Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Shima H

PROVIDER: S-EPMC4852369 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Shima Hiroki H Inagaki Akiko A Imura Takehiro T Yamagata Youhei Y Watanabe Kimiko K Igarashi Kazuhiko K Goto Masafumi M Murayama Kazutaka K

Journal of diabetes research 20160418

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 ...[more]

PMID: 27195301

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Project description:To penetrate host tissues, histotoxic clostridia secrete virulence factors including enzymes to hydrolyze extracellular matrix. Clostridium histolyticum, recently renamed as Hathewaya histolytica, produces two classes of collagenase (ColG and ColH). The high-speed AFM study showed that ColG collagenase moves unidirectionally to plane collagen fibril and rebundles fibril when stalled . The structural explanation of the roles for the tandem collagen-binding segment (CBDs) is illuminated by its calcium-bound crystal structure at 1.9 Å resolution (Rwork = 15.0%; Rfree = 19.6%). Activation may involve calcium-dependent domain rearrangement supported by both small-angle X-ray scattering and size exclusion chromatography. At pCa ? 5 (pCa = -log[Ca2+ ]), the tandem CBD adopts an extended conformation that may facilitate secretion from the bacterium. At pCa ? 4, the compact structure seen in the crystal structure is adopted. This arrangement positions the two binding surfaces ~ 55 Å apart, and possibly ushers ColG along tropocollagen molecules that allow for unidirectional movement. A sequential binding mode where tighter binding CBD2 binds first could aid in processivity as well. Switch from processive collagenolysis to fibril rearrangement could be concentration dependent. Collagen fibril formation is retarded at 1 : 1 molar ratio of tandem CBD to collagen. Tandem CBD may help isolate a tropocollagen molecule from a fibril at this ratio. At 0.1 : 1 to 0.5 : 1 molar ratios fibril self-assembly was accelerated. Gain of function as a result of gene duplication of CBD for the M9B enzymes is speculated. The binding and activation modes described here will aid in drug delivery design. ACCESSION CODES:The full atomic coordinates of the tandem CBD and its corresponding structure factor amplitudes have been deposited in the Protein Data Bank (PDB accession code 5IKU). Small-angle X-ray scattering data and corresponding ab initio models have been submitted to the Small Angle Scattering Biological Data Bank (SASBDB). Accession codes CL2, collagenase module 2, CN2, CP2 are assigned to envelopes for tandem CBD at -log[Ca2+ ] (pCa) 3, 4, 5, and 6, respectively. Accession code DC64 was assigned to the complex of polycystic kidney disease-CBD1-CBD2 with mini-collagen.

Project description:For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Dataset Information

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

Publications

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

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