Z-Gly-Pro-pNA

Two isoforms, Bmpop-a and Bmpop-b, were cloned and characterized, which were found to encode prolyl oligopeptidase (Pop) of the domestic silkworm Bombyx mori. The full lengths of Bmpop-a and Bmpop-b were 2497 and 2508 bp, deducing 707 and 740 amino acids, respectively. Both of them, possessing the typical characteristics of the Pop family of serine proteinase, were detected to be expressed among different tissues and development stages at the transcription and translation levels. Soluble recombinant BmPop-a (rBmPop-a) had oligopeptidase activity toward the substrates, Z-Gly-Pro-pNA, Z-Gly-Pro-AMC and angiotensin I. An inhibition assay showed that the activity of rBmPop-a was significantly inhibited by KYP-2047 and S17092 in vitro. BmPop-b was identified in the molting fluids at three different stages by Western blotting analysis, showing a predominant expression in the integument. Two isoforms of Bmpop gene and other three genes in the renin-angiotensin system (RAS) in the integument were down-regulated by starvation treatments but up-regulated by refeeding. These results suggested that BmPops may play an important role in balancing the molting fluid pressure to guarantee ecdysis normally. This study provides clues for further elucidating the function and regulation mechanisms of two isoforms of Bmpop gene.

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.