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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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Triple helix formation of procollagen occurs in the endoplasmic reticulum (ER) where the single-stranded α-chains of procollagen undergo extensive post-translational modifications. The modifications include prolyl 4- and 3-hydroxylations, lysyl hydroxylation, and following glycosylations. The modifications, especially prolyl 4-hydroxylation, enhance the thermal stability of the procollagen triple helix. Procollagen molecules are transported to the Golgi and secreted from the cell, after the triple helix is formed in the ER. In this study, we investigated the relationship between the thermal stability of the collagen triple helix and environmental temperature. We analyzed the number of collagen post-translational modifications and thermal melting temperature and α-chain composition of secreted type I collagen in zebrafish embryonic fibroblasts (ZF4) cultured at various temperatures (18, 23, 28, and 33 °C). The results revealed that thermal stability and other properties of collagen were almost constant when ZF4 cells were cultured below 28 °C. By contrast, at a higher temperature (33 °C), an increase in the number of post-translational modifications and a change in α-chain composition of type I collagen were observed; hence, the collagen acquired higher thermal stability. The results indicate that the thermal stability of collagen could be autonomously tuned according to the environmental temperature in poikilotherms.

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years., Competing Interests: The authors have declared that no competing interests exist.

In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47-/- mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.

Collagen is the most abundant protein in animals. Collagen hydrolysate has been found to have multiple functions in the skin, bones, joints, muscles, and blood vessels. Recently, it has been reported that the low molecular weight fraction of collagen hydrolysate exhibited anxiolytic activity, suggesting that collagen peptides affect brain functions. In the present study, we found that oral administration of ginger-degraded collagen hydrolysate (GDCH) significantly decreased depression-like behavior in a forced swim test, suggesting that GDCH exhibited antidepressant activity in mice. The antidepressant activity of GDCH was abolished by pre-treatment with an antagonist of the dopamine receptor, but not treatment with a serotonin receptor antagonist. GDCH significantly increased gene expression of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the hippocampus, molecules that affect the differentiation and survival of neurons, relative to that in the control condition. Meanwhile, there were no changes in the gene expression of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3, major factors related to depression-like behavior. We also found that GDCH exhibited antidepressant activity in corticosterone-administered mice in a model of stress. In addition, GDCH increased GDNF and CNTF expression in the stressed condition, suggesting that mechanisms of the antidepressant activity of GDCH were the same in unstressed and stressed conditions. These results imply that GDCH exhibits antidepressant activity in unstressed and stressed conditions in mice. The upregulation of neurotrophic genes in the hippocampus may contribute to the reduction of depression-like behavior via a dopamine signal pathway modulated by GDCH.

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 IC 50 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.

Introduction: 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., Methods: BALB/c mice fed a collagen-peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of collagen-derived oligopeptides, and the expression of IFN-γ, IL-4, and Foxp3 was analyzed., Results: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA-specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T-helper (Th) type 1 and regulatory T (Treg) cells. In vitro T-helper cell differentiation assays showed that Hyp-containing oligopeptides promoted Th1 differentiation by upregulating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3 + Treg cells in response to antigen stimulation in the presence of TGF-β., Conclusions: Collagen-peptide ingestion suppresses allergic responses by skewing the balance of CD4 + T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

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.

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys 933 , a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Medical-grade collagen peptide is used as an additive agent in pharmaceutical formulations; however, it is unknown as to whether the compound exerts antioxidant effects in vitro. In this study, we evaluated the antioxidant effects of medical-grade collagen peptide on reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen using electron spin resonance and spin trapping. We confirmed that medical-grade collagen peptide directly inhibited hydroxyl radical generated by the Fenton reaction or by ultraviolet irradiation of hydrogen peroxide, and singlet oxygen. In addition, an antioxidant effect of medical-grade collagen peptide on singlet oxygen was observed in peptide fractions 12-22. The total amount of antioxidant amino acids (Gly, Hyp, Glu, Ala, Cys, Met and His) constituted more than half of the total amino acids in these fractions. These results suggest that the observed antioxidant properties of medical-grade collagen peptide are due to the compound containing antioxidant amino acids. Medical-grade collagen peptide, which is used in pharmaceuticals, and especially in injectables, could provide useful antioxidant properties to protect the active ingredient from oxidation.

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting., (© 2013 Wiley Periodicals, Inc.)

Collagens are the most abundant proteins in animals and are involved in many physiological/pathological events. Although various methods have been used to quantify collagen and its post-translational modifications (PTMs) over the years, it is still difficult to accurately quantify type-specific collagen and minor collagen PTMs. We report a novel quantitative method targeting collagen using stable isotope-labeled collagen named "SI-collagen", which was labeled with isotopically heavy lysine, arginine, and proline in fibroblasts culture. We prepared highly labeled and purified SI-collagen for use as an internal standard in mass spectrometric analysis, particularly for a new approach using amino acid hydrolysis. Our method enabled accurate collagen analyses, including quantification of (1) type-specific collagen (types I and III in this paper), (2) total collagen, and (3) collagen PTMs by LC-MS with high sensitivity. SI-collagen is also applicable to other diverse analyses of collagen and can be a powerful tool for various studies, such as detailed investigation of collagen-related disorders.

Bone allografts are commonly used for the repair of critical-size bone defects. However, the loss of cellular activity in processed grafts markedly reduces their healing potential compared with autografts. To overcome this obstacle, we developed a healing system for critical-size bone defects that consists of overlaying an implanted bone graft with a collagen sheet (CS) loaded with basic fibroblast growth factor (bFGF) fused to the collagen-binding domain derived from a Clostridium histolyticum collagenase (CB-bFGF). In a murine femoral defect model, defect sites treated with CS/CB-bFGF had a significantly larger callus volume than those treated with CS/native bFGF. In addition, treatment with CS/CB-bFGF resulted in the rapid formation of a hard callus bridge and a larger total callus volume at the host-graft junction than treatment with CS/bFGF. Our results suggest that the combined use of CS and CB-bFGF helps accelerate the union of allogenic bone grafts.

Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting., (Copyright © 2013 The Authors. Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.)

Collagen sheets were used in a unique evaluation method to examine skin damage caused by ultraviolet (UV) light of short wavelength during a season of the Antarctic ozone hole. The collagen sheets were exposed outdoors for 25 and 50 d, in the spring when the ozone hole was formed and in the ozone-hole-free autumn. Extracts from the exposed collagen sheets were analyzed for total protein and terminal amino acid concentrations as an index of collagen fragmentation. The results show that the amount of extractable collagen and terminal amino acid concentration in the spring exposure were approximately double and five times higher, respectively, when compared with those in the autumn exposure. During the ozone hole occurrence, the terminal amino acid concentration of the extracted collagen was about five times higher when exposure lasted 50 d from mid-September to the end of October compared to when exposure lasted 25 d from mid-September to early October. This result could be attributed to a limited amount of short-wavelength UV radiation reaching the ground surface as a result of the low height of the sun in September, when the ozone hole occurred. In fact, UV radiation measurements taken at Syowa Station indicate that short-wavelength UV radiation in the range 290-295 nm was not detected until approximately 1-2 months after the beginning of the ozone hole occurrence.

A coiled-coil endoplasmic reticulum (ER) protein, p180, was originally reported as a ribosome-binding receptor on the rough ER and is highly expressed in secretory tissues. Recently, we reported new functions of p180 as a microtubule-bundling protein on the ER. Here, we investigated the specific roles of p180 in the Golgi complex organization following stimulated collagen secretion. Targeted depletion of p180 by siRNA transfection caused marked reduction of TGN, while other marker levels for the cis or medial Golgi were not markedly changed. Ascorbate stimulation resulted in trans-Golgi network (TGN) expansion to the periphery in control cells that is characterized by both increased membrane amounts and extended shape. In contrast, loss of p180 resulted in retraction of the TGN regardless of ascorbate stimulation. The TGN developed to the periphery along stabilized microtubule bundles, and overexpression of MTB-1 fragment caused dominant-negative phenotypes. Once disorganized, the retracted TGN did not recover in the absence of p180 despite elevated acetylated tubulin levels. TGN46 and p180 were co-distributed in epithelial basal layer cells of human mucosal and gastrointestinal tissues. Taken together, we propose a novel function of p180-abundant ER on the TGN expansion, both of which are highly developed in various professional secretory cells., (Copyright 2009 Elsevier Inc. All rights reserved.)

