A 113-Amino Acid Fragment of CD4 Produced in Escherichia Coli Blocks Human Immunodeficiency Virus-induced Cell Fusion

Chao, B H; Costopoulos, Donna; Curiel, Tyler J.; Bertonis, Jeanne M.; Chisholm, Patricia M.; Williams, Charlene J.; Schooley, R. T.; Rosa, Joseph J.; Fisher, Richard A.; Maraganore, John M.

doi:10.1016/s0021-9258(18)83622-5

Cited by 39 publications

(6 citation statements)

References 29 publications

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“…Truncated forms of CD4 or CD4 domains have been previously produced in bacteria (Chao et al, 1989) and in mammalian cells (Berger et al, 1988) by direct expression of truncated CD4 cDNA. However, the approach of using immunoglobulin fusion molecules to generate isolated domains of CD4 might have several advantages over direct-expression strategies.…”

Section: Resultsmentioning

confidence: 99%

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Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

Chamow¹,

Peers²,

Byrn³

et al. 1990

Biochemistry

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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)

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Section: Resultsmentioning

confidence: 99%

“…Previous studies (Chao et al, 1989;Berger et al, 1988) have shown that truncated forms of CD4 can be produced by direct expression. Here we describe an alternative approach for the production of isolated CD4 domains using enzymatic cleavage of CD4-immunoglobulin fusion molecules (CD4 immunoadhesins).…”

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confidence: 99%

Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

Chamow¹,

Peers²,

Byrn³

et al. 1990

Biochemistry

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“…CD4 contains four Ig-related domains with domain 1 resembling the variable domain of an Ig light chain (3,4). Domain 1 of CD4 is sufficient for high-affinity binding of gp120 (5,6), and mutation experiments resulting in the loss of gp120 binding have been undertaken in an attempt to map the gp120 binding site. The most informative studies are those where single amino acids were changed and initially CD4 residues 42-49 were suggested to encompass the gp120 site (7).…”

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confidence: 99%

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4.

Schockmel

Somoza

Davis

et al. 1992

The Journal of Experimental Medicine

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SummaryThe human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.

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“…McDougal et al (1986a) showed that the major envelope glycoprotein of HIV-1, gpl 20, forms a specific complex with CD4. The gpl20 binding region of CD4 has been localized to the one or two amino-terminal immunoglobulin-like domains of CD4 (Richardson et al, 1988;McDougal et al, 1986b;Traunecker et al, 1988;Chao et al, 1989) , since constructs containing just the amino-terminal region were shown to be effective at blocking HIV infectivity in vitro. Using mutational analysis, several groups have shown that a region encompassing residues 40-55 is important in gpl20 binding (Peterson & Seed, 1988;Clayton et al, 1988;Arthos et al, 1989;Brodsky et al, 1990;Ashkenazi et al, 1990) .…”

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confidence: 99%

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

Teshima¹,

Porter²,

Yim³

et al. 1991

Biochemistry

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High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.

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A 113-Amino Acid Fragment of CD4 Produced in Escherichia Coli Blocks Human Immunodeficiency Virus-induced Cell Fusion

Cited by 39 publications

References 29 publications

Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4.

Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120

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