Characterization of a Monoclonal Antibody Specific to Human Siah-1 Interacting Protein

인체 SIP 단백질에 특이적인 단일클론 항체의 특성

  • Yoon, Sun Young (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Joo, Jong Hyuck (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Joo Heon (Department of Pathology, Eulji University School of Medicine) ;
  • Kang, Ho Bum (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Jin Sook (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Younghee (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kwon, Do Hwan (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Chang Nam (Department of Surgery, Eulji University School of Medicine) ;
  • Choe, In Seong (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Jae Wha (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • 윤선영 (한국생명공학연구원 세포생물학연구실) ;
  • 주종혁 (한국생명공학연구원 세포생물학연구실) ;
  • 김주헌 (을지의과대학 병리학교실) ;
  • 강호범 (한국생명공학연구원 세포생물학연구실) ;
  • 김진숙 (한국생명공학연구원 세포생물학연구실) ;
  • 이영희 (한국생명공학연구원 세포생물학연구실) ;
  • 권두한 (한국생명공학연구원 세포생물학연구실) ;
  • 김창남 (을지의과대학 외과학교실) ;
  • 최인성 (한국생명공학연구원 세포생물학연구실) ;
  • 김재화 (한국생명공학연구원 세포생물학연구실)
  • Published : 2004.03.31

Abstract

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

Keywords

Acknowledgement

Supported by : 한국생명공학연구원

References

  1. Matsuzawa SI, Reed JC: Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses. Mol Cell 7;915-926, 2001 https://doi.org/10.1016/S1097-2765(01)00242-8
  2. Donato R: Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type. Biochim Biophys Acta 1450;191-231, 1999 https://doi.org/10.1016/S0167-4889(99)00058-0
  3. Donato R: S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int J Biochem Cell Biol 33;637-668, 2001 https://doi.org/10.1016/S1357-2725(01)00046-2
  4. Jastrzebska B, Filipek A, Nowicka D, Kaczmarek L, Kuznicki J: Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons. J Histochem Cytochem 48;1195-1202, 2000 https://doi.org/10.1177/002215540004800903
  5. Zimmer DB, Cornwall EH, Landar A, Song W: The S100 protein family: history, function, and expression. Brain Res Bull 37;417-429, 1995 https://doi.org/10.1016/0361-9230(95)00040-2
  6. Filipek A, Wojda U: p30, a novel protein target of mouse calcyclin (S100A6). Biochem J 320 (Pt 2);585-587, 1996 https://doi.org/10.1042/bj3200585
  7. Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman M, Surmacz L, Wyroba E, Kuznicki J: Ca2+ -dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J Biol Chem 277;21103-21109, 2002 https://doi.org/10.1074/jbc.M111010200
  8. Xia ZB, Dai MS, Magoulas C, Broxmeyer HE, Lu L:Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cDNA. J Hematother Stem Cell Res 9;651-658, 2000 https://doi.org/10.1089/15258160050196696
  9. Pircher TJ, Geiger JN, Zhang D, Miller CP, Gaines P, Wojchowski DM: Integrative signaling by minimal erythropoietin receptor forms and c-Kit. J Biol Chem 276;8995-9002, 2001 https://doi.org/10.1074/jbc.M007473200
  10. Nowotny M, Bhattacharya S, Filipek A, Krezel AM, Chazin W, Kuznicki J: Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein. J Biol Chem 275;31178-31182, 2000 https://doi.org/10.1074/jbc.M001622200
  11. Filipek A, Jastrzebska B, Nowotny M, Kuznicki J: CacyBP/ SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family. J Biol Chem, 277;28848-28852, 2002 https://doi.org/10.1074/jbc.M203602200
  12. Matsuzawa S, Li C, Ni CZ, Takayama S, Reed JC, Ely KR:Structural analysis of Siah1 and its interactions with Siahinteracting protein (SIP). J Biol Chem 278;1837-1840, 2003 https://doi.org/10.1074/jbc.M210263200
  13. Boni R, Heizmann CW, Doguoglu A, Ilg EC, Schafer BW, Dummer R, Burg G: Ca (2+) -binding proteins S100A6 and S100B in primary cutaneous melanoma. J Cutan Pathol 24;76-80, 1997 https://doi.org/10.1111/j.1600-0560.1997.tb01100.x
  14. Breen EC, Tang K: Calcyclin (S100A6) regulates pulmonary fibroblast proliferation, morphology, and cytoskeletal organization in vitro. J Cell Biochem 88;848-854, 2003 https://doi.org/10.1002/jcb.10398
  15. Maelandsmo GM, Florenes VA, Mellingsaeter T, Hovig E, Kerbel RS, Fodstad O: Differential expression patterns of S100A2, S100A4 and S100A6 during Progression of human malignant melanoma. Int J Cancer 74;464-469, 1997 https://doi.org/10.1002/(SICI)1097-0215(19970822)74:4<464::AID-IJC19>3.0.CO;2-9
  16. Yang Q, O'Hanlon D, Heizmann CW, Marks A: Demonstration of heterodimer formation between S100B and S10-0A6 in the yeast two-hybrid system and human melanoma. Exp Cell Res 246;501-509, 199 https://doi.org/10.1006/excr.1998.4314
  17. Komatsu K, Murata K, Kameyama M, Ayaki M, Mukai M, Ishiguro S, Miyoshi J, Tatsuta M, Inoue M, Nakamura H:Expression of S100A6 and S100A4 in matched samples of human colorectal mucosa, primary colorectal adenocarcinomas and liver metastases. Oncology 63;192-200, 2002 https://doi.org/10.1159/000063812
  18. Komatsu K, Kobune-Fujiwara Y, Andoh A, Ishiguro S, Hunai H, Suzuki N, Kameyama M, Murata K, Miyoshi J, Akedo H, Tatsuta M, Nakamura H: Increased expression of S100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma. Br J Cancer 83;769-774, 2000 https://doi.org/10.1054/bjoc.2000.1356
  19. Komatsu K, Andoh A, Ishiguro S, Suzuki N, Hunai H, Kobune-Fujiwara Y, Kameyama M, Miyoshi J, Akedo H, Nakamura H: Increased expression of S100A6 (Calcyclin), a calcium-binding protein of the S100 family, in human colorectal adenocarcinomas. Clin Cancer Res 6;172-177, 2000