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Enzymatic Cleavage of a CD4 Immunoadhesin Generates Crystallizable, Biologically Active Fd-like Fragments

Chamow, Steven M. and Peers, David H. and Byrn, Randal A. and Mulkerrin, Michael G. and Harris, Reed J. and Wang, Wen Ching and Bjorkman, Pamela J. and Capon, Daniel J. and Ashkenazi, Avi (1990) Enzymatic Cleavage of a CD4 Immunoadhesin Generates Crystallizable, Biologically Active Fd-like Fragments. Biochemistry, 29 (42). pp. 9885-9891. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170406-104919883

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Abstract

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 V_L, domains. The first of these (V_1) 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 V_1, 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 V_1 and V_2 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 V_L domains; this directly supports the hypothesis that the V_1 and V_2 domains of CD4 fold similarly to Ig V_L domains. Crystals of the Fd-like fragment diffract beyond 3-Å resolution and are suitable for detailed structural analysis. The approach described here may be useful, as an alternative to direct expression, in the study of receptors and other adhesion molecules which are members of the Ig gene superfamily.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi00494a019DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi00494a019PublisherArticle
ORCID:
AuthorORCID
Bjorkman, Pamela J.0000-0002-2277-3990
Additional Information:© 1990 American Chemical Society. Published in print 1 October 1990. This work was supported by Genentech, Inc., by Public Health Service Grants AI-28931 (W.-C.W. and P.J.B.), HL-43510, HL-42374, and HL-42112 (R.A.B.) from the National Institutes of Health, and by the Howard Hughes Medical Institute. P.J.B. is a Pew Scholar.
Funders:
Funding AgencyGrant Number
Genentech, Inc.UNSPECIFIED
NIHAI-28931
NIHHL-43510
NIHHL-42374
NIHHL-42112
Pew Charitable TrustUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Issue or Number:42
Record Number:CaltechAUTHORS:20170406-104919883
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170406-104919883
Official Citation:Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments Steven M. Chamow, David H. Peers, Randal A. Byrn, Michael G. Mulkerrin, Reed J. Harris, Wen Ching Wang, Pamela J. Bjorkman, Daniel J. Capon, and Avi Ashkenazi Biochemistry 1990 29 (42), 9885-9891 DOI: 10.1021/bi00494a019
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75803
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:06 Apr 2017 18:27
Last Modified:03 Oct 2019 16:54

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