The Globular Domain of the Proα1(I) N-Propeptide Is Not Required for Secretion, Processing by Procollagen N-Proteinase, or Fibrillogenesis of Type I Collagen in Mice

Paul Bornstein,Vanessa Walsh,Jennifer Tullis,Emily Stainbrook,John F Bateman,Sheriar G Hormuzdi

doi:10.1074/jbc.m106181200

Paul Bornstein, Vanessa Walsh + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m106181200

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Abstract

The globular domain in the NH(2)-terminal propeptide (N-propeptide) of the proalpha1(I) chain is largely encoded by exon 2 of the Col1a1 gene and has been implicated in a number of processes that are involved in the biogenesis, maturation, and function of type I collagen. These include intracellular chain association, transcellular transport and secretion, proteolytic processing of the precursor, feedback regulation of synthesis, and control of fibrillogenesis. However, none of these proposed functions has been firmly established. To evaluate the function of this procollagen domain we have used a targeted mutagenesis approach to generate mice that lack exon 2 in the Col1a1 gene. Mouse lines were established on both a mixed 129 OlaHsd/Sv and C57BL/6 background and a pure 129 OlaHsd/Sv background. Adult mice on the mixed background are normal in appearance and are fertile. To the extent that they have been studied, procollagen synthesis, secretion, and proteolytic processing are normal in these mice, and collagen fibrillogenesis is only slightly altered. However, breeding of heterozygous mutant mice on the 129 background generated homozygous mutants at only 64% of the expected frequency. These findings suggest that although the N-propeptide is not essential for collagen biogenesis in mice it may play some essential role during embryonic development.

Highlights

Type I collagen is synthesized as a precursor, procollagen, with NH2- and COOH-terminal non-triple helical extensions (N- and C-propeptides) that are released extracellularly by limited proteolysis with procollagen N- and C-proteinases [1,2,3,4]
The globular domain in the NH2-terminal propeptide (N-propeptide) of the pro␣1(I) chain is largely encoded by exon 2 of the Col1a1 gene and has been implicated in a number of processes that are involved in the biogenesis, maturation, and function of type I collagen
To the extent that they have been studied, procollagen synthesis, secretion, and proteolytic processing are normal in these mice, and collagen fibrillogenesis is only slightly altered

Summary

EXPERIMENTAL PROCEDURES

Generation and Diagnosis of Mutant Mice—Murine 129 genomic clones, containing fragments of the Col1a1 gene, were kindly provided by Dr H. Generation of cells containing the exon 2⌬ allele was performed essentially as described previously [26]. 2 ϫ 107 cells were electroporated with 30 ␮g of linearized targeting exon 2⌬ DNA and 24 h later were subjected to selection in media containing 400 ␮g/ml G418. Transfection of COS Cells and PCR Analysis—COS cells were cultured in Dulbecco’s modified Eagle’s medium, high glucose, supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, and nonessential amino acids (0.1 mM each). Cells were grown to confluence, washed with phosphate-buffered saline, and incubated at 37 °C for 24 h in Dulbecco’s modified Eagle’s medium, supplemented with 80 ␮g/ml ␤-aminopropionitrile, 50 ␮g/ml L-ascorbic acid, and 30 ␮Ci/ml [3H]proline. Five-␮m sections were stained with hematoxylin and eosin or with Sirius Red

RESULTS

Total Hyp

DISCUSSION