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.
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
G W
GW
Lynch
M
M
Dearden
A J
AJ
Sloane
I
I
Humphery-Smith
A L
AL
Cunningham
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