Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats.

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[(3)H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.

Pharmacokinetics of collagen dipeptides (Gly-Pro and Pro-Hyp) and tripeptides (Gly-Pro-Hyp) in rats.

Although systemic exposure to peptides, such as Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro, has been reported following administration of collagen hydrolysates from fish scale and porcine skin in vivo, the individual peptide pharmacokinetics remain unknown. We administered the three peptides individually to rats via the intravenous (5 mg/kg) and intragastric (100 mg/kg) routes and then monitored systemic exposure and urinary excretion. The peptides in biological samples were analyzed via liquid chromatography/tandem mass spectrometry. Gly-Pro-Hyp tended to exhibit higher first-pass metabolism than Pro-Hyp; the absolute oral bioavailabilities of Gly-Pro-Hyp and Pro-Hyp were 4.4% and 19.3%, respectively. Gly-Pro levels were very low in the systemic circulation. Pro-Hyp biotransformed from Gly-Pro-Hyp behaved similarly to Pro-Hyp alone when administered orally. Flip-flop kinetics (elimination rate ≫ absorption rate) were evident, probably reflecting transporter-mediated slow absorption. A double-peak phenomenon was observed for Gly-Pro-Hyp and Pro-Hyp when administered orally, and 5.9% ± 2.6% and 1.9% ± 0.3% of each dose were excreted in urine after intravenous administration, respectively. Urinary recovery of Gly-Pro was limited to 0.4% ± 0.5% of the intravenous dose. This work represents the first individual pharmacokinetics of Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro in vivo.

In Vitro and In Vivo Bone-Forming Effect of a Low-Molecular-Weight Collagen Peptide.

This study reveals that low-molecular-weight collagen peptide (LMWCP) can stimulate the differentiation and the mineralization of MC3T3-E1 cells in vitro and attenuate the bone remodeling process in ovariectomized (OVX) Sprague-Dawley rats in vivo. Moreover, the assessed LMWCP increased the activity of alkaline phosphatase (ALP), synthesis of collagen, and mineralization in MC3T3-E1 cells. Additionally, mRNA levels of bone metabolism-related factors such as the collagen type I alpha 1 chain, osteocalcin (OCN), osterix, bone sialoprotein, and the Runt family-associated transcription factor 2 were increased in cells treated with 1,000 µg/ml of LMWCP. Furthermore, we demonstrated that critical bone morphometric parameters exhibited significant differences between the LMWCP (400 mg/kg)-receiving and vehicle-treated rat groups. Moreover, the expression of type I collagen and the activity of ALP were found to be higher in both the femur and lumbar vertebrae of OVX rats treated with LMWCP. Finally, the administration of LMWCP managed to alleviate osteogenic parameters such as the ALP activity and the levels of the bone alkaline phosphatase, the OCN, and the procollagen type 1 N-terminal propeptide in OVX rats. Thus, our findings suggest that LMWCP is a promising candidate for the development of food-based prevention strategies against osteoporosis.

Oral Ingestion of AP Collagen Peptide Leads to Systemic Absorption of Gly-Pro-Hyp, Alleviating H2O2-Induced Dermal Fibroblast Aging.

Collagen-derived dipeptides and tripeptides have various physiological activities. In this study, we compared the plasma kinetics of free Hyp, peptide-derived Hyp, Pro-Hyp, cyclo(Pro-Hyp), Hyp-Gly, Gly-Pro-Hyp, and Gly-Pro-Ala after ingestion of four different collagen samples: AP collagen peptide (APCP), general collagen peptide, collagen, and APCP and γ-aminobutyric acid (GABA) combination. Each peptide was measured by high-performance liquid chromatography and triple quadrupole mass spectrometer. We found that, among all the peptides that were analyzed, only Gly-Pro-Hyp was significantly increased after ingestion of APCP compared with that of general collagen peptides and collagen. In addition, ingestion of the APCP and GABA combination improved the absorption efficiency of Gly-Pro-Ala. Finally, we reveal that Gly-Pro-Hyp was effective for preventing H2O2-induced reduction in extracellular matrix (ECM)-related genes, COL1A, elastin, and fibronectin, in dermal fibroblasts. Taken together, APCP significantly enhances the absorption of Gly-Pro-Hyp, which might act as an ECM-associated signaling factor in dermal fibroblasts, and the APCP and GABA combination promotes Gly-Pro-Ala absorption. Clinical Trial Registration number: UMIN000047972.

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses.

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active-site zinc ion. Since this water was not observed in the product (GPO)-complexed Ghcol, it was hypothesized that the GPOGPO-complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active-site geometry and site-directed mutagenesis of the active-site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

Complexation of fish skin gelatin with glutentin and its effect on the properties of wheat dough and bread.

This study aimed to investigate the effect of fish skin gelatin (Gadus morhua, 0.5%, or 1.0%, flour basis) on the properties of wheat (Triticum aestivum) dough and bread. Compared with the control group, the addition of 1.0% gelatin increased the storage modulus and the maximum resistance of dough, resulting in a longer rupture time and a larger final gas-retention volume of the dough. Bread characteristics showed that the specific loaf volume and crumb cell size both increased. Molecular dynamics simulation indicated that gelatin and glutenin segments formed a complex, where a large amount of hydroxyl groups on the surface retarded water mobility in bread. Gelatin-glutentin complexes with the high water-holding capacity inhibited water diffusion from marginal crumb to crust, and decreased starch retrogradation enthalpy and firming rate of crumb. Thus, fish skin gelatin might be a good improver of wheat dough and bread.

Oral Ingestion of Collagen Hydrolysate Leads to the Transportation of Highly Concentrated Gly-Pro-Hyp and Its Hydrolyzed Form of Pro-Hyp into the Bloodstream and Skin.

Collagen hydrolysate is a well-known dietary supplement for the treatment of skin aging; however, its mode of action remains unknown. Previous studies have shown that the oral ingestion of collagen hydrolysate leads to elevated levels of collagen-derived peptides in the blood, but whether these peptides reach the skin remains unclear. Here, we analyzed the plasma concentration of collagen-derived peptides after ingestion of high tripeptide containing collagen hydrolysate in humans. We identified 17 types of collagen-derived peptides transiently, with a particular enrichment in Gly-Pro-Hyp. This was also observed using an in vivo mouse model in the plasma and skin, albeit with a higher enrichment of Pro-Hyp in the skin. Interestingly, this Pro-Hyp enrichment in the skin was derived from Gly-Pro-Hyp hydrolysis, as the administration of pure Gly-Pro-Hyp peptide led to similar results. Therefore, we propose that functional peptides can be transferred to the skin by dietary supplements of collagen.

Orally Available Collagen Tripeptide: Enzymatic Stability, Intestinal Permeability, and Absorption of Gly-Pro-Hyp and Pro-Hyp.

Collagen-derived small peptides, such as Gly-Pro-Hyp (GPH) and Pro-Hyp (PH), play a role in various physiological functions. Although collagen degrades in the gastrointestinal tract randomly and easily, it is not readily cleaved into bioactive peptides. To increase the bioavailability of bioactive peptides, a collagen tripeptide (CTP) was prepared from fish scales by the digestion method using collagenase from nonpathogenic Bacillus bacteria. It was demonstrated that Hyp-containing peptides-GPH and PH-were better absorbed and reached higher plasma levels after the oral administration of CTPs in rats compared to high molecular weight collagen peptide (H-CP). GPH and PH were stable in gastrointestinal fluid and rat plasma for 2 h, and GPH was able to be transported across the intestinal cell monolayer. These results suggest that the ingestion of CTP is an efficient method for taking bioactive peptides orally due to the enzymatic stability and intestinal permeability of GPH and PH.