Interactions of human lacrimal and salivary cystatins with adenovirus endopeptidase

Over 100 serotypes of adenoviruses have been implicated in a variety of hum an and domesticated animal pathologies and some serotypes are widely used a s gene transfer vectors. Aside from the limited use of vaccines for specifi c serotypes, little effort has been expended in the development of antivira ls. The objective here was to study the effect of cystatins from human sali va (CS) and tears (CT), two points of viral entry, on adenain, the adenovir us type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both flu ids 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 CSA 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 con sensus sites specific to adenain in the human cystatins could drive the ade nain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denatura tion and by highly active fresh enzyme preparations. Our experiments design ed to test the nature of the interaction between adenain and cystatins sugg est 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 saliv a are unlikely to play a significant role in inhibiting adenovirus infectio ns. (C) 2001 Elsevier Science B.V. All rights reserved.