Masashi Kusubata

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen

Background: The physiological role of 3-hydroxyproline (3-Hyp) rarely found in collagen is unclear. Results: Significant increases in 3-Hyp were observed at specific sites in tendon type I collagen at early ages. Conclusion: The tendon-specific alterations in the 3-Hyp level were correlated with tissue development, rather than aging. Significance: Prolyl 3-hydroxylation is suggested to contribute to fibril diameter regulation in tendon collagen. 3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5–18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro707 and newly identified sites at Pro716 and Pro719, at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease

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 P2 position 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 IC50 values 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.

Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

Collagen-derived peptides modulate CD4+ T-cell differentiation and suppress allergic responses in mice: Suppression of allergic responses by collagen peptides

Collagen peptides have been widely used as a food supplement. After ingestion of collagen peptides, oligopeptides containing hydroxyproline (Hyp), which are known to have some physiological activities, are detected in peripheral blood. However, the effects of collagen‐peptide administration on immune response are unclear. In the present study, we tested the effects of collagen‐peptide ingestion on allergic response and the effects of collagen‐derived oligopeptides on CD4+ T‐cell differentiation.

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation

Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 μM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.