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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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Immunohistochemical detection of cell-associated TSP1 in  human gingival mucosa. Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSP1 and 2 (lower  panel), or with preimmune serum (upper panel), followed by biotinylated  second antibody, and developed using avidin-conjugated peroxidase.  Brown deposits indicate sites of thrombospondin reactivity. Original  magnification, ×200.
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Figure 6: Immunohistochemical detection of cell-associated TSP1 in human gingival mucosa. Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSP1 and 2 (lower panel), or with preimmune serum (upper panel), followed by biotinylated second antibody, and developed using avidin-conjugated peroxidase. Brown deposits indicate sites of thrombospondin reactivity. Original magnification, ×200.

Mentions: As shown in Table 1, levels of TSP1 in whole saliva from either HIV+ or HIV− donors were at least 10-fold greater (1–12 μg/ml) compared to plasma. Amounts of TSP1 in parotid saliva fractions were equivalent to that of plasma, with the bulk of TSP1 found in the submandibular secretions. These values are consistent with previous reports of anti-HIV activity predominantly in submandibular and not parotid gland fluids (11, 19) although only a single saliva fractionation was performed. To document that elevated levels of TSP1 in saliva may be secondary to local production, rather than leakage and concentration from plasma, we performed immunohistochemical staining of fixed tissue from oral mucosa. Moderate to intense levels of TSP1-directed immunoreactivity were evident on gingival epithelium (Fig. 6), confirming that cell-associated TSP1 may correspond to significantly high local concentrations in the oral cavity.


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)

Immunohistochemical detection of cell-associated TSP1 in  human gingival mucosa. Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSP1 and 2 (lower  panel), or with preimmune serum (upper panel), followed by biotinylated  second antibody, and developed using avidin-conjugated peroxidase.  Brown deposits indicate sites of thrombospondin reactivity. Original  magnification, ×200.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199189&req=5

Figure 6: Immunohistochemical detection of cell-associated TSP1 in human gingival mucosa. Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSP1 and 2 (lower panel), or with preimmune serum (upper panel), followed by biotinylated second antibody, and developed using avidin-conjugated peroxidase. Brown deposits indicate sites of thrombospondin reactivity. Original magnification, ×200.
Mentions: As shown in Table 1, levels of TSP1 in whole saliva from either HIV+ or HIV− donors were at least 10-fold greater (1–12 μg/ml) compared to plasma. Amounts of TSP1 in parotid saliva fractions were equivalent to that of plasma, with the bulk of TSP1 found in the submandibular secretions. These values are consistent with previous reports of anti-HIV activity predominantly in submandibular and not parotid gland fluids (11, 19) although only a single saliva fractionation was performed. To document that elevated levels of TSP1 in saliva may be secondary to local production, rather than leakage and concentration from plasma, we performed immunohistochemical staining of fixed tissue from oral mucosa. Moderate to intense levels of TSP1-directed immunoreactivity were evident on gingival epithelium (Fig. 6), confirming that cell-associated TSP1 may correspond to significantly high local concentrations in the oral cavity.

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