2011 - IPITA - Prague
Parallel session 4 – Open oral presentations Topic: Islet transplantation: Technical aspects
4.5 - 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
Presenter: A., Breite, Indianapolis, USA
Authors: A. Breite, R. McCarthy, F. Dwulet
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
A. Breite, R. McCarthy, F. Dwulet
VitaCyte, Indianapolis, USA
Objectives: C.histolyticum contains two genes expresssing C1 and C2 with masses of114 and 112 kDa, respectively. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to inconsistent performance of different products or lots to release 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 physical (HPLC, SDS-PAGE) and enzymatic analysis(collagen degradation activity - CDA). Q-tof mass spectrometry (MS) was used to determine the exact molecular weight of major collagenase molecular forms and associated enzyme activity.
Results: MS was used to confirm the full-length sequences for C2 112kDa and C1 114kDa from the reported gene sequence. These data were correlated with the molecular weights observed on the SDS-PAGE and elution after analytical anion-exchange HPLC. HPLC peaks labeled as C1b and C1c were both confirmed to be C1 with the terminal CBD missing. The only difference being 12 amino acids between the two CBDs. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. This synergy was not observed between any of the C1 molecular forms.
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. While the implications of this are not yet understood, it is fundamental in advancing the understanding of how collagenases work together and with the neutral protease to breakdown the extra cellular matrix for islet isolation.
Disclosure: Author Breite is an empolyee of VitaCyte LLC. Authors McCarthy and Dwulet have a financial interest in VitaCyte LLC.
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