Animal collagenases: Specificity of action, and structures of the substrate cleavage site

Gross, Jerome; Harper, Elvin; Harris, Edward D.; McCroskery, Peter; Highberger, John H.; Corbett, Clare; Kang, Andrew H.

doi:10.1016/0006-291x(74)91000-6

Cited by 144 publications

(76 citation statements)

References 17 publications

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“…It has not been reported whether zinc is required for the unwinding function, which prompted us to examine whether MMP-1 could unwind collagen in the presence of EDTA to expose sites otherwise not accessible for cleavage by rFAP or APCE. As expected (41) and shown in Figure 3, EDTA did prevent the cleavage of collagen I by MMP-1. Even at a significantly higher concentration of MMP-1 (1.5 μg), but with EDTA still in the digestion mixture, no new bands were noted in analyses of the reduced digestion mixture on SDS-PAGE as evidenced by the gel pattern being identical to the control sample in Figure 1a.…”

Section: Cleavage Of Collagen Isupporting

confidence: 87%

“…Chung et al [43] demonstrated that the unwinding and catalytic activities of MMP-1 are clearly distinct, yet both are required for collagen cleavage by MMP-1. Importantly, MMP-1 promotes only a local unwinding of collagen and the overall collagen triple helix is not denatured (41). It has not been reported whether zinc is required for the unwinding function, which prompted us to examine whether MMP-1 could unwind collagen in the presence of EDTA to expose sites otherwise not accessible for cleavage by rFAP or APCE.…”

Section: Cleavage Of Collagen Imentioning

confidence: 99%

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Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Christiansen

Jackson

Lee

et al. 2007

Archives of Biochemistry and Biophysics

111

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The circulating enzyme, α 2 -antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α 2 -antiplasmin to a derivative more easily crosslinked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Aminoterminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.

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Section: Cleavage Of Collagen Isupporting

confidence: 87%

Section: Cleavage Of Collagen Imentioning

confidence: 99%

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Christiansen

Jackson

Lee

et al. 2007

Archives of Biochemistry and Biophysics

111

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“…In contrast, cleavage of collagen with tadpole collagenase (Fig. 2) produced the fragments expected from the known specificity of this enzyme (16). The high-molecular-weight chains, migrating more rapidly than al represent the al and a2 segments in TCA*, and the lower molecular weight peptides near al-CB7 are the fragments in TCB.…”

Section: Methodsmentioning

confidence: 90%

Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain.

Byers¹,

Click²,

Harper³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Tadpole collagenase (EC 3.4.24.3) cleaved chick cranial bone procollagen into two triple-stranded fragments, PCA and PCB. Only pCB, with an estimated molecular weight of about 60,000 for each component chain after reduction, was found to contain interchain disulfide bonds. The analogous cleavage of collagen is known to produce a large NH2-terminal fragment with a molecular weight of 70 Since the initial identification of a biosynthetic precursor to collagen, it has become apparent that the nontriple-helical portion of the molecule is important for several functions, which may include molecular assembly, maintenance of intracellular solubility, transmembrane transport, and regulation of fibril formation (see ref. 1 for a review). Early structural studies of collagen precursors suggested, on the basis of electron micrographs of segment-long-spacing crystallites, that there was a single "noncollagenous" domain at the NH-terminus of each precursor chain (2, 3). Subsequent chemical studies of these precursors and of those procollagen-derived proteins which accumulated in the tissues of animals affected with dermatosparaxis tended to confirm the structural studies (4, 5).More recent studies of (a) peptides derived from procollagen secreted into culture medium by cells, (b) precursor chains protected from proteolysis during purification, and (c) peptides secreted into culture medium by cranial bone have suggested that the nonhelical, noncollagenous domain is considerably larger than was previously thought (6-9). Tanzer and his colleagues (10) have recently indicated that there is a noncollagenous peptide extension at the COOHterminus of the procollagen molecule, in addition to that at the NH2-terminus. In this report, we present data to show that the COOH-terminal domain of procollagen is larger than the NH2-terminal extensions, that the composition is different, and that interchain disulfide bonds occur exclusively in the COOH-terminal region. MATERIALS AND METHODSProcollagen, pulse labeled with L-[2,3-3H]proline (43.1 Ci/ mmol, New England Nuclear) for 18 min or with L-[G-3H]tryptophan (7.9 Ci/mmol, New England Nuclear) for 2 hr (7, 36) was extracted from 17-day-old chick embryo cranial bones with 1 M NaCl, 50 mM Trip-HCl, pH 7.5, to which 25 mM EDTA, 10 mM N-ethylmaleimide, 1 mM diisopropylfluorophosphate or phenylmethylsulfonylfluoride, and 1 mM benzamidine-HCl had been added to inhibit proteolysis. The bones were homogenized in that solvent, centrifuged at 39,000 X g for 15 min, and the labeled procollagen together with coextracted collagen was precipitated from the supernatant with 20% NaCl. The precipitate was collected by centrifugation, redissolved in extraction buffer, precipitated with 5% cold trichloroacetic acid and then dissolved in extraction buffer containing 0.1 M Tris-HCl, pH 7.5. Acid-extracted procollagen was obtained as previously described (4), except that extraction was limited to 1 hr, and was further purified by chromatography on DEAE-cellulose by minor modifications of the method of S...

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“…Currently, common procedures for preparation of single-cell suspensions from adult mouse pancreata include subjecting the excised tissue to digestive fluid supplemented with collagenase alone or a combination of collagenase and trypsin at 37 °C, followed by mechanical shearing (Kayali et al, 2003;Suzuki et al, 2004;Inada et al, 2008). Collagenase provides gentle, selective digestion of the intercellular matrix, but is unable to completely disassociate organ tissue into single cells (Gross et al, 1974). Trypsin can disassociate tissue fragments at 37 °C more effectively than collagenase, but can also activate pancreatic zymogens and induce widespread tissue damage and impair viability (Amsterdam and Jamieson, 1974;Ji et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion

Peng

Zhang

et al. 2013

J. Zhejiang Univ. Sci. B

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Abstract:The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.

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Animal collagenases: Specificity of action, and structures of the substrate cleavage site

Cited by 144 publications

References 17 publications

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain.

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion

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