Characterization and functional assessment of clostridium histolyticum class i (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase

摘要

Objectives: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. Methods: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. Results: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. Conclusions: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.