The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed <20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ϳ60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ϳ4-fold greater activity than that of C. histolyticum collagenase.Bacterial collagenases are metalloproteases containing a consensus motif for zinc proteases, the HEXXH sequence and are capable of digesting both native and denatured collagen. They make multiple cleavages at the Y-Gly bond in repeating X-Y-Gly sequences within triple helical regions, where proline and hydroxyproline residues are most common in the X and Y positions, respectively (17). Because of their characteristics, bacterial collagenases have been widely used in biological experiment as tissuedispersing enzymes, as well as in medical procedures such as the isolation of pancreatic islet cells for transplantation (14) and the treatment for Dupuytren's disease (6).Much of our knowledge of bacterial collagenases has come from studies of the enzymes produced by Clostridium histolyticum (13,(15)(16)(17)34). Analysis of the primary structure of the gene product from C. histolyticum has revealed that clostridial collagenases consist of three domains (catalytic domain, polycystic kidney disease [PKD] domain, and collagen-binding domain [CBD]) in their molecules. Moreover, CBD has utilized for anchoring molecule that growth factors fused to CBD can be functional to bind to collagen fibrils and maintain biological activities (21). On the other hand, one of the other well-investigated bacterial collagenases is Vibrio alginolyticus collagenase (7, 10, 11, 28). The collagenase activity of V. alginolyticus collagenase is higher than that of any other bacterial collagenase, and it was found highly efficient in debridement of necrotic burns, ulcers and decubitus. To date, bacterial collagenases have been purified from various species, and their genes have been cloned and sequenced (8,12,18,24,35). However, many collagenases have not yet been both enzymatically and structurally characterized.Vibrio hollisae is a Gram-negative bacterium first described in 1982 (4) and recently reclassified ...

The collagenase activity and the fpcol gene were examined in Flavobacterium psychrophilum isolates from cold-water disease (CWD)-affected ayu, Plecoglossus altivelis. Collagenase expression was closely related to the accumulated mortality of CWD-affected ayu. RT-qPCR and bacterial challenge experiments showed that F. psychrophilum ayu isolate WA-1 expressed the fpcol gene more actively and was more virulent than ayu isolate WA-2. The amago (Oncorhynchus masou) isolate WB-1, which possesses a pseudo-fpcol gene, was not harmful to ayu. Hitherto, the well-studied metalloproteases Fpp1 and Fpp2 have been considered virulence factors. However, the most virulent isolate against ayu (WA-1) showed no Fpp activity because of a deletion mutation or an insertion of a transposon in the fpp genes. The less virulent WA-2 isolate showed only Fpp1 activity. Taken together, these results suggest that collagenolytic activity, but not Fpp activity, is related to the virulence of F. psychrophilum isolates in CWD-affected ayu.

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 ( CBD s) derived from Hathewaya histolytica ( Clostridium histolyticum ) collagenases; these CBD s 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 CBD s 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 CBD s from H. histolytica . As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBD s 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 .

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.

collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. in this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells. Collagenase has been widely used to isolate a variety of specialised cell types from attendant connective tissue where collagen is a major component. The isolation of primary cells is necessary to study cell function in basic research 1,2 ; isolation of cancer stem cells of solid organs is also important for the investigation of cancer pathophysiology in clinical research 3,4. Moreover, techniques for isolation of primary cells are essential aspects of therapeutic procedures in the fields of transplantation 5,6 and regenerative medicine 7,8. Collagenase products commonly used for these purposes are derived from Clostridium histolyticum. Clostridial collagenase products are known to exhibit lot-to-lot and intra-lot variability even when collagenase is highly purified, resulting in variable isolation outcomes 9-12 ; thus, there is room to improve these collagenase products. Clostridial collagenase products contain two components: class I (ColG) and class II (ColH) collagenases. These components play different roles in collagen digestion 13,14 and both are necessary for efficient isolation of primary cells 15. The composition of the enzyme blend can be modified to achieve optimisation for specific protocols and organ characteristics 16. However, the combination of these two components in a single enzyme product impairs its homogeneity and might induce an auto-degradation process, leading to lot-to-lot and even intra-lot variability in clostridial collagenase products 9. Grimontia hollisae is a Gram-negative bacterium that was previously classified in the genus Vibrio 17 ; s...