Detection of 3-nitrotyrosine-modified human leukocyte antigen-G in biological fluids.
Human leukocyte antigen (HLA)-G is a suppressive molecule that can be shed to the medium by a metalloprotease-dependent mechanism. Nitric oxide increases HLA-G shedding and also causes tyrosine nitration. As the presence of nitrated HLA-G has not been demonstrated in vivo, the aim of this work was to investigate whether this post-translational modification in the HLA-G molecule could also occur in vivo. Exudates and blood samples were collected during an analytical routine. HLA-G was analyzed by enzyme-linked immunoabsorbent assay and nitrites plus nitrates by colorimetry. Samples were immunoprecipitated with anti-nitrotyrosine antibody. After nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot with anti-HLA-G antibody, a band at 45 kDa was visualized and assigned to nitrated HLA-G. In addition, there were several bands at higher molecular weight that disappeared in the immunoblot performed after electrophoresis under reducing conditions, where only the band at 45 kDa remained. This indicates that nitrated HLA-G was also present as multimers. The degree of HLA-G nitration did not correlate with the concentrations of nitrites plus nitrates or HLA-G. These results indicate that HLA-G could circulate as nitrotyrosine modified protein, both as monomer and as multimer.
Díaz-Lagares
Angel
A
Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain.
Alegre
Estibaliz
E
Gonzalez
Alvaro
A
D001826:Body Fluids / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006680:HLA Antigens / Q000032:analysis / Q000378:metabolism*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine