Kimiko Watanabe

Collagenase H is Crucial for Isolation of Rat Pancreatic Islets

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

ABSTRACT The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca2+ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca2+ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca2+ was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca2+ chelation, in comparison with the thermal melting point (Tm ). The melting point changed from 54 to 49°C by the Ca2+ chelation, and it was restored to 54°C by the addition of excess Ca2+. These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca2+.

Solution Structure of Clostridial Collagenase H and Its Calcium-Dependent Global Conformation Change

Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

Background Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. Methods The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. Results The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. Conclusions Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

NO NEED OF TRYPTIC-LIKE ACTIVITY FOR SUCCESSFUL PORCINE ISLET ISOLATION: 1291

M. Goto1, Y. Yamagata2, K. Watanabe3, K. Murayama4, T. Imura5, A. Inagaki5, H. Yamaya6, K. Fujimori7, Y. Kurokawa8, S. Satomi9 1Division Of Advanced Surgical Science And Technology/international Advanced Research And Education Organization, Tohoku University, Sendai/JAPAN, 2) Department Of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Tokyo/JAPAN, 3Niche, Tohoku University, Sendai/JAPAN, 4Biomedical Measurements And Diagnostics, Tohoku University, Sendai/JAPAN, 5International Advanced Research And Education Organization, Tohoku University, Sendai/JAPAN, 6Division Of Advanced Surgical Science And Technology, Tohoku University, Sendai/JAPAN, 7, Tohoku University, Sendai/JAPAN, 8Innovation Of New Biomedical Engineering Center, Tohoku University, Sendai/JAPAN, 9Transplantation, Reconstruction And Endoscopic Surgery, Tohoku University, Sendai/JAPAN