Maturation Processing and Characterization of Streptopain

Abstract

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis-and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn 356 might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10-and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.

Streptococcal Mitogenic Exotoxin, SmeZ, Is the Most Susceptible M1T1 Streptococcal Superantigen to Degradation by the Streptococcal Cysteine Protease, SpeB

“…The consensus target for cleavage by SpeB has been suggested to contain a hydrophobic residue (isoleucine or valine) at the P 2 site and a positively charged residue (preferably lysine) at P 1 (23,24). Bioinformatics analysis of the primary .…”

Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis

“…The expression and purification of recombinant SPE B (rSPE B) have been previously described (5). The gene encoding ProSPE B was amplified using a PCR with six histidine tags and a BamH1 recognition site.…”

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

“…12 The RGD motif is located at the apex of a long loop, between two b strands of the protein, protruding 10-17 Å from the protein core. [6][7][8][9][10][11][12] The flexibility and solvent exposure of the RGD loop are responsible for the recognition and fitting the interface between integrin a and b subunits. Studies 35,37,38 also show that the dynamic properties of the RGD motif may be important in its interaction with integrins.…”

“…The synergy between structure and dynamics is essential to the function of integrin complexes. [6][7][8][9][10][11][12] Many studies have compared the binding affinity of RGD-containing peptide with their 3D conformations and backbone dynamics. 27,36,38,43 As shown in Table IV, the residues of the RGD loop of these proteins exhibited extensive flexibility on fast motion on the picasecond per nanosecond time scale.…”

“…[3][4][5] Many toxins are obtained from a variety of species, such as snakes, ticks, and leeches, and contain an arginylglycyl-aspartic acid (RGD) motif with different protein scaffolds. [6][7][8][9][10][11] They specifically inhibit the integrin-binding function and are potent integrin antagonists. Many of these proteins have potential as therapeutic agents that can directly target integrins.…”