Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments.
CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)
Chamow
S M
SM
Department of Recovery Process Research and Development, Genentech, Inc., South San Francisco, California 94080.
Peers
D H
DH
Byrn
R A
RA
Mulkerrin
M G
MG
Harris
R J
RJ
Wang
W C
WC
Bjorkman
P J
PJ
Capon
D J
DJ
Ashkenazi
A
A
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism*; D016860:CD4 Immunoadhesins; D002942:Circular Dichroism; D003460:Crystallization; D006678:HIV / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015728:Receptors, HIV / Q000378:metabolism*; D014961:X-Ray Diffraction
D000888:Antibodies, Anti-Idiotypic; D015703:Antigens, CD; D015704:CD4 Antigens; D016860:CD4 Immunoadhesins; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D010446:Peptide Fragments; D015728:Receptors, HIV; D010206:Papain