1 Tel. 02-2164-4512 / Fax. 042-865-3419 / E-mail. yongil382@catholic.ac.kr 2 Tel. 042-860-4457 / E-mail. dboh@kribb.re.kr 3 Tel. 042-860-4378 / Fax. 042-860-4594 / E-mail. hyunkang@kribb.re.kr 4 Tel. 042-865-3425 / Fax. 042-865-3419 / E-mail. shkim@kbsi.re.kr.. danger..,,, 4,.,. 20. Raymond A. Dwek 1988 glycobiology..,. Consortium for Functional Glycomics (NIH) $34M..? (glyco-), -ome, -ics 18
1. ( Introduction to Glycobiology Maureen E. Taylor, Kurt Drickamer (Eds)), ( ).,,..,.,,,,..,,.. (oligosaccharides, glycan)... DNA mrna (transcription), (translation),. (glycosylation) 30 200. (glycosyltransferase) (glycosidase). (heterogeneity) glycoform. (1, 1),,,,... (Congenital Disorder of Glycosylation, CDG) (2),., (3).,.. $3. Pfizer, Merck. 19
2. 2003. Ernst&Young, Resilience: Americas Biotechnology Report 2003.. 2003 10% $300 2-30% ( 2).,.,...,.,. 1... ( ),,,,,.,. DNA 3. ( Introduction to Glycobiology Maureen E. Taylor, Kurt Drickamer (Eds))...,.,,?.,. glycoform. (endoplasmic reticulum) (Golgi apparatus). chaperon vesicle. 20
(4).,.. sorting (5, 6). ( 3).,.,.. -.., glycoform.., (mucus) extracellular matrix..,.,. O-. 20,,. cystic fibrosis.,. (lysosome)., N- α-.. protein body.. protein body. protein body. 2. N- (ribosome). GDP- (mannose), UDP-, CMP- (sialic acid) (nucleotide-sugar). dolichol. GDP-N-acetylglucosamine (GlcNAc) GlcNAc-phosphate dolichol-pp-glcnac, dolichol-pp-man 5GlcNAc 2 flipase. 4 dolichol-pp-. 3., 14 Glc 3 Man 9 Glc- NAc 2 3 dolichol (7). Asparagine N-. Asn-Xaa (proline )-Ser/Thr. Glycine alanine lysine methionine. 21
. conformation. 10-30%. Asn-Xaa-Cys N- (8). Translation oligosaccharyltransferase. post-traslational modification N- co-translational modification..,..,..... high-,,. N- Man 3 GlcNAc 2 5. 3. O- O-.,., post-translational modification. N- serine threonine O- serine threonine proline.,,, O- (9). O- N-acetylgalactosamine. 4-8., N-,. N- O-,,.., O-. GlcNAc O-. O- GlcNAcylation. O-Glc- NAc antagonist (10). mannosylation, glucosylation, fucosylation O-.. 1970,,.,, proteoglycan glycoconjugates ( ), 1975 Nathan Sharon (polymer) (specificity), 22
1. Biological functions of glycoprotein glycans Type Phycochemical Biological Functions Modify solubility, electrical charge, mass, size, and viscosity in solution Control protein folding Stabilize protein conformation confer thermal stability and protection against proteolysis Regulate intracellualr traffic and localization of glycoproteins Determine lifetime of glycoproteins in circulation Modify immunological properties modulate activity of enzymes and hormones Act as cell surface receptors for lectins, antibodies, toxins, etc. Participate in cell-cell recognition, interaction and signal transduction (Lis H, Sharon N. 1993. Eur. J. Biochem. 218, 1-27.),.,, NMR, MS, 80. 1980, Montreuil (11), Kornfeld (12), (N- and O- ),, review,. 1985 (CCRC), (functional glycomics). 1990,,,,. 3 (protein folding and targeting), - (protein-protein recognition and interaction), (cellcell recognition and interaction), host,,,,,,, (13, 14) ( 1).,. (lectin),... 1., 3,. 3. β 2 -glycoprotein I 2 3,., (heat stability)., (13). 2. (targeting) (15)( 4). Ribosome 23
