Aggregation of a Type I Collagen Precursor Containing N-Terminal Propeptides

Miyahara, Michinori; Brückner, Peter; Helle, O.; Prockop, Darwin J.

doi:10.1016/s0174-173x(83)80010-7

Cited by 22 publications

(6 citation statements)

References 25 publications

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“…Microscopic examinations confirmed that the aggregates formed under higher concentrations of NaCl were indeed fibrillar rather than amorphous. Interestingly, a similar Vshaped dependence of the rate of fibril assembly (reciprocal of the reaction half-time) of pN-collagen, a collagen with the N-terminal propeptides remaining on the molecule, has been reported by Miyahara et al (1983). The minimum of the kinetic curve was also found near 0.35 M ionic strength.…”

Section: Discussionsupporting

confidence: 75%

In vitro collagen fibril assembly: thermodynamic studies

Na¹,

Phillips²,

Freire³

1989

Biochemistry

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The in vitro fibril assembly of calf skin collagen was examined as a function of ionic strength and temperature. In a 0.03 M NaPi, pH 7.0, buffer, fibril assembly required a minimum critical concentration of collagen. At nearly physiological ionic strengths and temperatures, the critical concentration was less than 1 microgram/mL and required a very sensitive method for measurement. Raising the ionic strength of the buffer resulted first in higher and then lower critical concentrations. Raising the temperature led to lower critical concentrations. A van't Hoff plot of the fibril growth constant calculated from the critical concentration gave positive enthalpy changes and positive heat capacity changes which indicate that the fibril growth is driven by both hydrophobic and ionic inter-collagen interactions. Sedimentation equilibrium studies showed the collagen to be monomeric at subcritical concentrations. Differential scanning microcalorimetric studies showed only one very sharp heat absorption peak for the fibril assembly which coincided with the appearance of solution turbidity. Within experimental error, the enthalpy changes of the fibril assembly measured with the microcalorimeter were of the same magnitude as the van't Hoff enthalpy changes. These results are discussed in light of a cooperative nucleation-growth mechanism of collagen fibril assembly proposed earlier.

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Section: Discussionsupporting

confidence: 75%

In vitro collagen fibril assembly: thermodynamic studies

Na¹,

Phillips²,

Freire³

1989

Biochemistry

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“…Fleischmajer et al (2,3), using specific anti-pN-collagen antibodies, have shown that newly formed thin fibrils possess pN-collagen but not pC-collagen. Several workers have shown that pN-collagen can assemble in vitro to give thin fibrils with the characteristic collagen banding pattern (68,69), while pC-collagen will not (61). Hulmes (4) recently proposed that biological fibril assembly requires pN-collagen as an intermediate, followed by the trimming of NH2 propeptides from the fibrils by procollagen NH2-terminal proteinase.…”

