ANALYSIS OF RECOMBINANT AND NATIVE CD4 BY ONE-DIMENSIONAL AND 2-DIMENSIONAL GEL-ELECTROPHORESIS

Knowledge of CD4 conformation within the membranes of human lymphoid a nd monocytoid cells is essential for a clear understanding of its func tion as a ligand for major histocompatibility complex II (MHC) molecul es in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-D E) electrophoresis antigen mapping and silver staining. Recombinant CD 4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHG E) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electro phoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kD a, consistent with a maximal incorporation of similar to 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degre es C) off cell-(CEM-T4, THP-1) expresses CD4 was not accompanied by an y apparent alteration in molecular weight, nor abrogation of CD4 antig enicity. This was determined by isolation of nCD4 by immunoprecipitati on and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a. OKT4A, Q41 20, T4, OKT4, Q425) and by flow cytometry (leu4a. T4). The immunopreci pitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell e xtracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation , 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.