Interactions of human lacrimal and salivary cystatins with adenovirus endopeptidase.
Over 100 serotypes of adenoviruses have been implicated in a variety of human and domesticated animal pathologies and some serotypes are widely used as gene transfer vectors. Aside from the limited use of vaccines for specific serotypes, little effort has been expended in the development of antivirals. The objective here was to study the effect of cystatins from human saliva (CS) and tears (CT), two points of viral entry, on adenain, the adenovirus type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both fluids at a yield of 5 mg/l. In vitro adenain activity was inhibited to 50% at a molar ratio of 5 CS:1 adenain and 3 CT:1 adenain. By comparison, papain was inhibited to 50% at a molar ratio of 2 CS:1 papain and 1.5 CT:1 papain. Adenain differed from papain in response to CS and chicken egg white (CEW) cystatin in being stimulated at low concentrations, and in being inhibited only at very high concentrations of cystatins. The presence of cleavage consensus sites specific to adenain in the human cystatins could drive the adenain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denaturation and by highly active fresh enzyme preparations. Our experiments designed to test the nature of the interaction between adenain and cystatins suggest a docking model for the adenain-human cystatin interaction, similar to that proposed for papain and CEW. At equilibrium the dissociation constant, K(d), between adenain and CT was 1.2 nM. The kinetic parameters determined here suggest a simple reversible mechanism for the inhibition of adenain by human cystatins. We conclude that the cystatins present in tears and saliva are unlikely to play a significant role in inhibiting adenovirus infections.
Ruzindana-Umunyana
A
A
Departement de Microbiologie et d'Infectiologie, Faculte de Medecine, Universite de Sherbrooke, Quebec, J1H 5N4, Sherbrooke, Canada.
Weber
J M
JM
D000260:Adenoviruses, Human / Q000737:chemistry; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry; D012463:Saliva / Q000737:chemistry*; D013379:Substrate Specificity; D013666:Tears / Q000737:chemistry*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010206:Papain