mentioning

confidence: 99%

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Helseth¹,

Veis²

1984

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

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The proteolytic removal of the extension COOH-terminal propeptide from procollagen has been examined in vitro. A crude enzyme activity was identified in a whole-chicken-embryo extract that acted at acid pH and appeared to be similar to one identified previously [Davidson, J. M., McEneany, L. S. G. & Bornstein, P. (1979) Eur. J. Biochem. 100,[551][552][553][554][555][556][557][558]. This activity was inhibitable by pepstatin but not by leupeptin, suggesting that it might be cathepsin D. Cathepsin D was purified 907-fold from chicken livers by affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and was found to remove the COOH propeptides from procollagen. At pH 6.0, the site of cleavage appeared to shift from the COOH telopeptide to the COOH telopeptide/propeptide junction, based upon the difference in electrophoretic migration of the cleavage products, although determining the actual cleavage site will require end-group analysis. A model for the involvement of cathepsin D in the in vivo processing of procollagen is presented.The enzymes responsible for the sequential removal of the extension propeptides from procollagen, procollagen NH2-terminal proteinase and procollagen COOH-terminal proteinase, have been actively studied, but only the former has been well characterized (1). In vivo it appears that the COOH-terminal propeptide (COOH propeptide) is removed first, with the NH2-terminal propeptide (NH2 propeptide) removed later, possibly after the intermediate has assembled into extracellular fibrils (2-4). The site of COOH propeptide removal has not been well defined. In cell culture experiments, the apparent in vivo pathway is reversed. The COOH propeptide is not cleaved from the product that accumulates in cell culture media, yet conversion is complete in organ culture (5), suggesting that an intact extracellular matrix is required for conversion.In searching for a neutral metalloproteinase that is a COOH-terminal proteinase (6), we observed a cathepsin Dlike activity, which removed the COOH propeptide from procollagen at acid pH. We report here that the cleavage of procollagen with purified cathepsin D is specific for the COOH propeptide and that cleavage occurs at or near the authentic cleavage site when digestion is carried out near pH 6.0. We postulate a role for cathepsin D in the in vivo conversion of procollagen to collagen. MATERIALS AND METHODSProcollagen was isolated from 17-day chicken-embryo tendon fibroblasts (7,8). After tendon dissection and dissociation with collagenase and trypsin, fibroblasts were incubated (2 x 107 cells per ml) for 6 hr in modified Krebs medium II in the presence of 1 ,uCi (1 Ci = 37 GBq) of a mixture of 15 14C-labeled amino acids (New England Nuclear) per ml. Procollagen and partially processed collagen from which the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3302NH2 propeptide was removed (...

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“…Initial C-propeptide removal is consistent with the finding of pNcollagens in normal collagen fibrils in vivo (Veis et al, 1973;Fleischmajer et al, 1981Fleischmajer et al, , 1983). Studies performed in vitro have also demonstrated that pN-collagen can form fibrils (Miyahara et al, 1983), but that pC-collagen does not (Miyahara et al, 1982). Since pN-collagen forms thin fibrils (Miyahara et al, 1983), it has been suggested that N-terminal propeptide cleavage during fibrillogenesis may act as a regulatory mechanism of collagen fibril diameter (Hulmes, 1983).…”

Section: Resultsmentioning

confidence: 99%

Assessment of procollagen processing defects by fibroblasts cultured in the presence of dextran sulphate

Bateman

Golub

1990

Biochemical Journal

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The culture of skin fibroblasts in the presence of 0.01% (w/v) dextran sulphate results in complete proteolytic processing of procollagen to collagen. Processing occurs predominantly via a pN-collagen intermediate, suggesting that C-propeptide cleavage occurs early during the processing pathway. The processed collagen is associated with the cell-layer fraction. This method of inducing procollagen processing was evaluated for use in detecting procollagen processing abnormalities in heritable connective-tissue diseases. Abnormal type I procollagen processing was clearly demonstrated in two cases with known defects of pN-propeptide cleavage. In one, the cleavage deficiency was due to diminished N-proteinase activity (dermatosparaxis) and in the other case (Ehler's-Danlos syndrome type VIIA) the cleavage site was deleted. In a case of osteogenesis imperfecta (type II) the slow electrophoretic migration of type I collagen alpha-chains due to over-modification of lysine was readily demonstrated. Inefficient procollagen processing was also evident in this patient, as had been previously reported [de Wet, Pihlanjaniemi, Myers, Kelly & Prockop (1983) J. Biol. Chem. 258, 7721-7728]. Thus this method of culture in the presence of dextran sulphate provides a simple and rapid procedure for the detection of procollagen processing defects and electrophoretic abnormalities.

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Aggregation of a Type I Collagen Precursor Containing N-Terminal Propeptides

Cited by 22 publications

References 25 publications

In vitro collagen fibril assembly: thermodynamic studies

In vitro collagen fibril assembly: thermodynamic studies

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Assessment of procollagen processing defects by fibroblasts cultured in the presence of dextran sulphate

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