Chemical shift assignments of the fibronectin III like domains 7–8 of type VII collagen

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain. The NC1 domain has subdomains with homology to adhesion molecules and mediates important interactions within the extracellular matrix. An 185 amino acid long part of the NC1-subdomain termed fibronectin III like domains 7 and 8 (FNIII7-8) was investigated. Antibodies against this region are pathogenic in a mouse model of EBA and one reported missense mutations of Col7 lies within these domains. The nearly complete NMR resonance assignment of recombinant FNIII7-8 of Col7 is reported.     

Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa

In animal models it has been shown that mesenchymal stromal cells (MSC) contribute to skin regeneration and accelerate wound healing. We evaluated whether allogeneic MSC administration resulted in an improvement in the skin of two patients with recessive dystrophic epidermolysis bullosa (RDEB; OMIM 226600). Patients had absent type VII collagen immunohistofluorescence and since birth had suffered severe blistering and wounds that heal with scarring. Vehicle or 0.5 × 10⁶ MSC were infused intradermally in intact and chronic ulcerated sites. One week after intervention, in MSC-treated skin type VII collagen was detected along the basement membrane zone and the dermal–epidermal junction was continuous. Re-epithelialization of chronic ulcerated skin was observed only near MSC administration sites. In both patients the observed clinical benefit lasted for 4 months. Thus intradermal administration of allogeneic MSC associates with type VII collagen replenishment at the dermal–epidermal junction, prevents blistering and improves wound healing in unconditioned patients with RDEB.     

Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR

Type VII collagen as component of anchoring fibrils plays an important role in skin architecture, however, no detailed structural information is available. Here, we describe the recombinant expression, isotope labeling, and (1)H, (15)N, (13)C chemical shift assignment of a subdomain of the murine type VII collagen - the von-Willebrand-factor-A-like domain 2 (mvWFA2). vWFA2 interacts with type I collagen and plays a central role in certain skin blistering diseases. Based on these assignments a secondary structure prediction was performed showing a properly folded protein. An interaction of mvWFA2 with its neighboring domain mFNIII-9 was characterized with NMR spectroscopy and SPR.     

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT–loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72 h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT–loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p < 0.01) and IL-1β (p < 0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p < 0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.     

Molecular cloning and characterization of type VII collagen cDNA.

Type VII collagen, located in human epidermal basement membrane, is the primary pathogenic target molecule in epidermolysis bullosa acquisita and epidermolysis bullosa dystrophica. Using a monoclonal antibody against the non-collagenous domain of type VII collagen, approximately 1 Kb cDNA was isolated from human keratinocyte library. The deduced primary structure of this clone thus reflects the non-collagenous domain of type VII collagen that may be involved in cell attachment. This region shows a weak homology (approximately 23%) to the cell attachment domain of fibronectin. Northern blot revealed approximately 9.5 Kb single band.     

Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2 months of life with an approximate 20% penetrance (~ 35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9 nm versus exon 2-deleted: 88.2 nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2 nm, SPARC-null/exon 2-deleted: 40.8 nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.     

Isolation and characterization of thermostable collagen from the marine eel-fish (Evenchelys macrura)

Aim and methodsCollagen is the most abundant protein found in animal body, which is widely used for biomedical and pharmaceutical applications. In the present study, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin wastes of marine eel fish (Evenchelys macrura) were isolated and characterized.ResultsASC and PSC extracted from eel fish skin showed the yields of 80 and 7.10 percent (based on dry weight), respectively. ASC and PSC comprising different α-chains (α1, α2 and α3) were characterized as type I and exhibited high solubility in acidic pH (1–4) and were soluble in the presence of NaCl at concentration up to 3.0 and 4.0 percent (w/v) for ASC and PSC, respectively. Amino acids analysis of both ASC and PSC contained imino acid of 190 and 200 residues per 1000 residues, respectively. The present results of ASC and PSC from eel fish skin exhibited higher thermal stability of 39 °C and 35 °C, respectively. Similar, Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and suggesting that pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.ConclusionThese results suggest that the marine eel fish skin collagen close to the T_d (denaturation temperature) of mammalian collagen which could be used in the biomedical materials, food and pharmaceutical industries as an alternative source.     

Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders that are caused by mutations in the type VII collagen gene and for which ex vivo gene therapy has been considered. To develop a simpler approach for treating DEB, we evaluated the feasibility of protein-based therapy by intradermally injecting human recombinant type VII collagen into mouse skin and a DEB human skin equivalent transplanted onto mice. The injected collagen localized to the basement membrane zone of both types of tissues, was organized into human anchoring fibril structures and reversed the features of DEB disease in the DEB skin equivalent.     

Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2)

An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20–40% following single-point substitutions, by 60–75% after double-point modifications, and by > 90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k_cat/K_m) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.     

Isolation and characterization of collagen from the cartilage of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon cartilage was isolated using sodium chloride (salt-solubilized collagen, SSC, 2.18%), followed by acetic acid (acid-solubilized collagen, ASC, 27.04%) and then pepsin (pepsin-solubilized collagen, PSC, 55.92%). These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC (35.71 and 123.90 °C) were significantly higher than SSC (32.64 and 114.51 °C) and ASC (32.98 and 120.72 °C) assessed by circular dichroism and differential scanning calorimetry, which could be attributed to its high imino acid content (22.57%) and degree of hydroxylation (47.29%). Electrophoresis pattern showed that the SSC and ASC were type I collagen, while PSC was predominantly type II collagen along with other minor types. Infrared spectra confirmed their triple helical structure, and indicated more hydrogen bonding in ASC and more intermolecular crosslinks in PSC. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.     

Supramolecular interactions in the dermo-epidermal junction zone: anchoring fibril-collagen VII tightly binds to banded collagen fibrils

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.     

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO₄. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α₁, α₂ of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.     

Basement membrane reactivity of antisera to type IV collagen and sera from patients with bullous pemphigoid and epidermolysis bullosa acquisita

The basement membrane antigenic specificities of antibodies to Type IV collagen were compared to naturally occurring antibodies in sera from patients with bullous pemphigoid and epidermolysis bullosa acquisita (EBA) by indirect immunofluorescence, mixed immunofluorescence and immunoabsorption. Results suggested that the three sera reacted with three different basement membrane antigens. In addition, absorption with Types I, II, III, or IV collagen failed to reduce the basement membrane reactivities of bullous pemphigoid or EBA sera. The antibodies to the basement membrane components should be useful in studying skin and mucous membrane diseases including periodontal diseases.     

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1^+ ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1^+ ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.     

The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.     

Directional dependence of hydroxyapatite-collagen interactions on mechanics of collagen

Bone is a biological nanocomposite composed primarily of collagen and hydroxyapatite. The collagen molecules self-assemble to from a structure known as a fibril that comprises of about 85–95% of the total bone protein. In a fibril, the molecular level interactions at the interface between molecular collagen and hydroxyapatite nanocrystals have a significant role on its mechanical response. In this study, we have used molecular dynamics and steered molecular dynamics to study directional dependence of deformation response of collagen with respect to the hydroxyapatite surface. We have also studied mechanical response of collagen in the proximity of (0 0 0 1) and (1 0 1¯0) surfaces of hydroxyapatite. Our simulations indicate that the mechanics of collagen pulled in different directions with respect to hydroxyapatite is significantly different. Similar results were obtained for collagen pulled in the proximity of different crystallographic surfaces of hydroxyapatite.