Effect of the GPI anchor of human Thy-1 on antibody recognition and function.
Thymocyte differentiation antigen-1 (Thy-1) is a glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein expressed on numerous cell types, which regulates signals affecting cell adhesion, migration, differentiation, and survival. In addition, Thy-1 has been detected in the serum, cerebral spinal fluid, wound fluid from venous ulcers, synovial fluid from joints in rheumatoid arthritis, and, more recently, urine. We previously detected Thy-1 in the conditioned media of cytokine-stimulated lung fibroblasts, suggesting that Thy-1 shedding may be a response to cellular stress. Soluble and membrane-bound forms of Thy-1 from in vivo sources have been shown to be identical in size when deglycosylated, suggesting that soluble Thy-1 is separated from the diacyl glycerol portion of its GPI anchor by hydrolysis within the GPI moiety. For Thy-1- and other GPI-anchored proteins, delipidation induces a stable change in conformation that manifests itself in a change in antibody affinity for soluble forms. Using epitope-tagged recombinant soluble Thy-1, we report that widely available monoclonal antibodies to human Thy-1 are unable to detect soluble Thy-1 by immunoblotting. We re-evaluated the Thy-1 that we previously reported in the conditioned media of normal human lung fibroblasts and found it to be entirely insoluble. These findings suggest that most Thy-1 reported in body fluids retains its GPI anchor and may be associated with membrane fragments or vesicles. This phenomenon should be considered in the generation of antibodies and controls for Thy-1 bioassays. Furthermore, the changes in Thy-1 conformation with delipidation, beyond affecting antibody affinity, likely affect the ligand affinity and biological function of soluble vs released membrane-associated forms.
Department of Medicine, Division of Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, USA.
John E
JE
Bradley
Joy M
JM
Chan
James S
JS
Hagood
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D002498:Centrifugation; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D017830:Octoxynol; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011092:Polyethylene Glycols; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D018800:Thy-1 Antigens / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase