Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of Egg Yolk Antibody Specific to the Pancreatic Lipase Domain for Anti-Obesity

비만 억제를 위한 췌장 리파아제 도메인에 대한 특이 난황항체의 개발

  • 우승은 ((주)단바이오텍 생명과학연구소) ;
  • 권진혁 ((주)단바이오텍 생명과학연구소) ;
  • 양시용 ((주)단바이오텍 생명과학연구소) ;
  • 박현주 (가톨릭대학교 생명공학) ;
  • 김형권 (가톨릭대학교 생명공학)
  • Published : 2008.12.28
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Abstract

Human pancreatic lipase is a digestive enzyme which is synthesized in pancreas, secreted into small intestine, and there hydrolyze the fat in food. Pancreatic lipase protein composes of catalytic domain and colipase-binding domain. In this research, the gene segments corresponding to total protein, catalytic domain, and co lipase-binding domain were cloned by PCR method, inserted into an expression vector, and then used to transform Escherichia coli BL21 (DE3). The recombinant proteins produced were purified and injected intramuscularly three times into laying hens. The egg yolk antibodies (IgY) were obtained from the egg yolks and tested for their antibody titer. Among three IgY, the IgY against colipase-binding domain showed the highest antibody titer. All three IgY had inhibitory effects on the porcine pancreatic lipase. Among them, the IgY against colipase-binding domain showed the highest inhibition effects. The fat diet with corn oil and IgY was administrated to the experimental rats and their blood compositions were examined with time course. The triglyceride concentration of treated rats was decrease meaningfully when compared with those of control rats. This suggested that the IgY against colipase-binding domain antigen inhibited pancreatic lipase in vivo.

사람 췌장 리파아제는 췌장에서 합성되어 소장으로 분비된 후, 음식물속의 지방을 가수분해하는 소화효소이다. 췌장 리파아제 단백질은 촉매활성도메인과 코리파아제 결합도메인의 2개의 도메인으로 구성되어 있다 본 연구에서는 리파아제 전체단백질과 촉매활성도메인 및 코리파아제 결함도메인 유전자를 PCR 방법으로 증폭하여 발현벡터에 넣은 후, 대장균에서 발현시켜 얻은 3가지 재조합 단백질을 산란계에 3차례 주사하여 면역반응을 유도하였다. 난황단백질을 분리하여 항체가를 측정한 결과, 코리파아제 결합도메인의 항원성이 가장 높은 것으로 밝혀졌다. 또한, 돼지 췌장 리파아제의 활성저해 실험에서도 코리파아제 결합도메인에 의해 만들어진 난황항체가 지방분해활성 저해작용이 가장 큰 것으로 밝혀졌다. 코리파아제 결합도메인 난황항체가 첨가된 지방을 실험동물에게 급여하고 혈중 주요 성분을 분석한 결과, 코리파아제 결합도메인에 의한 난황항체를 섭취한 경우에 혈중 중성지방의 수치가 대조군에 비해서 유의적으로 감소했음을 화인하였다. 결론적으로 코리파아제 결합도메인을 항원단백질로 사용하여 얻은 난황항체가 생체 내에서 췌장 리파아제의 활성을 억제할 수 있으며 비만억제제로의 개발 가능성이 있음을 보여주었다.

