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Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.

Crombie R, Silverstein RL, MacLow C, Pearce SF, Nachman RL, Laurence J - J. Exp. Med. (1998)

Bottom Line: In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines.Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity.Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The New York Hospital-Cornell Medical Center, New York 10021, USA.

ABSTRACT
Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

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(A) Concentration-dependent binding of 125I-TSP1 to gp160. Increasing concentrations of soluble 125I-labeled TSP1 (1 nM–1 μM) were added  to immobilized recombinant HIV-1 gp160 for 3 h at 22°C, and bound TSP1 was measured after extensive washing. Nonlinear curve fit was generated  with ExcelTM version 5.0. Apparent affinity was estimated from Scatchard analysis. (n = 2; error calculated as SD). (B) Competitive inhibition demonstrates specificity of the TSP1–HIV interaction. A fixed concentration of 125I-labeled TSP1 (50 nM) was added to immobilized rgp160 in the absence or  presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, fusion protein LFP75–155 containing the LIMPII TSP-binding domain (aa 75–155), or  downstream fusion protein LFP156–243. Samples were incubated and bound TSP1 was measured as in A. Plots represent single data sets of triplicate  samples. (n = 3, error as SD).
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Figure 2: (A) Concentration-dependent binding of 125I-TSP1 to gp160. Increasing concentrations of soluble 125I-labeled TSP1 (1 nM–1 μM) were added to immobilized recombinant HIV-1 gp160 for 3 h at 22°C, and bound TSP1 was measured after extensive washing. Nonlinear curve fit was generated with ExcelTM version 5.0. Apparent affinity was estimated from Scatchard analysis. (n = 2; error calculated as SD). (B) Competitive inhibition demonstrates specificity of the TSP1–HIV interaction. A fixed concentration of 125I-labeled TSP1 (50 nM) was added to immobilized rgp160 in the absence or presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, fusion protein LFP75–155 containing the LIMPII TSP-binding domain (aa 75–155), or downstream fusion protein LFP156–243. Samples were incubated and bound TSP1 was measured as in A. Plots represent single data sets of triplicate samples. (n = 3, error as SD).

Mentions: We used solid-phase binding assays to assess direct binding of TSP1 to the HIV-1 envelope complex. Fig. 2 A shows concentration-dependent, saturable binding of radiolabeled soluble TSP1 to immobilized recombinant gp160. An apparent affinity of ∼250 nM was comparable to that demonstrated for binding of TSP1 to purified platelet CD36. Binding was effectively quenched in the presence of 10-fold molar excess unlabeled soluble TSP1 (Fig. 2 B, 94 ± 6% inhibition), demonstrating specificity. In addition, a LIMPII fusion protein LFP75–155 containing the TSP-binding domain partially blocked binding (47 ± 14% inhibition), whereas control fusion protein LFP156–243 representing downstream LIMPII sequences did not, supporting the existence of a functionally similar domain in HIV-1 env.


Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.

Crombie R, Silverstein RL, MacLow C, Pearce SF, Nachman RL, Laurence J - J. Exp. Med. (1998)

(A) Concentration-dependent binding of 125I-TSP1 to gp160. Increasing concentrations of soluble 125I-labeled TSP1 (1 nM–1 μM) were added  to immobilized recombinant HIV-1 gp160 for 3 h at 22°C, and bound TSP1 was measured after extensive washing. Nonlinear curve fit was generated  with ExcelTM version 5.0. Apparent affinity was estimated from Scatchard analysis. (n = 2; error calculated as SD). (B) Competitive inhibition demonstrates specificity of the TSP1–HIV interaction. A fixed concentration of 125I-labeled TSP1 (50 nM) was added to immobilized rgp160 in the absence or  presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, fusion protein LFP75–155 containing the LIMPII TSP-binding domain (aa 75–155), or  downstream fusion protein LFP156–243. Samples were incubated and bound TSP1 was measured as in A. Plots represent single data sets of triplicate  samples. (n = 3, error as SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199189&req=5

Figure 2: (A) Concentration-dependent binding of 125I-TSP1 to gp160. Increasing concentrations of soluble 125I-labeled TSP1 (1 nM–1 μM) were added to immobilized recombinant HIV-1 gp160 for 3 h at 22°C, and bound TSP1 was measured after extensive washing. Nonlinear curve fit was generated with ExcelTM version 5.0. Apparent affinity was estimated from Scatchard analysis. (n = 2; error calculated as SD). (B) Competitive inhibition demonstrates specificity of the TSP1–HIV interaction. A fixed concentration of 125I-labeled TSP1 (50 nM) was added to immobilized rgp160 in the absence or presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, fusion protein LFP75–155 containing the LIMPII TSP-binding domain (aa 75–155), or downstream fusion protein LFP156–243. Samples were incubated and bound TSP1 was measured as in A. Plots represent single data sets of triplicate samples. (n = 3, error as SD).
Mentions: We used solid-phase binding assays to assess direct binding of TSP1 to the HIV-1 envelope complex. Fig. 2 A shows concentration-dependent, saturable binding of radiolabeled soluble TSP1 to immobilized recombinant gp160. An apparent affinity of ∼250 nM was comparable to that demonstrated for binding of TSP1 to purified platelet CD36. Binding was effectively quenched in the presence of 10-fold molar excess unlabeled soluble TSP1 (Fig. 2 B, 94 ± 6% inhibition), demonstrating specificity. In addition, a LIMPII fusion protein LFP75–155 containing the TSP-binding domain partially blocked binding (47 ± 14% inhibition), whereas control fusion protein LFP156–243 representing downstream LIMPII sequences did not, supporting the existence of a functionally similar domain in HIV-1 env.

Bottom Line: In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines.Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity.Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The New York Hospital-Cornell Medical Center, New York 10021, USA.

ABSTRACT
Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

Show MeSH
Related in: MedlinePlus