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. 1990 May 1;267(3):573–577. doi: 10.1042/bj2670573

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

J F Bateman 1, S B Golub 1
PMCID: PMC1131335  PMID: 1692701

Abstract

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bateman J. F., Chan D., Mascara T., Rogers J. G., Cole W. G. Collagen defects in lethal perinatal osteogenesis imperfecta. Biochem J. 1986 Dec 15;240(3):699–708. doi: 10.1042/bj2400699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bateman J. F., Cole W. G., Pillow J. J., Ramshaw J. A. Induction of procollagen processing in fibroblast cultures by neutral polymers. J Biol Chem. 1986 Mar 25;261(9):4198–4203. [PubMed] [Google Scholar]
  3. Bateman J. F., Harley V., Chan D., Cole W. G. Comprehensive analysis of collagen metabolism in vitro using [4(3H)]/[14C]proline dual-labeling and polyacrylamide gel electrophoresis. Anal Biochem. 1988 Jan;168(1):171–175. doi: 10.1016/0003-2697(88)90025-5. [DOI] [PubMed] [Google Scholar]
  4. Bateman J. F., Mascara T., Chan D., Cole W. G. Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem J. 1984 Jan 1;217(1):103–115. doi: 10.1042/bj2170103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bateman J. F., Pillow J. J., Mascara T., Medvedec S., Ramshaw J. A., Cole W. G. Cell-layer-associated proteolytic cleavage of the telopeptides of type I collagen in fibroblast culture. Biochem J. 1987 Aug 1;245(3):677–682. doi: 10.1042/bj2450677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonadio J., Byers P. H. Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II. Nature. 1985 Jul 25;316(6026):363–366. doi: 10.1038/316363a0. [DOI] [PubMed] [Google Scholar]
  7. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  8. Cole W. G., Chan D., Chambers G. W., Walker I. D., Bateman J. F. Deletion of 24 amino acids from the pro-alpha 1(I) chain of type I procollagen in a patient with the Ehlers-Danlos syndrome type VII. J Biol Chem. 1986 Apr 25;261(12):5496–5503. [PubMed] [Google Scholar]
  9. Davidson J. M., McEneany L. S., Bornstein P. Intermediates in the conversion of procollagen to collagen. Evidence for stepwise limited proteolysis of the COOH-terminal peptide extensions. Eur J Biochem. 1977 Dec 1;81(2):349–355. doi: 10.1111/j.1432-1033.1977.tb11958.x. [DOI] [PubMed] [Google Scholar]
  10. Eyre D. R., Paz M. A., Gallop P. M. Cross-linking in collagen and elastin. Annu Rev Biochem. 1984;53:717–748. doi: 10.1146/annurev.bi.53.070184.003441. [DOI] [PubMed] [Google Scholar]
  11. Fessler L. I., Morris N. P., Fessler J. H. Procollagen: biological scission of amino and carboxyl extension peptides. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4905–4909. doi: 10.1073/pnas.72.12.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fleischmajer R., Olsen B. R., Timpl R., Perlish J. S., Lovelace O. Collagen fibril formation during embryogenesis. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3354–3358. doi: 10.1073/pnas.80.11.3354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fleischmajer R., Timpl R., Tuderman L., Raisher L., Wiestner M., Perlish J. S., Graves P. N. Ultrastructural identification of extension aminopropeptides of type I and III collagens in human skin. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7360–7364. doi: 10.1073/pnas.78.12.7360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gerstenfeld L., Beldekas J. C., Sonenshein G. E., Franzblau C. Processing of procollagen types III and I in cultured bovine smooth muscle cells. J Biol Chem. 1984 Jul 25;259(14):9158–9162. [PubMed] [Google Scholar]
