Dataset Information

The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

ABSTRACT: Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

SUBMITTER: Chahal G

PROVIDER: S-EPMC4482499 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

Chahal Gurdeep G Thorpe Michael M Hellman Lars L

PloS one 20150625 6

Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of nega ...[more]

PMID: 26110612

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Project description:BACKGROUND: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB). RESULTS: In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I29?>?T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2. CONCLUSIONS: Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.

Dataset Information

The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

Publications

The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

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