Analysis of recombinant and native CD4 by one- and two-dimensional gel electrophoresis.
Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules 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-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) 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 electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, 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.
Lynch
G W
GW
Department of Virology, ICPMR, University of Sydney, Westmead Hospital, Westmead and National Centre for HIV Virology Research, Australia. GARRYL@Westmed.WH.SU.E-DU.AU
Dearden
M
M
Sloane
A J
AJ
Humphery-Smith
I
I
Cunningham
A L
AL
D015704:CD4 Antigens / Q000097:blood*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D015699:HIV Envelope Protein gp120 / Q000032:analysis*; D015497:HIV-1; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008970:Molecular Weight; D011233:Precipitin Tests; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000097:blood
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins