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1 Tel. 02-2164-4512 / Fax. 042-865-3419 / E-mail.

042-860-4457 / E-mail. dboh@kribb.re.kr 3 Tel.

hyunkang@kribb.re.kr 4 Tel. 042-865-3425 / Fax.

.,,, 4,.,. 20. Raymond A. Dwek 1988 glycobiology.

1 1 Tel / Fax / . yongil382@catholic.ac.kr 2 Tel / . dboh@kribb.re.kr 3 Tel / Fax / . hyunkang@kribb.re.kr 4 Tel / Fax / . shkim@kbsi.re.kr.. danger..,,, 4,.,. 20. Raymond A. Dwek 1988 glycobiology..,. Consortium for Functional Glycomics (NIH) $34M..? (glyco-), -ome, -ics 18

2 1. ( Introduction to Glycobiology Maureen E. Taylor, Kurt Drickamer (Eds)), ( ).,,..,.,,,,..,,.. (oligosaccharides, glycan)... DNA mrna (transcription), (translation),. (glycosylation) (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.

3 Ernst&Young, Resilience: Americas Biotechnology Report % $ % ( 2).,.,...,., ( ),,,,,.,. DNA 3. ( Introduction to Glycobiology Maureen E. Taylor, Kurt Drickamer (Eds))...,.,,?.,. glycoform. (endoplasmic reticulum) (Golgi apparatus). chaperon vesicle. 20

4 (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

5 . conformation %. Asn-Xaa-Cys N- (8). Translation oligosaccharyltransferase. post-traslational modification N- co-translational modification..,..,..... high-,,. N- Man 3 GlcNAc 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 ,,.,, proteoglycan glycoconjugates ( ), 1975 Nathan Sharon (polymer) (specificity), 22

6 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 Eur. J. Biochem. 218, 1-27.),.,, NMR, MS, , Montreuil (11), Kornfeld (12), (N- and O- ),, review, (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).

7 4. Glycoconjugate biosynthesis and cell surface recognition.(bertozzi CR, Kiessling LL. Chemical glycobiology. Science. 291, ) (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

8 , (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 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.

9 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 ~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

10 .,. ( ), EPO Aranesp ( 5).,..,.,..,.,,, , % (23). EPO 70%. (24). EPO (erythropoietin): EPO, 100. Amgen Epogen N- AranespTM. AranespTM , ( 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

11 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)., (Gaucher) (Fabry) (US $ Millions) ( ) Ceredase Genzyme 443 N/A Glucocerebrosidase Glucocerebrosidase Cerezyme Genzyme 932(2005) 1048 (CHO cell) 3 Fabrazyme Genzyme Replagal TKT alpha-galactosidase MPS-1 Aldurazyme Genzyme alpha-l-iduronidase (Pompe) Myozyme Genzyme Approved (2006) aglucosidase alfa 28

12 .,,. 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

13 N- Sf9 (MimicTM cells, Invitrogen). :, (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

14 7. Vancomycin epivancomycin Genzyme 1990, Fidia., LG : (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

15 ,. (2-O-α-glucopyranosyl-L-ascorbic acid).,, CDG (informational biomacromolecules)., functional glycomics, NIH James Paulson Consortium for Functional Glycomics 2007.,.,, 20,., MIT 21, 10 BT.,,,.., ( ).. (built-in), 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 (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, (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, (2005) 4. Miura Y, Kim S, Etchison JR, Ding Y, Hindsgaul O, 32

