Binding of GC (VDBP) to membranes of human B lymphocytes following stripping of extant protein

• Published in: Journal of endocrinological investigation Vol. 18; no. 8; pp. 630 - 637
• Main Authors: PETRINI, M, ALLEGRINI, A, AMBROGI, F, VALENTINI, P, SABBATINI, A, ARNAUD, P, GALBRAITH, R. M
• Format: Journal Article
• Language: English
• Published: Milano Kurtis 01-09-1995
• Subjects: B-Lymphocytes - metabolism; Biological and medical sciences; Cell Extracts - chemistry; Cell Membrane - metabolism; Humans; Immunoglobulin M - blood; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Protein Binding; Solubility; Vitamin D-Binding Protein - blood; Vitamin D-Binding Protein - isolation & purification; Antigen; Human; Binding; Flow cytometry; Binding protein; Vitamin D; Immunological investigation; B-Lymphocyte; Cell surface; Blood plasma
• ISSN: 0391-4097; 1720-8386
• DOI: 10.1007/BF03349781

The presence of Gc (vitamin D binding protein) has been consistently demonstrated on the membrane of B lymphocytes. This protein appears to be spatially associated with surface immunoglobulins. The origin of this surface protein has not yet been determined and the purpose of the present paper was to investigate if Gc may bind to human lymphocytes after immunoglobulin (Ig) capping. For this purpose the presence of Gc on B lymphocytes was examined by three different approaches. First, when cells were examined by immunofluorescence and quantified by flow cytometry, membrane Ig capping was followed by a dramatic decrease in positivity for Gc when compared to native cells. In addition, incubation of capped cells with purified Gc was followed by a significant increase in fluorescence, indicating that this protein had been able to bind again. Second, analysis of solubilized lymphocytes by Western blotting showed that native lymphocytes and capped cells incubated with purified Gc contained a large quantity of a 56kDa protein which was immunoreactive with anti Gc antibodies. This protein band was much weaker on blots from capped cells not treated with Gc. Third, radiobinding assays indicated that, following capping, cells were able to bind Gc in saturable fashion. These results suggest that membrane Gc could play a role in the entry of vitamin D metabolites into lymphocytes.