnification reveals that E coli K5 cells with pPmHAS produce a substantial capsule that appears as a white halo around the cells Panel B the capsular material could be removed from the E coli K5 pPmHAS cells by brief treatment with Streptomyces HA lyase PmHAS directs polymerization of the HA polysaccharide Pasteurella multocida is a prevalent gram negative animal pathogen It produces hyaluronan to avoid host defenses Pasteurella HAS is a class II HAS DeAngelis et al 1998 J Biol Chem 273 8454 6 Proteoglycans proteins that contain one or more covalently bound glycosaminoglycans GAGs Proteoglycan protein core 1 GAG To date 30 genes for protein core of different proteglycans identified Secreted proteoglycans SLRPS small leucine rich proteoglycans Modular proteoglycans Basement Membrane Proteoglycans perlecan agrin bamacan Hyalectans versican aggrecan neurocan brevican 7 SLRP Modular Iozzo Murdoch 1996 FASEB 10 598 SLRPs Iozzo Murdoch 1996 FASEB 10 598 8 Iozzo Murdoch 1996 FASEB 10 598 Functions of proteoglycans tissue organizers influence cell growth maturation of specialized tissues biological filters modulate growth factor activities regulate collagen fibrillogenesis skin tensile strength affect tumor cell growth invasion influence corneal transparency influence neurite outgrowth some proteoglycans are essential for growth others may be redundant 9 Heparin and Heparan Sulfate as examples Definitions Heparanoids glycoconjugates that contain heparin or heparan sulfate HSPG heparan sulfate proteoglycan FGF fibroblast growth factor bFGF basic fibroblast growth factor Location on cell surface and basement membrane of animals cells mast cells mucosa lung Functions anticoagulant heparin binds FGF heparin heparan sulfate free HS fragments bind bFGF and may extend half life of bFGF binds laminin fibronectin cell surface phenomenon heparan sulfate bind to binding proteins on cell surface heparin and heparan sulfate binding to clusters of Arg Lys on protein surface many heparinoids must bind two proteins simultaneously to carry out their physiological role e g antithrombin thrombin interaction with heparin requires at least 18 consecutive glycosyl residues bind selectins protease inhibitors lipoprotein lipase HS implicated in cell proliferation differentiation adhesion migration morphogenesis blood coaggulation viral bacterial infection tumor cell malignancy 10 Structure HS is a complex mammalian GAGs whose molecular fine structure varies in a cell type specific fashion Basic repeat of alternating GlcN and uronic acids that contain N and Olinked sulfate Heparin is a member of the HS family Heparin higher N and O sulfate content than HS greater proportion of the uronic acid as iduronic acid HS greater proportion of uronic acid is GlcA Generalized structures Heparin L iduronic acid 2S 1 4 GlcN NS 6S 1 4 n HS D GlcNAc 1 4 D GlcA 1 4 D GlcNAc or GlcN NS GlcA GlcNAc Heparan sulfate block structures composed of