16 Freeze HH. Aglycone structure influences alpha-fucosyltransferase III activity using N-acetyllactosamine glycoside acceptors. Glycoconj J. 16, (1999) 5. Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-golgi network. Nat Rev Mol Cell Biol. 7, (2006) 6. Shibata Y, Voeltz GK, Rapoport TA. Rough sheets and smooth tubules. Cell 126, (2006) 7. Parodi AJ. Protein glucosylation and its role in protein folding. Annu Rev Biochem. 69, (2000) 8. Satomi Y, Shimonishi Y, Takao T. N-glycosylation at Asn(491) in the Asn-Xaa-Cys motif of human transferrin. FEBS Lett. 576, (2004) 9. Julenius K, Mφlgaard A, Gupta R and Brunak S. Prediction, conservation analysis and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology, 15, , (2005) 10. Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS, Cho JW. Modification of p53 with O-linked N- acetylglucosamine regulates p53 activity and stability. Nat Cell Biol. 8, (2006) 11. Montreuil J. Glycoproteins, In: A. Neuberger and L.L.M. Van Deenen (Eds), Comprehensive Biochemistry. Elsevier, Amsterdam, pp (1982). 12. Kornfeld R, Kornfeld S. Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54, (1985). 13. Lis H, Sharon N. Protein glycosylation: Structural and functional aspects. Eur. J. Biochem. 218, (1993). 14. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, (1993). 15. Bertozzi CR, Kiessling LL. Chemical glycobiology. Science. 291, (2001). 16. Higuchi M, Oh-eda M, Kuboniwa H, Tomonoh K, Shimonaka Y, Ochi N. Role of sugar chains in the expression of the biological activity of human erythropoietin. J. Biol. Chem. 267, (1992). 17. Fedok VA, Bratton DL, Frasch SC, Warner ML, Henson PM. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death and Differentiation, 5, (1998). 18. Howard SC, Wittwer AJ, Welply JK. Oligosaccharides at each glycosylation site make structure-dependent contributions to biological properties of human tissue plasminogen activator. Glycobiology 1, (1991). 19. Hooper LV, Gordon JI. Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity. Glycobiology 11, 1R-10R. (2001). 20. Hakomori SI. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco)lipid metabolism. Cancer Res., 56, (1996) 21. Shriver, Z., Raguram, S., and Sasisekharan, R., Glycomics: A Pathway to a Class of New and Improved Therapeutics. Nature Reviews on Drug Discovery 3, (2004) 22.,,. CEO information 473 ( ) 23. Medicines in Biotechnology PhRMA Report (2006) 24. Datamonitor Biogenetics (2005) Ref. code: DMHC Datamonitor Recombinant Therapeutic Proteins ( ) Ref. code: DMHC Umana P, Jean-Mairet J, Moudry R, Amstutz H, and Bailey J. E, Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nature Biotechnology 17, (1999) 27. Shinkawa T. et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharide shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278, (2003) 28. Niwa R. et al. Defucosylated chimeric anti-cc chemokine receptor 4 IgG1 with enhanced antibodydependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cacer Research 64, (2004) 29. Li H. et al. Optimization of humanized IgGs in glycoenginnered Pichia pastoris. Nat. Biotechnol. 24, (2006) 30. Kaneko Y. et al. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, (2006) 31. Werber Y. Lysosomal storage diseases market. Nature 33

17 Reviews on Drug Discovery 3, 9-10 (2004) 32. Wurm F. M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotech. 22, (2004) 33. Hamilton, S. R. et al. Production of complex human glycoproteins in yeast. Science 301, (2003) 34. Gerngross T. U. Advances in the production of human therapeutic proteins in yeast and filamentous fungi. Nature Biotech. 22, (2004) 35. Hamilton, S. R. et al. Humuanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, (2006) 36. Kim, M. W. et al. Functional characterization of the Hansenula polymorpha HOC1, OCH1 and OCR1 genes as members of yeast OCH1 mannosyltransferase family involved in protein glycosylation. J. Biol. Chem. 281, (2006) 37. Baker, K. N. et al. Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol. Bioeng. 73, (2001) 38. Yang, M. and Butler, M. Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture. Biotechnol. Prog. 16, (2000) 39. Senger, R. S. and Karim, M. N. Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein. Biotechnol Prog. 19, (2003) 40. Shimma Y.-I. and Jigami Y. Expression of human glycosyltransferase genes in yeast as a tool for enzymatic synthesis of sugar chain. Glycoconjugate J. 21, (2004) 41. Dove A. Redesigner Drugs. Nature Biotech (2004) Kren V. and Martinkova L. Glycosides in medicine: the role of glycosidic residue in biological activity. Current Medicinal Chemistry 8, (2001) 44. Glycomics (2005) 34

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,