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Report No. 010 Development of recombinant collagenase from Grimontia hollisae
Report No. Development of recombinant collagenase from Grimontia hollisae
010

Abstract

Collagen molecule is heterotrimeric molecule with three polypeptide strands called α chain. The primary structural feature of collagen is repeating Gly-Xaa-Yaa amino acid sequences. Glycine (Gly) is found at almost every third residue. Proline and hydroxyproline are frequently found at the Xaa and Yaa positions, respectively. Type I collagen molecule is a rod-like molecule with a triple helical structure, approximately 300 nm long and 1.5 nm in diameter. Collagen can be solubilized with proteolytic enzyme such as pepsin because triple helix of collagen is resistant to enzymatic hydrolysis. On the other hand, collagenase can digest triple helix of collagen. We isolated G. hollisae strain 1706B from seashore sand collected from the Shin-Kiba coast in Tokyo. This organism produces a collagenase with a very high specific activity in the presence of gelatin. Moreover, we cloned a novel collagenase gene from G. hollisae 1706B and produced a high yield of recombinant enzyme by using the Brevibacillus expression system. This recombinant collagenase is commercialized as Brightase-C , an enzyme for tissue dissociation.

Bacterial 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 Yaa-Gly bond in repeating Gly-Xaa-Yaa sequences within triple helical regions and produce tripeptides (ref. 1). Bacterial collagenases are classified into the M9 subfamily in the MEROPS database, which is divided into two subgroups (M9A and M9B) based on the amino acid sequence and substrate specificity. According to this classification, Vibrio collagenase is classified into the M9A subgroup while Clostridial collagenase is classified into the M9B subgroup (Fig. 1).
*One of well-investigated bacterial collagenases is Clostridium histolyticum collagenase. C. histolyticum, which has been recently reclassified into Hathewaya histolytica, produces two classes of collagenases: type I collagenase (ColG) and type II collagenase (ColH). Clostridial collagenases are multidomain enzymes and consist of three domains (collagenase module, polycystic kidney disease [PKD] domain, and collagen-binding domain [CBD]) in their molecules. ColG contains one PKD domain and two CBDs, and ColH contains two PKD domains and one CBD (ref. 2). These two collagenases play different roles in collagen digestion such as substrate specificity (ref. 3). Clostridial collagenases have been widely used in biological experiment as tissue dispersing enzymes, as well as in medical procedures such as the isolation of pancreatic islet cells for transplantation, the treatment for Dupuytren’s disease, the debridement of necrotic burns, ulcers and decubitus (ref. 2).

Collagenase derived from Grimontia (Vibrio) hollisae 1706B

Vibrio hollisae is a Gram-negative bacterium first described in 1982 and recently reclassified as the novel genus Grimontia. G. hollisae strain 1706B was isolated from seashore sand collected from the Shin-Kiba coast in Tokyo (ref. 4). This organism produces a collagenase with a very high specific activity in the presence of gelatin, and this purified enzyme has a molecular mass of ~ 60 kDa (ref. 5). We have purified the collagenase from G. hollisae strain 1706B, cloned its gene, and determined its complete nucleotide sequence. Subsequently, we deduced its complete amino acid sequence, and found that this collagenase originally possesses a prepro region (aa 1–87), a collagenase module (aa 88–615), and a bacterial pre-peptidase C-terminal (PPC) domain (aa 688–749). This collagenase is secreted as a 74 kDa protein consisting of two parts: the collagenase module and the C-terminal segment including the PPC domain. N-terminal sequence analysis reveals that most of the 74 kDa collagenase spontaneously becomes truncated to 62 kDa collagenase which consists of only collagenase module (ref. 6). Moreover, based on this deduced amino acid sequence, we have classified this collagenase into the M9A subgroup of the MEROPS database (Fig. 1).


Fig. 1 Schematic representation of the domain architecture of bacterial collagenases.

Development of recombinant collagenase from G. hollisae 1706B

Bacterial collagenases are used to enzymatically dissociate tissues and organs in which collagen is a major component and to isolate various tissue- and organ-specific cells, including stem cells. 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 (ref. 7). The combination of two collagenases (ColG and ColH) 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. On the other hand, collagenase from G. hollisae strain 1706B was expected to be used for tissue dissociation as a single-component collagenase product (ref. 8). However, the 74 kDa enzyme consisting of a collagenase module and a PPC domain was found to have high collagenolytic activity, but its activity decreased by auto-degradation and turned to be a 62 kDa enzyme consisting only of the collagenase module (ref. 6, 9).
In order to produce a stable recombinant collagenase product, we designed and directly expressed the recombinant 62 kDa collagenase from G. hollisae using the Brevibacillus Expression System. Using synthetic substrates for analysis of collagenolytic activity, we found that the 62-kDa recombinant protein exhibited optimal activity in the pH range of 7.5–9.0, and in the temperature range of 30–40 °C, indicating that the recombinant collagenase has the same property as the original collagenase produced by G. hollisae strain 1706B (Fig. 2). Moreover, we analyzed the stability of the 62-kDa recombinant protein and observed that it remained intact without degradation for up to 24 hours at 37 °C; during the 24-hour incubation period, it retained stable collagenolytic activity (ref. 10).

Fig. 2 Characterization of recombinant 62 kDa collagenase. (A) Purified recombinant 62 kDa collagenase was analyzed by SDS-PAGE (lane 1) and real-time gelatin zymography (lane 2). (B) pH-dependence of recombinant 62 kDa collagenase. (C) Temperature-dependence of recombinant 62 kDa collagenase.
When determining specific activity of the recombinant G. hollisae collagenase using two types of substrates, FITC-labelled collagen and a synthetic peptide of furylacryloyl-Leu-Gly-Pro-Ala (FALGPA), we found that its specific activity was more than three-fold higher than that of the purified collagenase product from C. histolyticum (Liberase MTF C/T, Roche) against both substrates (Fig. 3A). Moreover, we confirmed that the recombinant collagenase cleaved types I, II, III, IV, V, and type VI collagen (Fig. 3B). Since the recombinant G. hollisae collagenase is able to digest type VI collagen which the purified clostridial collagenase is not, the ability to digest type VI collagen could enable effective isolation of primary cells from fibrous tissues because type VI collagen may increase during fibrous changes (e.g., liver fibrosis). Furthermore, the recombinant G. hollisae collagenase, unlike clostridial collagenase, favors substrates that contain glutamic acid at the Xaa position of collagen and produces tripeptides, indicating that recombinant G. hollisae collagenase degrades collagen more efficiently than clostridial collagenase (ref. 1).



Fig. 3 Collagenolytic activity of recombinant 62 kDa collagenase. (A) The collagenolytic activity of the recombinant collagenases were determined using FITC-collagen and FALGPA. (B) Collagen cleavage assay. Recombinant 62 kDa collagenase was incubated with type I, type II , type III, type IV, type V and type VI collagens at 30 °C. Liberase MTF C/T was used as the purified collagenase product from C. histolyticum.

Application for islet isolation

To evaluate the potency of the recombinant protein to isolate primary cells, we adopt isolation of mouse pancreatic islets. Isogeneic islets were isolated using the recombinant 62-kDa collagenase from G. hollisae and thermolysin at concentrations of 0.15 mg/ml and 0.012 mg/ml, respectively (Fig. 4A). Three hundred purified islets were transplanted under the kidney capsule of each mouse recipient; diabetes had been induced in all mice by prior administration of streptozotocin (Fig. 4B). The blood glucose concentrations gradually decreased in all mice that received islets, such that they returned to normal within 3 days after transplantation; conversely, diabetic mice that did not receive islets continued to exhibit high concentrations of blood glucose. All mice that had achieved normoglycaemia reverted to hyperglycaemia immediately after the removal of kidneys bearing islet grafts, at 38 days after transplantation (Fig. 4C). Based on these results, the recombinant 62-kDa collagenase from G. hollisae is able to isolate functional murine islets.

Fig. 4 Transplantation of primary islet cells into diabetic mice.
(A) Optical image of primary islet cells. Scale bar, 500 μm. (B) Transplantation of 300 islet cells into the subrenal capsular space of a recipient mouse. Scale bar, 2 mm (right). (C) Change in blood glucose concentrations of five diabetic mice that received 300 islet cells (solid lines) and three diabetic mice that did not undergo transplantation (dashed lines). Nephrectomy of the graft-bearing kidney was performed at 38 days after transplantation.

Conclusion

Using the Brevibacillus expression system, we succeeded in producing a stable recombinant 62 kDa collagenase and commercialized it as Brightase-C, a recombinant collagenase product. Similarly, we also produced thermolysin from Bacillus thermoproteolyticus using the Brevibacillus expression system and commercialized it as Brightase-TH. These enzymes are available as a tissue dissociation enzyme kit (Brightase-C/TH) for isolating primary cells. The Brevibacillus expression system is suitable to produce recombinant proteins for medical applications because this system can produce them with low endotoxin and without using animal-derived materials. We showed that Brightase-C, a recombinant collagenase, can digest tissue as well as C. histolyticum collagenase product and has different substrate specificity from C. histolyticum collagenases, such as degrading activity to type VI collagen. In addition, since Brightase-C consists only of a collagenase module without CBD, the collagenase is not expected to remain in isolated pancreatic islets, and even if residual enzyme exists, its collagenolytic activity would be reduced by wash process (ref. 11). For the above reasons, Brightase-C/TH is useful as an enzyme for clinical tissue dispersion such as islet transplantation and stem cell transplantation (ref. 12). In addition to tissue dissociation, Brightase-C/TH can also be used as a tool for basic research such as analysis of collagen telopeptides (ref. 13), analysis of collagenase-digested tripeptides (ref. 14), and analysis of the distribution of tendon cells in tissues (ref. 15).

References

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