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The Korean Association of Immunobiologists (대한면역학회)
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Role of N-terminal Hydrophilic Amino Acids in Molecular Translocation of CTLA-4 to Cell Surface

CTLA-4 항원의 세포막 도달 기작에서 친수성 N말단 아미노산 잔기의 역할

  • Han, Ji-Woong (Biomedical Research Institute of MedExGen Inc.) ;
  • Lee, Hye-Ja (Biomedical Research Institute of MedExGen Inc.) ;
  • Kim, Jin-Mi (Biomedical Research Institute of MedExGen Inc.) ;
  • Choi, Eun-Young (Department of Microbiology, College of Medicine, Hanyang University) ;
  • Chung, Hyun-Joo (Department of Microbiology, College of Medicine, Hanyang University) ;
  • Lim, Soo-Bin (Department of Microbiology, College of Medicine, Hanyang University) ;
  • Choi, Jang-Won (Division of Life Resources, College of Natural Resources, Taegu University) ;
  • Chung, Yong-Hoon (Department of Microbiology, College of Medicine, Hanyang University)
  • 한지웅 (메덱스젠 생명공학연구소) ;
  • 이혜자 (메덱스젠 생명공학연구소) ;
  • 김진미 (메덱스젠 생명공학연구소) ;
  • 최은영 (한양대학교 의과대학 미생물학교실) ;
  • 정현주 (한양대학교 의과대학 미생물학교실) ;
  • 임수빈 (한양대학교 의과대학 미생물학교실) ;
  • 최장원 (대구대학교 자연자원대학 생명자원학부) ;
  • 정용훈 (한양대학교 의과대학 미생물학교실)
  • Published : 2002.06.30
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Abstract

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.

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