4. Glycoconjugate biosynthesis and cell surface recognition.(bertozzi CR, Kiessling LL. Chemical glycobiology. Science. 291, 2357-2364.) (molecular chaperon) calnexin glucose, calnexin,. (hydrolases). mannose 6-phosphate (M6P), M6P ( ) M6P lysosome (12, 14)., M6P,, lysosome I- (I-cell disease). (Gaucher s disease). 3.. N- β- galactose (hepatic asialoglycoprotein receptor).,. EPO in vivo, N- β-galactose (16)., apoptosis., (17). 4. (tpa) serine protease 3 N-, Asn117, Asn184 Asn448 (complextype N-glycans). Type I 3, type II Asn117 Asn448,, CHO cell tpa, Asn448 tpa (18). 5. hcg, site-directed mutagenesis α-chain Asn52, β-chain Asn13,, Asn30., (deglycosylation) hcg hcg 24
, (13). EPO, in vitro (affinity), in vivo (16). in vivo, N- β-gal. EPO, biantennary tetraantennary in vivo, O- in vitro in vivo (16). 6. -,,,,, ( 2)., ( 4). haemagglutinin (glycophorin A) O-, ganglioside G M1. Helicobacter pylori Le b (blood group antigen, Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ) (19).,, (14, 2. Carbohydrates as attachment sites for infectious agents Carbohydrate Organism Target tissue Predominant Structure form Viruses Influenza type A respiratory tract NeuAc(α2-6)Gal GP Influenza type B respiratory tract NeuAc(α2-3)Gal GP Influenza type C respiratory tract 9-O-aceuAc(α2-3)Gal GP Bacteria E. coli type 1 urinary tract Man(α1-3){Man(α1-3)[Man(α1-6)]Man(α1-6)}Man(β1-4)- GP E. coli type P urinary tract Gal(α1-4)Gal GSL E. coli type S neural NeuAc(α2-3)Gal(β1-3)GalNAc GSL E. coli type CFA/1 intestine NeuAc(α2-8)- GSL E. coli type K99 Intestine NeuGc(α2-3)Gal(β1-4)Glc GSL Actinomyces naeslundii oral GalNAc(β1-3)Galβ GP Neisseria gonorrhoea genital Gal(β1-4)Glcβ GSL NeuAc(α2-3)Gal(β1-4)GlcNAc GP Streptococcus pneumonia respiratory tract GlcNAc(β1-3)Gal GP Fungi Candida albicans skin and mucosa Gal(β1-4)Glc GSL Protozoa Entamoeba histolytica intestine Gal(β1-4)GlcNAc GP Giardia lamblia intestine Man-6-P GP = glycoprotein; GSL = glycosphingolipid. (Lis H, Sharon N. 1993. Eur. J. Biochem. 218, 1-27.) 25
19). 7.,, ABO.. Anti-(α-1,6 dextran) MAb, heavy chain (variable region) Asn58, 15. IgG constant region, Fc.. Galα-1,3Galβ4GlcNAc (13, 20). NK,, NK CHO (13). B Galα-1,3Galβ4GlcNAc., β-1,4 galactosyltransferase ZP3 O- α-galactose. polysialic acid poly-kdn.,. T Galβ1-3GalNAc.,., neural cell adhesion molecule (NCAM) polysialic acid (PSA) NCAM PSA., PSA NCAM (axon) NCAM-PSA. 2001 ~omics, 3 (Glycan) (Glycomics).. Glycome (21)., 20% (22)., 5. EPO (A) Amgen Epogen 2 N-glycosylation AranespTM. (B) (Aventis Lovenox, Pfizer Frgmin, DuPont Innohep ) (Sanofi Arixtra ). 26
.,. ( ), EPO Aranesp ( 5).,..,.,..,.,,,. 1. 10 4, 2005 500 10% (23). EPO 70%. (24). EPO (erythropoietin): EPO, 100. Amgen Epogen N- AranespTM. AranespTM 2001 2003 15, 2005 33 ( 50% ) (25)., 2010 Epogen Procrit EPO 30%., Neose GlycoPEGylation platform PEG Glyco-PEGylated EPO,. : 1980, 1990 display mouse., (affinity maturation) Fc engineering., (Fc), (, ) ADCC (Antibody Dependent Cell Cytotoxicity) 27
