Glycosaminoglycans and protein disulfide isomerase-mediated reduction of HIV Env

Conformational changes within the human immunodeficiency virus-1 (HIV-1) surface glycoprotein gp120 result from binding to the lymphocyte surface receptors and trigger gp41-mediated virus/cell membrane fusion. The triggering of fusion requires cleavage of two of the nine disulfide bonds of gp120 by a cell-surface protein disulfide-isomerase (PDI). Soluble glycosaminoglycans such as heparin and heparan sulfate bind gp120 via V3 and, possibly, a CD4-induced domain. They exert anti-HIV activity by interfering with the HIV envelope glycoprotein ( Env)/cell-surface interaction. Env also binds cell-surface glycosaminoglycans. Here, using surface plasmon resonance, we observed an inverse relationship between heparin binding by gp120 and its thiol content. In vitro, and in conditions in which gp120 could bind CD4, heparin and heparan sulfate reduced PDI-mediated gp120 reduction by approximately 80%. Interaction of Env with the surface of lymphocytes treated using sodium chlorate, an inhibitor of glycosaminoglycan synthesis, led to gp120 reduction. We conclude that besides their capacity to block Env/cell interaction, soluble glycosaminoglycans can effect anti-HIV activity via interference with PDI- mediated gp120 reduction. In contrast, their presence at the cell surface is dispensable for Env reduction during the course of interaction with the lymphocyte surface. This work suggests that the reduction of exofacial proteins in various diseases can be inhibited by compounds targeting the substrates ( not by targeting PDI, as is usually done), and that glycosaminoglycans that primarily protect proteins by preserving them from proteolysis also have a role in preventing reduction.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.