Production of multimeric forms of CD4 through a sugar-based cross-linking strategy.
We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.
Chen
L L
LL
Biogen, Inc., Cambridge, Massachusetts 02142.
Rosa
J J
JJ
Turner
S
S
Pepinsky
R B
RB
D000596:Amino Acids / Q000032:analysis; D015704:CD4 Antigens / Q000737:chemistry*; D002241:Carbohydrates / Q000737:chemistry*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D013379:Substrate Specificity
D000596:Amino Acids; D015704:CD4 Antigens; D002241:Carbohydrates; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide