Recombinant Expression and Characterization of CD2-binding Domain of <i>Macaca mulatta</i> Lymphocyte Function-associated Antigen 3 in <i>Pichia pastoris</i>_Journal of Biomedical Engineering

Authors：

ZHUJian , ZHUShengyun , YANGHao , LUXiaofeng ,  WANLin

Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

WANLin, Email: linwan@scu.edu.cn

Keywords：

lymphocyte function-associated antigen 3; immunosuppression; Rhesus monkey; Pichia pastoris

DOI：

10.7507/1001-5515.20150022

Video：

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Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.

Citation： ZHUJian, ZHUShengyun, YANGHao, LUXiaofeng, WANLin. Recombinant Expression and Characterization of CD2-binding Domain of Macaca mulatta Lymphocyte Function-associated Antigen 3 in Pichia pastoris. Journal of Biomedical Engineering, 2015, 32(1): 120-125. doi: 10.7507/1001-5515.20150022 Copy

1.	DENGLER T J, HOFFMANN J C, KNOLLE P, et al. Structural and functional epitopes of the human adhesion receptor CD58 (LFA-3)[J]. Eur J Immunol, 1992, 22(11): 2809-2817.
2.	VELTRONI M, DE ZEN L, SANZARI M C, et al. Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia[J]. Haematologica, 2003, 88(11): 1245-1252.
3.	HAIDER A S, LOWES M A, GARDNER H, et al. Novel insight into the agonistic mechanism of alefacept in vivo: differentially expressed genes May serve as biomarkers of response in psoriasis patients[J]. J Immunol, 2007, 178(11): 7442-7449.
4.	SELVARAJ P, PLUNKETT M L, DUSTIN M, et al. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3[J]. Nature, 1987, 326(6111): 400-403.
5.	CRAWFORD K, STARK A, KITCHENS B, et al. CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation[J]. Blood, 2003, 102(5): 1745-1752.
6.	KAIZUKA Y, DOUGLASS A D, VARDHANA S, et al. The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells[J]. J Cell Biol, 2009, 185(3): 521-534.
7.	ARIEL O, LEVI Y, HOLLANDER N. Signal transduction by CD58: the transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform[J]. Cell Signal, 2009, 21(7): 1100-1108.
8.	LEITNER J, GRABMEIER-PFISTERSHAMMER K, STEINBERGER P. Receptors and ligands implicated in human T cell costimulatory processes[J]. Immunol Lett, 2010, 128(2): 89-97.
9.	PILAT N, SCHWARZ C, WEKERLE T. Modulating T-cell costimulation as new immunosuppressive concept in organ transplantation[J]. Curr Opin Organ Transplant, 2012, 17(4): 368-375.
10.	KATO K, KOYANAGI M, OKADA H, et al. CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation[J]. J Exp Med, 1992, 176(5): 1241-1249.
11.	YAMASHITA K, PARISH C R, WARREN H S, et al. A multimeric form of soluble recombinant sheep LFA-3 (CD58) inhibits human T-cell proliferation[J]. Immunology, 1997, 92(1): 39-44.
12.	BROSSAY A, HUBÉ F, MOREAU T, et al. Porcine CD58: cDNA cloning and molecular dissection of the porcine CD58-human CD2 interface[J]. Biochem Biophys Res Commun, 2003, 309(4): 992-998.
13.	何玉林, 邓玲.CD58分子研究概况[J].中国医药指南, 2012, 10(2): 50-52.
14.	芦晓立, 颜新敏, 吴国华, 等.绵羊CD58基因的克隆与原核表达[J].甘肃农业大学学报, 2010, 45(05): 11-14.
15.	窦永喜, 景志忠, 侯俊玲, 等.猪CD58分子基因克隆、表达及其结构功能预测[J].畜牧兽医学报, 2007, 38(4): 388-394.
16.	MACRI S C, BAILEY C C, DE OCA N M, et al. Immunophenotypic alterations in resident immune cells and myocardial fibrosis in the aging rhesus macaque (Macaca mulatta) heart[J]. Toxicol Pathol, 2012, 40(4): 637-646.
17.	SHIN I S, CHOI E W, CHUNG J Y, et al. Cloning, expression and bioassay of canine CTLA4Ig[J]. Vet Immunol Immunopathol, 2007, 118(1/2): 12-18.
18.	WALLNER B P, FREY A Z, TIZARD R, et al. Primary structure of lymphocyte function-associated antigen 3 (LFA-3). The ligand of the T lymphocyte CD2 glycoprotein[J]. J Exp Med, 1987, 166(4): 923-932.
19.	YAMASHITA K, KAKUTANI T, OHASHI T, et al. A dimeric form of soluble recombinant sheep LFA-3(CD58) inhibits human T-cell proliferation by generating regulatory T cells[J]. Immunopharmacology, 1997, 37(2/3): 209-220.
20.	DA SILVA A J, BRICKELMAIER M, MAJEAU G R, et al. Alefacept, an immunomodulatory recombinant LFA-3/IgG1 fusion protein, induces CD16 signaling and CD2/CD16-dependent apoptosis of CD2(+) cells[J]. J Immunol, 2002, 168(9): 4462-4471.
21.	LEE S, YAMADA Y, TONSHO M, et al. Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells[J]. Am J Transplant, 2013, 13(12): 3223-3229.
22.	MILLER G T, HOCHMAN P S, MEIER W, et al. Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses[J]. J Exp Med, 1993, 178(1): 211-222.

1. DENGLER T J, HOFFMANN J C, KNOLLE P, et al. Structural and functional epitopes of the human adhesion receptor CD58 (LFA-3)[J]. Eur J Immunol, 1992, 22(11): 2809-2817.
2. VELTRONI M, DE ZEN L, SANZARI M C, et al. Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia[J]. Haematologica, 2003, 88(11): 1245-1252.
3. HAIDER A S, LOWES M A, GARDNER H, et al. Novel insight into the agonistic mechanism of alefacept in vivo: differentially expressed genes May serve as biomarkers of response in psoriasis patients[J]. J Immunol, 2007, 178(11): 7442-7449.
4. SELVARAJ P, PLUNKETT M L, DUSTIN M, et al. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3[J]. Nature, 1987, 326(6111): 400-403.
5. CRAWFORD K, STARK A, KITCHENS B, et al. CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation[J]. Blood, 2003, 102(5): 1745-1752.
6. KAIZUKA Y, DOUGLASS A D, VARDHANA S, et al. The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells[J]. J Cell Biol, 2009, 185(3): 521-534.
7. ARIEL O, LEVI Y, HOLLANDER N. Signal transduction by CD58: the transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform[J]. Cell Signal, 2009, 21(7): 1100-1108.
8. LEITNER J, GRABMEIER-PFISTERSHAMMER K, STEINBERGER P. Receptors and ligands implicated in human T cell costimulatory processes[J]. Immunol Lett, 2010, 128(2): 89-97.
9. PILAT N, SCHWARZ C, WEKERLE T. Modulating T-cell costimulation as new immunosuppressive concept in organ transplantation[J]. Curr Opin Organ Transplant, 2012, 17(4): 368-375.
10. KATO K, KOYANAGI M, OKADA H, et al. CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation[J]. J Exp Med, 1992, 176(5): 1241-1249.
11. YAMASHITA K, PARISH C R, WARREN H S, et al. A multimeric form of soluble recombinant sheep LFA-3 (CD58) inhibits human T-cell proliferation[J]. Immunology, 1997, 92(1): 39-44.
12. BROSSAY A, HUBÉ F, MOREAU T, et al. Porcine CD58: cDNA cloning and molecular dissection of the porcine CD58-human CD2 interface[J]. Biochem Biophys Res Commun, 2003, 309(4): 992-998.
13. 何玉林, 邓玲.CD58分子研究概况[J].中国医药指南, 2012, 10(2): 50-52.
14. 芦晓立, 颜新敏, 吴国华, 等.绵羊CD58基因的克隆与原核表达[J].甘肃农业大学学报, 2010, 45(05): 11-14.
15. 窦永喜, 景志忠, 侯俊玲, 等.猪CD58分子基因克隆、表达及其结构功能预测[J].畜牧兽医学报, 2007, 38(4): 388-394.
16. MACRI S C, BAILEY C C, DE OCA N M, et al. Immunophenotypic alterations in resident immune cells and myocardial fibrosis in the aging rhesus macaque (Macaca mulatta) heart[J]. Toxicol Pathol, 2012, 40(4): 637-646.
17. SHIN I S, CHOI E W, CHUNG J Y, et al. Cloning, expression and bioassay of canine CTLA4Ig[J]. Vet Immunol Immunopathol, 2007, 118(1/2): 12-18.
18. WALLNER B P, FREY A Z, TIZARD R, et al. Primary structure of lymphocyte function-associated antigen 3 (LFA-3). The ligand of the T lymphocyte CD2 glycoprotein[J]. J Exp Med, 1987, 166(4): 923-932.
19. YAMASHITA K, KAKUTANI T, OHASHI T, et al. A dimeric form of soluble recombinant sheep LFA-3(CD58) inhibits human T-cell proliferation by generating regulatory T cells[J]. Immunopharmacology, 1997, 37(2/3): 209-220.
20. DA SILVA A J, BRICKELMAIER M, MAJEAU G R, et al. Alefacept, an immunomodulatory recombinant LFA-3/IgG1 fusion protein, induces CD16 signaling and CD2/CD16-dependent apoptosis of CD2(+) cells[J]. J Immunol, 2002, 168(9): 4462-4471.
21. LEE S, YAMADA Y, TONSHO M, et al. Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells[J]. Am J Transplant, 2013, 13(12): 3223-3229.
22. MILLER G T, HOCHMAN P S, MEIER W, et al. Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses[J]. J Exp Med, 1993, 178(1): 211-222.

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Recombinant Expression and Characterization of CD2-binding Domain of Macaca mulatta Lymphocyte Function-associated Antigen 3 in Pichia pastoris

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