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Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum , which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic p K a (p K e1), while no residue was assigned to contribute alkaline p K a (p K e2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[ N 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for p K e2. [ABSTRACT FROM AUTHOR]

The collagenase produced by a gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently than that produced by a gram-positive bacterium Clostridium histolyticum (Chcol), which is currently the most widely used collagenase in industry [Teramura et al. (Cloning of a novel collagenase gene from the gram-negative bacterium Grimotia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol 2011;193:3049–3056)]. Here, we compared the Ghcol and Chcol activities using two synthetic substrates. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[ N 3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropioyl]-L-Ala-L-Arg-NH2, Ghcol exhibited 350-fold higher activity than Chcol in the absence of CaCl2 and NaCl. The Ghcol activity markedly decreased with increasing concentrations of buffer, CaCl2 or NaCl, while the Chcol activity did not, suggesting that the Ghcol activity was sensitive to solvent components. In the hydrolysis of N -[3-(2-furyl)acryloyl]-L-Leu-Gly-L-Pro-Ala, Ghcol exhibited 16-fold higher activity than Chcol in the absence of CaCl2 and NaCl, and both enzyme activities did not decrease with increasing concentrations of buffer, CaCl2 or NaCl. pH dependences of activity revealed that the ionizable group responsible for acidic p K e may be Glu for Ghcol and Chcol, while that for alkaline p K e may be His for Ghcol and Tyr for Chcol. These striking differences suggest that the catalytic mechanism of Ghcol might be considerably different from that of clostridial collagenases. [ABSTRACT FROM AUTHOR]

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P2position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC50values of 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes. Production of DPP-IV inhibitory tripeptides from gluten using ginger protease. [ABSTRACT FROM PUBLISHER]

Flavobacterium psychrophilum(F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayuPlecoglossus altivelis. Formalin-killed cell ofF. psychrophilumhas long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility ofF. psychrophilumcollagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somaticF. psychrophilumand fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed inEscherichia coliandBrevibacillus chosinensis(B. chosinensis). The culture supernatant of theB. chosinensiswas used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking intoF. psychrophilumculture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD. [ABSTRACT FROM PUBLISHER]

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH2was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH2(13−16) were readily identified, pQLSK(MOCAc)GY (1−6) and DEKSTGISK(Dnp)P-NH2(7−16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not. Pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. The former cleavage requires native collagen structure, while the latter cleavage does not. [ABSTRACT FROM PUBLISHER]

The collagenase activity and thefpcolgene were examined inFlavobacterium psychrophilumisolates from cold-water disease (CWD)-affected ayu,Plecoglossus altivelis. Collagenase expression was closely related to the accumulated mortality of CWD-affected ayu. RT-qPCR and bacterial challenge experiments showed thatF. psychrophilumayu isolate WA-1 expressed thefpcolgene more actively and was more virulent than ayu isolate WA-2. The amago (Oncorhynchus masou) isolate WB-1, which possesses a pseudo-fpcolgene, was not harmful to ayu. Hitherto, the well-studied metalloproteases Fpp1 and Fpp2 have been considered virulence factors. However, the most virulent isolate against ayu (WA-1) showed no Fpp activity because of a deletion mutation or an insertion of a transposon in thefppgenes. The less virulent WA-2 isolate showed only Fpp1 activity. Taken together, these results suggest that collagenolytic activity, but not Fpp activity, is related to the virulence ofF. psychrophilumisolates in CWD-affected ayu. Comparative study ofFlavobacterium psychrophilumisolates from ayu and amago suggests that the collagenase is one of the pathogenic factors for ayu. [ABSTRACT FROM PUBLISHER]

The article discusses the results of a study which examined the improvement in the physical and biochemical characteristics of a pepsin-solubilized collagen (PSC) membrane by conjugating with soluble egg shell membrane protein (SEP). The conjugated membrane was found to have thicker collagen fibrils and higher stability than PSC membrane alone. An SEP-PSC coating accelerated the growth of fibroblasts. However, the cell growth of keratinocytes did not change.

When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas µ-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, α-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel. [ABSTRACT FROM PUBLISHER]

Investigates the effects of (-)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts. Interaction between EGCG and platelet-derived growth factor; Affinity chromatography; Enzyme-linked immunoassay; Mechanism of inhibition of collagen gel contraction by EGCG.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1(MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37°C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30°C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation. [ABSTRACT FROM AUTHOR]