Breite A G, McCarthy R C, Dwulet F E
VitaCyte LLC, Indianapolis, Indiana 46202, USA.
Transplant Proc. 2011 Nov;43(9):3171-5. doi: 10.1016/j.transproceed.2011.09.059.
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.
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 sulfate-polyacrylamide 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.
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.
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.
溶组织梭菌表达两类胶原酶，即C1和C2。然而，在发酵或纯化过程中这些酶被蛋白酶降解可能会导致产生多种分子形式，从而导致从人胰腺中分离胰岛的释放情况不一致。本报告确定了含有单个胶原结合结构域（CBD）的C1截短形式（C1b或C1c）的氨基酸序列，并研究了不同形式的C1胶原酶和C2之间协同降解天然胶原的作用。
使用既定的柱色谱技术从溶组织梭菌培养上清液中纯化高度纯化的胶原酶同工型，并使用高压液相色谱（HPLC）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和质谱（MS）进行分析。采用胶原降解活性（CDA）测定法研究不同胶原酶分子形式之间的协同作用。
利用MS从报道的基因序列确认了全长C2和C1的序列。这些结果与SDS-PAGE上观察到的分子量以及分析性阴离子交换HPLC后的洗脱情况相关。指定为C1b和C1c的HPLC峰均被确认为缺少末端CBD的C1。唯一的区别在于切割位点，导致两种形式之间有12个氨基酸的差异。对于C2与三种C1分子形式中的每一种，观察到CDA相对于单个胶原酶活性具有非加性协同作用。在没有C2的情况下，C1分子形式未显示出这种协同作用。
这些观察结果支持了早期的报告，即两种胶原酶与胶原的不同部分结合，并且对切割天然胶原具有不同的特异性。尽管其影响尚不清楚，但它们对于推进对胶原酶如何与中性蛋白酶协同作用以分解细胞外基质用于胰岛分离的理解至关重要。