Elucidating the mechanisms and factors regulating multimerization is biologically important in order to modulate the biological activities of functional proteins, especially adhesive proteins in the extracellular matrix (ECM). Vitronectin (VN) is a multifunctional glycoprotein present in plasma and ECM. Linkage of cellular adhesion and fibrinolysis by VN plays an essential role during tissue remodeling. Our previous study determined that the collagen-binding activity of VN was markedly enhanced with the decreased glycosylation during liver regeneration. This study demonstrated how alternations of glycans modulate the biological activity of VN. Human and rat VNs were used because of their similarities in structure and activities. The binding affinity of human VN to immobilized collagen was shown to be higher at pH 4.5 than at 7.5, at 37 degrees C than at 4 degrees C. Sedimentation velocity studies indicated that the greater the multimerization of human VN, the better it bound to collagen. The results indicate that the collagen binding of VN was modulated through its multimerization. Stepwise trimming of glycan with various exoglycosidases increased both the multimer size and the collagen binding of human VN, indicating that they are modulated by changes in glycosylation. The multimer sizes of VN purified from plasma of partially hepatectomized (PH) rats and sham-operated (SH) rats increased by about 45 and 31%, respectively, compared with those of nonoperated (NO) rats. In accordance with this, PH-VN exhibited remarkably enhanced collagen binding than SH-VN and NO-VN on surface plasmon resonance. In the PH rat sera, the multimer VN was increased in both amount and size compared with those in SH- and NO-sera. The results demonstrate that glycan alterations during tissue remodeling induce increased multimerization state to enhance the biological activity of VN.

Soluble egg shell membrane protein (SEP) prepared from the egg shell membrane by performic acid oxidation and pepsin digestion was applied to prepare a membrane conjugated with pepsin-solubilized collagen (PSC) by cross-linking with water-soluble carbodiimide. SEP enabled a conjugated membrane to be constructed with a well-developed network structure having thicker collagen fibrils and higher stability than the PSC membrane alone, as indicated by reduced solubility and collagenase-digestion, increased denaturation temperature, and superior flexibility. The SEP-PSC membrane (SEP:PSC of 1:1, w/w) had lower sweatiness than the PSC membrane. Fibroblasts and keratinocytes adhered to the SEP-PSC-coated wells (SEP:PSC of 1:10, w/w) as a fundamental model of an artificial skin similar to that to the PSC-coated wells due to similar expression level of several cell-adhesion proteins. An SEP-PSC coating accelerated the cell growth of fibroblasts, whereas the cell growth of keratinocytes did not change, or decreased slightly due to more rapid differentiation indicated by a more substantial expression of differentiation marker proteins than a PSC-coating without SEP.

Purpose: To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs., Methods: Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations., Results: Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells., Conclusions: The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet., (Copyright Association for Research in Vision and Ophthalmology)

The effects of (-)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts were investigated. EGCG inhibited collagen gel contraction dose-dependently. On the basis of the fact that platelet-derived growth factor (PDGF) is one of the serum components with stimulatory activity in collagen gel contraction, we examined the possibility that interaction between EGCG and PDGF may be involved in this inhibition mechanism. We confirmed this by recombinant PDGF-BB in the present system and we found that EGCG inhibited PDGF-stimulated collagen gel contraction. The results of affinity chromatography indicated that PDGF was bound by EGCG immobilized on agarose gel as detected by enzyme-linked immunoassay and Western blotting. These findings suggest that binding of EGCG to PDGF is at least partly involved in the mechanism of inhibition of collagen gel contraction by EGCG.

Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25 degrees C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen alpha chain. The purified enzymes degraded squid collagen at 25 degrees C, which is 2.5 degrees lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20 degrees C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2-3 degrees C below the denaturation temperature.

Background: Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain., Objective: To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children., Methods: Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE-reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients., Results: We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain., Conclusions: We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis to gelatin in vaccines might be reduced by digestion of this IgE-binding site in gelatin.

Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.

Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) plays an important role in tissue repair, cell proliferation, and differentiation. Our previous study showed that collagen I and collagen V differently regulate the proliferation of rat pancreatic β cells (INS-1 cells) through opposite influences on the nuclear translocation of β-catenin. In this study, we investigated the β-catenin pathway in INS-1 cells on dishes coated with collagen I or V. We found that nuclear translocation of the transcription factor Yes-associated protein (YAP) was enhanced by collagen I and suppressed by collagen V, but had no effect on INS-1 cell proliferation. Morphologically, INS-1 cells on collagen V-coated dishes showed stronger cell-to-cell adhesion, while the cells on collagen I-coated dishes showed weaker cell-to-cell adhesion in comparison with the cells on non-coated dishes. E-cadherin played an inhibitory role in the proliferation of INS-1 cells cultured on collagen I or collagen V coated dishes via regulation of the nuclear translocation of β-catenin. Integrin β1 was enhanced with collagen I, while it was repressed with collagen V. The integrin β1 pathway positively regulated the cell proliferation. Inhibition of integrin β1 pathway restored the protein level of E-cadherin and inhibited the nuclear translocation of β-catenin in the cells on collagen I-coated dishes, but no effect was observed in the cells on collagen V-coated dishes. In conclusion, collagen I enhances the proliferation of INS-1 cells via the integrin β1 and E-cadherin/β-catenin signaling pathway. In INS-1 cells on collagen V-coated dishes, both integrin β1 and E-cadherin/β-catenin signal pathways are involved in the inhibition of proliferation. [ABSTRACT FROM AUTHOR]

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]

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3‐L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3‐L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3‐L1 cell migration via stimulating the nuclear translocation of yes‐associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high‐fat diet fed mice. Increased expression of YAP in the cells cultured on col I‐coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small‐interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic β-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of β-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of β-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of β-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of β-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear β-catenin could be important for understanding fibrosis where collagen I and V may have differential effects. [ABSTRACT FROM AUTHOR]

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin. [ABSTRACT FROM AUTHOR]

The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C‐terminal segment that includes the bacterial pre‐peptidase C‐terminal domain. Here, we produced a recombinant C‐terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen‐binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C‐terminal segment binds to collagen only when the collagen is in its triple‐helical conformation. Moreover, the C‐terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C‐terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate‐labeled type I collagen to show that the C‐terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) participate in various cell responses in association with cell proliferation, migration, differentiation, and death. Extracellular matrix (ECM) serves as cellular microenvironments for many kinds of cells, affecting cell activities. However, whether or not ECM influences cellular ROS levels has not been well studied. In this study, cells are cultured on collagen I molecule-coated (mol. coated) dishes and collagen I fibrous gel-covered (gel) dishes to explore their influence on cell behaviours. We found that the levels of ROS in murine 3T3-L1 preadipocytes increased both in cells on mol. coated and those on the gel. Much higher ROS levels were found in the cells cultured on the gel. Cell proliferation and migration were stimulated to opposite directions between the cells on mol. coated and the cells on gel. ROS in a moderate level were positive regulators in the proliferation and migration of cells on mol. coated; however, ROS in a high level served as negative regulators in the cells on gel. These opposite effects on cell proliferation and migration affected by different ROS levels are in parallel with opposite levels of NF-κB p65 activation. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: 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. Methods: BALB/c mice fed a collagen‐peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4+ T cells were stimulated with anti‐CD3 and anti‐CD28 in the presence of collagen‐derived oligopeptides, and the expression of IFN‐γ, IL‐4, and Foxp3 was analyzed. Results: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA‐specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T‐helper (Th) type 1 and regulatory T (Treg) cells. In vitro T‐helper cell differentiation assays showed that Hyp‐containing oligopeptides promoted Th1 differentiation by upregulating IFN‐γ‐induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3+ Treg cells in response to antigen stimulation in the presence of TGF‐β. Conclusions: Collagen‐peptide ingestion suppresses allergic responses by skewing the balance of CD4+ T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases. [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]

Purpose:Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel.Material and methods:We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy.Results:L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed.Conclusion:NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death. [ABSTRACT FROM AUTHOR]

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]

