Abstract
Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K(i app)) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans.
Highlights
The plasma kallikrein-kinin system is a paradigm of a tightly controlled pro-inflammatory proteolytic cascade activated by vascular injury [1]
Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS)
Heparan Sulfate Modulates the Endopeptidase Activity of Cruzipain—Because the catalytic efficiency of some papainlike proteases is modulated by GAGs [25,26,27], HS may likewise alter the kinetic properties of cruzipain, a member of the C1 peptidase family [32], affecting its ability to function as a kininogenase [21]
Summary
Purified Proteases, Kininogen, and GAG—Natural cruzipain (n-cruzipain) was isolated from crude aqueous extracts of Dm28c epimastigotes as described [28]. McKerrow from the University of California, San Francisco), designated as r-cruzipain 1, was expressed in Escherichia coli [24]; r-cruzipain 2 (80% sequence similarity with r-cruzipain 1) was recombinantly expressed in Saccharomyces cerevisiae and purified as described elsewhere [29] These recombinant proteases differ from their natural enzymes by: (i) having a truncated C terminus where residues 216 –346 are deleted; (ii) glycosylation in yeast (r-cruzipain 2); and (iii) lack of glycosylation in E. coli (r-cruzipain 1). The measurements were performed at 37 °C in the same buffer, and the kinetic parameters were determined by measuring the initial rate of hydrolysis at various substrate concentrations in the presence or absence of different concentrations of sulfated GAGs
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