Keywords

References

  1. Aoublal, M., L. de la Fourniere, I. Douchet, A. Abousalham, C. Daniel, M. Him, Y. Gargouri, R. Verger, and A. De Caro. 1995. Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies. J. Biol. Chem. 270: 3932-3937 https://doi.org/10.1074/jbc.270.8.3932
  2. Ayvazian, L., I. Crenon, S. Granon, C. Chapus, and B. Kerfelec, 1996. Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase. Protein Eng. 9: 707-711 https://doi.org/10.1093/protein/9.8.707
  3. Bezzine, S., F. Ettato, M. G. Ivanova, V. Lopez, R. Verger, and F. Carriere. 1999. Human pancreatic lipase: colipase dependence and interfacial binding of lLid domain mutants. Biochemistry 38: 5499-5510 https://doi.org/10.1021/bi982601x
  4. Carriere, F., K. Thirstrup, S. Hjorth, F. Ferrato, P. F. Nielsen, C. Withers-Martinez, C. Cambillau, E. Boel, L. Thim, and R. Verger. 1997. Pancreatic Lipase Structure-function relationships by domain exchange. Biochemistry 36: 239-248 https://doi.org/10.1021/bi961991p
  5. Hatta, H., J. S. Sim, and S. Nakai. 1998. Separation of phospholipids from egg yolk and recovery of water soluble protein. J. Food. Sci. 53: 425-432 https://doi.org/10.1111/j.1365-2621.1988.tb07721.x
  6. Jennens, M. L. and M. E. Lowe. 1995. C-terminal domain of human pancreatic lipase is required for stability and maximal activity but not colipase reactivation. J. Lipid Res. 36: 1029-1036
  7. Kim, J. W., D. K. Kim, and C. Kim. 2000. Production of specific egg yolk antibody against K99 (F5) fimbriae from enterotoxigenic Escherichi. J. Anim. Sci. Technol. 42: 371-378
  8. Kim, M. H. 2004. Updates in treating obesity. Kor. J. Health Psychol. 9: 493-509
  9. Kim, Y. S., 1990. Classification and evaluation of obesity. Kor. J. Nutrition. 23: 337-340
  10. Lee, E. H., Y. J. Lee, O. B. Cho, and S. M. Kang. 2007. Effects of a combined diet of Jerusalem artichoke's Inulin, Lotus leaf and herb extracts in obesity induced white rat with fat diet. J. Kor. Soc. Appl. Biol. Chem. 50: 295-303
  11. Lowe, M. E. 1997. Structure and function of pancreatic lipase and colipase. Annu. Rev. Nutr. 17: 141-158 https://doi.org/10.1146/annurev.nutr.17.1.141
  12. Lowe, M. E. 1997. Molecular mechanisms of rat and human pancreatic triglyceride lipases. J. Nutr. 127: 549-557
  13. Lowe, M. E. 2002. The triglyceride lipases of the pancreas. J. Lipid Res. 43: 2007-2016 https://doi.org/10.1194/jlr.R200012-JLR200
  14. Lowe, M. E., J. L. Rosenblum, and A. W. Strauss. 1989. Cloning and characterization of human pancreatic lipase cDNA. J. Biol. Chem. 33: 20042-20048
  15. Moreno D. A., N. Ilic, A. Poulev, D. L. Brasaemle, S. K. Fried and I. Raskin, 2003, Inhibitory effects of grape seed extract on lipases, Nutrition 19: 876-879 https://doi.org/10.1016/S0899-9007(03)00167-9
  16. Park, H. S. 2001. Pharmacological therapy of obesity, J. Kor. Soc. Study Obesity, 10: 118-127
  17. Prpkins, B. M. and C. M. Doak, 1998, The obesity prevalence is a worldwide phenomenon. Nut. Rev. 56: 104-114
  18. Reddy, P. M. and S. S. Chow, 1998, Focus on orlistat: a nonsystemic inhibitor of gastrointestinal lipase for weight reduction in the management of obesity. Formulary 33: 943-959
  19. Shin, M. K. and S. H. Han, 2006, Effects of Lotus (Nelumbo Nucifera Gaertner) leaf powder on lipid concentration in rat fed high fat diet rats. Kor. Food Culture 21: 202-208
  20. Sharma, N., V. K. Sharma, and S. Y. Seo, 2005. Screening of some medicinal plants for anti-lipase activity. J. Ethnopharmacol. 97: 453-456 https://doi.org/10.1016/j.jep.2004.11.009
  21. Shimizu, M., R. C. Frtzsimmons, and S. Nakai. 1998. Anti- E. coli immunoglobulin Y isolated from egg yolk of immunized chickens as a potential food ingredient. J. Food Sci. 53: 1360-1366 https://doi.org/10.1111/j.1365-2621.1988.tb09277.x
  22. Van Tilbeurgh, H., L. Sarda, R. Verger, and C. Cambillau, 1992. Structure of the pancreatic lipase-procolipase complex. Nature 359: 159-162 https://doi.org/10.1038/359159a0
  23. Whitcomb, D. C. and M. E. Lowe. 2007. Human pancreatic digestive enzymes. Dig. Dis. Sci. 52: 1-17 https://doi.org/10.1007/s10620-006-9589-z
  24. Yamamoto, M., S. Shimura, Y. Itoh, T. Ohsaka, M. Egawa, and S. Ionue. 2000. Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs, Nomame Herba, on rats fed a high-fat diet. Int. J. Obes. Relat. Metab. Disord. 24: 758-764 https://doi.org/10.1038/sj.ijo.0801222
  25. Yoon, Y. S., S. M. Choi, S. B. Hong, J. M. Hong, J. W. Kim, H. S. Lee, and S. G. Hong. 2002. Development of an antiobesity dietary supplement inhibiting the digestion of carbohydrate and lipid. Kor. J. Soc. Food cookery Sci. 18: 319-324