Short-term culture of surface-biotinylated cells: Application in non-radioactive analysis of surface protein shedding

Abstract

As an alternative to radiolabelling, cell surface biotinylation followed by immunoprecipitation and enhanced chemiluminescence detection can be used to characterize membrane-bound surface proteins. Since many membrane proteins are known to be shed from the surface, we tested whether surface biotinylation can also be used to characterize these soluble proteins released into culture supernatant. First, we found that biotinylated cells could be cultured for up to 7 days without any influence on viability and 3H-thymidine incorporation. Moreover, the PHA response ( 3H-thymidine incorporation) of peripheral blood mononuclear cells was affected only by high concentrations of the biotinylation reagent N-hydroxysuccinimidobiotin and the LPS response (cytokine release) was unaffected by surface biotinylation. Second, we were able to monitor the shedding of biotinylated HLA class I antigens from Balm 1 cells by immunoprecipitation, SDS-PAGE, and enhanced chemiluminescence detection on blots. Membrane-bound HLA class I antigens disappeared from the cell surface during 5-day culture and, simultaneously, two forms of soluble HLA class I heavy chains (36 and 44 kDa) accumulated in the culture supernatant. Thus, short-term culture of surface biotinylated cells can be used in the analysis of the shedding processes of leukocyte antigens.