Bovine dermis was exposed outdoors in the Antarctic in 2013 to study the skin damage caused by short-wavelength ultraviolet light under the ozone hole. Collagen was extracted from the exposed dermis with pepsin. The amount of solubilized collagen in the exposed dermis was only 20%–40% of that in dermis shielded from ultraviolet light. The dermis was most difficult to extract when exposed in summer, and then when exposed in spring. Differential scanning calorimetry was used to determine the melting endothermic behavior of the dermal tissue. The peak temperature was highest for the dermis exposed in summer. The exposed dermis was degraded with cyanogen bromide to determine whether cross-linking had occurred. Cross-linked peptides were detected in the dermis exposed in summer or spring, but the dermis exposed in autumn did not differ markedly from the light-shielded dermis. These data show that cross-linkages were readily formed in the collagen molecule chains in dermis exposed to ultraviolet light in summer, when solar elevation is highest and the period of sunshine is longest. A comparison of the dermis exposed in spring and that exposed in autumn showed that cross-linkages were formed more readily by ultraviolet light in spring, when the ozone hole occurred. [ABSTRACT FROM AUTHOR]

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Here, we report the behavior of three kinds of human cancer cell lines (Caco-2, MCF-7, HT-1080) on type I collagen substrates, which are in two-dimensional coated collagen or three-dimensional fibrils form. All tested cells on coated collagen adhered and proliferated. However, in the case of collagen fibrils, the proliferation of cancer cells was suppressed. Furthermore, Akt activation, which is known as a cell-survival signal, was inhibited in cells on collagen fibrils. But the activation of ERK1//2 was not completely inhibited. In Caco-2 cells, delay of cell cycle progression and cell death occurred at the same time. Thus, cell division and cell death occurred at equivalent rates on the collagen fibrils, and cell growth seemed to be stopped. These results imply that the fibril form of collagen plays a potential role in inhibiting the growth of cancer cells. [ABSTRACT FROM AUTHOR]

We examined the behavior of human foreskin keratinocytes (HFKs) on reconstituted type IV collagen gel. HFKs survived for several days and the upper layer cells expressed a differentiation marker, involucrin. Apoptosis was induced after involucrin expression while cell proliferation was suppressed. On molecular type IV collagen, integrins shifted from α2β1 to α3β1 during HFK culture. On type IV collagen gel, HFKs initially expressed integrin α2β1, and later expressed integrin α3β1 in the presence of α2β1 did not disappear. Using synthetic peptides, we examined integrin α2-mediated adhesion to type IV collagen gel. Addition of synthetic peptide dose-dependently inhibited cell adhesion both on type IV collagen gel and on molecular type IV collagen. On type IV collagen gel, weaker phosphorylation of focal adhesion kinase, paxillin, and Akt was observed compared with the molecular forms. Based on these observations, we think type IV collagen gel is a novel culture substrate that mimics the physiological environment for HFKs. [ABSTRACT FROM AUTHOR]

Keratinocytes adhere and proliferate well on collagen-coated surfaces, but they undergo apoptosis without differentiation on collagen gels according to our past research. In the current studies, we investigated the necessary conditions for keratinocyte survival on fibrous collagen gels. We found that keratinocytes survived on collagen gels when the medium contains elevated levels (1.8 mM) of calcium. Under this high calcium condition, cells formed multicellular colonies and differentiated. Akt was not activated in cells cultured on collagen gels regardless of the calcium concentration, whereas it was activated in cells cultured on nonfibrous collagen. On the other hand, Erk1/2, key kinases of MAPK pathway, were phosphorylated in cells cultured under high calcium condition but not in cells cultured on collagen gels under low calcium condition. The necessity of Erk1/2 activation for keratinocyte survival on collagen gel was confirmed with experiment using U0126, an inhibitor for Erk1/2. These studies show that activation of Akt depends on collagen assembly, whereas activation of Erk1/2 is induced by increased extracellular calcium concentration. Thus, activation of the Erk1/2 by increasing calcium concentration in the incubation medium may compensate for the loss of Akt activation, allowing keratinocyte survival on collagen gels. [ABSTRACT FROM AUTHOR]

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]

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin α2β1. During incubation, the receptors used for adhesion are changed to integrins α3β1 and α6β4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin α2β1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins α2β1 and α3β1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin α3β1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival. [Copyright &y& Elsevier]

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]