Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent.
Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.
Chamow
S M
SM
Department of Process Science, Genentech, Inc., South San Francisco, California 94080.
Kogan
T P
TP
Peers
D H
DH
Hastings
R C
RC
Byrn
R A
RA
Ashkenazi
A
A
D015704:CD4 Antigens / Q000737:chemistry* / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008301:Maleimides; D010084:Oxidation-Reduction; D011485:Protein Binding; D012794:Sialic Acids
D015704:CD4 Antigens; D002241:Carbohydrates; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120; D006454:Hemoglobins; D008301:Maleimides; D012794:Sialic Acids; C075837:4-(4-N-maleimidophenyl)butyric acid hydrazide