Molecular cloning, recombinant expression and characterization of GMCSF from the rhesus monkey, Macaca mulatta.
Recent studies have found that, in addition to hematopoiesis, granulocyte-macrophage colony-stimulating factor (GMCSF) plays pivotal roles in multiple immune disorders. The gene encoding Macaca mulatta GMCSF (mmGMCSF) was cloned from stimulated peripheral blood mononuclear cells (PBMCs). Concanavalin A (Con A) and mismatched allogeneic antigen-stimulation significantly increased the production of mmGMCSF by monkey PBMCs. The gene encoding mature mmGMCSF was first expressed as a soluble fusion protein in Escherichia coli, and native mmGMCSF was further prepared by protease cleavage. The recombinant mmGMCSF induced antigen-presenting dendritic cells from monkey PBMCs, suggesting a central role of mmGMCSF in the immune system of the rhesus monkey. Although the predicted mature mmGMCSF protein differs from human GMCSF (hGMCSF) at six amino acid residues, mmGMCSF showed a strong ability to support human TF-1 cell survival. Additional co-immunoprecipitation experiments revealed that mmGMCSF cross reacts with the hGMCSF receptor (hGMCSFR). In addition, the hGMCSF-neutralizing agents hGMCSFR-Fc and anti-hGMCSF antibody reduced the biological effects of mmGMCSF on TF-1 cells. These results suggest that recombinant mmGMCSF might be used for the in vitro evaluation of novel hGMCSF-neutralizing agents prior to the in vivo preclinical evaluation of these agents in the rhesus monkey model.
Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, China.
Ze
Z
Tao
Hao
H
Yang
Dianlong
D
Jia
Lin
L
Wan
Jingqiu
J
Cheng
Xiaofeng
X
Lu
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology*; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006649:Histocompatibility Antigens / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D007959:Lymphocyte Culture Test, Mixed; D008253:Macaca mulatta / Q000276:immunology; D008969:Molecular Sequence Data; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment
D019138:Antibodies, Blocking; D006649:Histocompatibility Antigens; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D003208:Concanavalin A; D016178:Granulocyte-Macrophage Colony-Stimulating Factor