6. (Fc) (A) Fc (glycoengineering) (Biowa ) (B) biantenary, G2. (S1 S2 ), Fc Fc anti-inflammatory., N-acetylglucosamine (Glc- NAc) G0, (Burton DR & Dwek RA,(2006) Sugar determines antibody activity Science 313, p628 ). ( 6A). ETH bisecting GlcNAc ADCC, (GlycoMAbTM) Glycart (26). Kowa Hakko fucose 50~100 ADCC, Biowa fucose (POTELLE- GENTTM) (27-28)., G0 (cell killing) (29), 9 Fc S2 ( 6B, 30).,. : Genzyme (Enzyme Replacement Therapy) 1988 Ceredase Cerezyme, Fabrazyme, Aldurazyme Myozyme ( 3, 31)., 1 1-2 3. (Gaucher) (Fabry) (US $ Millions) 2004 2007 ( ) Ceredase Genzyme 443 N/A Glucocerebrosidase Glucocerebrosidase Cerezyme Genzyme 932(2005) 1048 (CHO cell) 3 Fabrazyme Genzyme 209 397 Replagal TKT 57 168 alpha-galactosidase MPS-1 Aldurazyme Genzyme 12 204 alpha-l-iduronidase (Pompe) Myozyme Genzyme Approved (2006) aglucosidase alfa 28
.,,. Genzyme Cerezyme, N- glucocerebrosidase. 2.,,,.,, in vitro. :,,.,,, ( 4)., (32).,,,. Glycofi Pichia pastoris (33-34), (35)., B Hansenula polymorpha (36)., 4. Characteristics E. coli Yeast/Fungi Insect cells Mammalian cells Cell growth rapid(30 min) rapid(90 min) slow(18-24 h) slow(24 h) Cost of growth medium low low high high Expression level high low to high low to high low to moderate secretion to secretion to secretion to secretion to Extracellular expression periplasm medium medium medium Protein folding refolding usually required proper folding proper folding proper folding Post-translational modifications N-glycosylation none high mannose simple, no sialic acid complex O-glycosylation no yes yes yes Phosphorylation no yes yes yes Acetylation no yes yes yes Acylation no yes yes yes - : Nevalainene et al. TRENDS in Biotechnology 23, 468 (2005) 29
N- Sf9 (MimicTM cells, Invitrogen). :, 20 100 (32)..,, glucosamine,, ph,, CO 2 shear stress (37-39)., (microheterogeniety) in vitro. : Neose in vitro GlycoAdvanceTM Bristol-Myers Squibb, Glaxo- SmithKline Novozyme., GlycoGene, 35% (40)., GMP in vitro. 3.,.,. : 60, glucosamine iduronic acid alpha-1,4 5,000-30,000 Da.. (Aventis Lovenox, Pfizer Frgmin, DuPont Innohep ), (Sanofi Arixtra ) (41). (21). : (GAG, Glycosaminoglycan) (glucuronic acid) GlcNAc. extracellular matrix,,,.,.,, filler,, scaffold, matrix. 30
7. Vancomycin epivancomycin Genzyme 1990, Fidia., LG 2003. : (42)., thymus 2. (conjugated polysaccharide). : Aminoglycoside aminosugar aminocyclitol cyclitol streptomycin, dihydrosterptomycin, gentamycin. Glycopeptides mono-, di- tetrasacchrides, vancomycin, teicoplanin, bleomycin, ristocetin 100., vancomycin epivancomycin ( 7). : Ergot alkaloids(ea),,, EA glycoside elymoclavine-o-β-d-fructofuranoside (Claviceps sp.) invertase. 4. (glycoside) ( ) OH,,,.,,,, (43, 44). :. Pyridoxine ( B6) pyridoxine-α-glucoside, Rutin ( B2) ( E). Ascorbic ( C), 31
,. (2-O-α-glucopyranosyl-L-ascorbic acid).,, CDG 16.. 3 (informational biomacromolecules)., functional glycomics, NIH James Paulson Consortium for Functional Glycomics 2007.,.,, 20,.,. 2003 MIT 21, 10 BT.,,,.., ( ).. (built-in),..... 1. Varki A. Evolution of glycan diversity, In: Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J (Eds) Essentials of glycobiology. Cold Spring Harbor, New York, pp.31-40. (1999) 2. Kim S, Westphal V, Srikrishna G, Mehta DP, Peterson S, Filiano J, Karnes PS, Patterson MC, Freeze HH. Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie). J Clin Invest. 105, 191-8. (2000) 3. Jung HI, Kim YH, Kim S. Structural basis for the presence of a monoglucosylated oligosaccharide in mature glycoproteins. Biochem Biophys Res Commun. 331, 100-6. (2005) 4. Miura Y, Kim S, Etchison JR, Ding Y, Hindsgaul O, 32
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