  15. Goldberg B., Sherr C. J. Secretion and extracellular processing of procollagen by cultured human fibroblasts. Proc Natl Acad Sci U S A. 1973 Feb;70(2):361–365. doi: 10.1073/pnas.70.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helseth D. L., Jr, Veis A. Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3302–3306. doi: 10.1073/pnas.81.11.3302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hulmes D. J. A possible mechanism for the regulation of collagen fibril diameter in vivo. Coll Relat Res. 1983 Jul;3(4):317–321. doi: 10.1016/s0174-173x(83)80013-2. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  20. Layman D. L., Ross R. The production and secretion of procollagen peptidase by human fibroblasts in culture. Arch Biochem Biophys. 1973 Aug;157(2):451–456. doi: 10.1016/0003-9861(73)90661-9. [DOI] [PubMed] [Google Scholar]
  21. Leung M. K., Fessler L. I., Greenberg D. B., Fessler J. H. Separate amino and carboxyl procollagen peptidases in chick embryo tendon. J Biol Chem. 1979 Jan 10;254(1):224–232. [PubMed] [Google Scholar]
  22. Limeback H. F., Sodek J. Procollagen synthesis and processing in periodontal ligament in vivo and in vitro. A comparative study using slab-gel fluorography. Eur J Biochem. 1979 Oct 15;100(2):541–550. doi: 10.1111/j.1432-1033.1979.tb04200.x. [DOI] [PubMed] [Google Scholar]
  23. Minor R. R., Sippola-Thiele M., McKeon J., Berger J., Prockop D. J. Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes. J Biol Chem. 1986 Jul 25;261(21):10006–10014. [PubMed] [Google Scholar]
  24. Miyahara M., Bruckner P., Helle O., Prockop D. J. Aggregation of a type I collagen precursor containing N-terminal propeptides. Coll Relat Res. 1983 Jul;3(4):279–293. doi: 10.1016/s0174-173x(83)80010-7. [DOI] [PubMed] [Google Scholar]
  25. Miyahara M., Njieha F. K., Prockop D. J. Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases. J Biol Chem. 1982 Jul 25;257(14):8442–8448. [PubMed] [Google Scholar]
  26. Peltonen L., Halila R., Ryhänen L. Enzymes converting procollagens to collagens. J Cell Biochem. 1985;28(1):15–21. doi: 10.1002/jcb.240280104. [DOI] [PubMed] [Google Scholar]
  27. Ramshaw J. A. A mild form of ovine dermatosparaxis. Coll Relat Res. 1984 Dec;4(6):441–451. doi: 10.1016/s0174-173x(84)80011-4. [DOI] [PubMed] [Google Scholar]
  28. Ramshaw J. A., Bateman J. F., Cole W. G. Precipitation of collagens by polyethylene glycols. Anal Biochem. 1984 Sep;141(2):361–365. doi: 10.1016/0003-2697(84)90056-3. [DOI] [PubMed] [Google Scholar]
  29. Sonohara S., Brentani R. R., Andrade H. F., Jr, Machado-Santelli G. M. Procollagen biosynthesis and processing in guinea pig fibroblast culture medium. Braz J Med Biol Res. 1981 Oct;14(4-5):267–276. [PubMed] [Google Scholar]
  30. Taubman M. B., Goldberg B. The processing of procollagen in cultures of human and mouse fibroblasts. Arch Biochem Biophys. 1976 Apr;173(2):490–494. doi: 10.1016/0003-9861(76)90286-1. [DOI] [PubMed] [Google Scholar]
  31. Tromp G., Prockop D. J. Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5254–5258. doi: 10.1073/pnas.85.14.5254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Veis A., Anesey J., Yuan L., Levy S. J. Evidence for an amino-terminal extension in high-molecular-weight collagens from mature bovine skin. Proc Natl Acad Sci U S A. 1973 May;70(5):1464–1467. doi: 10.1073/pnas.70.5.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Wet W. J., Pihlajaniemi T., Myers J., Kelly T. E., Prockop D. J. Synthesis of a shortened pro-alpha 2(I) chain and decreased synthesis of pro-alpha 2(I) chains in a proband with osteogenesis imperfecta. J Biol Chem. 1983 Jun 25;258(12):7721–7728. [PubMed] [Google Scholar]

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