Key points are not available for this paper at this time.
The globular domain in the NH2-terminal propeptide (N-propeptide) of the proα1(I) chain is largely encoded by exon 2 of the Col1a1gene 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 Col1a1gene. 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. The globular domain in the NH2-terminal propeptide (N-propeptide) of the proα1(I) chain is largely encoded by exon 2 of the Col1a1gene 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 Col1a1gene. 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. wild-type embryonic stem Col1a1 gene with a deletion of exon 2 phosphoglycerate kinase promoter-neomycin resistance gene procollagen N-proteinase reverse transcription 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 (1Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 2Prockop D.J. Sieron A.L. Li S.W. Matrix Biol. 1998; 16: 399-408Crossref PubMed Scopus (144) Google Scholar, 3Hojima Y. Morgelin M.M. Engel J. Boutillon M.-M. van der Rest M. McKenzie J. Chen G.-C. Rafi N. Ramanic A.M. Prockop D.J. J. Biol. Chem. 1994; 269: 11381-11390Abstract Full Text PDF PubMed Google Scholar, 4Colige A. Li S. Sieron A.L. Nusgens B.V. Prockop D.J. Lapière C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2374-2379Crossref PubMed Scopus (155) Google Scholar). The C-propeptide domain of procollagen participates in the association of the two proα1 and one proα2 chains to initiate triple helix formation from the COOH terminus of the protein (5Doege K.J. Fessler J.H. J. Biol. Chem. 1986; 261: 8924-8935Abstract Full Text PDF PubMed Google Scholar, 6Lees J.F. Tasab M. Bulleid N.J. EMBO J. 1997; 16: 908-916Crossref PubMed Scopus (123) Google Scholar, 7McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (97) Google Scholar). A number of functions have been proposed for the α1(I) N-propeptide in the biogenesis of type I collagen, including prevention of premature intracellular molecular association and facilitation of transcellular transport and secretion, conversion of procollagen to collagen, regulation of extracellular fibrillogenesis, and feedback regulation of procollagen synthesis. However, none of these functions has been established unequivocally, and some have been questioned. Lee et al. (8Lee S.T. Lee S. Peters D.P. Hoffman G.G. Stacey A. Greenspan D.S. J. Biol. Chem. 1992; 267: 24126-24133Abstract Full Text PDF PubMed Google Scholar) studied the secretion of mutated type I procollagen, generated from human cDNA genes that were transfected into Chinese hamster lung, Mov-13, and COS-7 cells. Whereas wild-type (WT)1 procollagen was secreted efficiently, proteins lacking either the entire N-propeptide (139 amino acids) or the majority of it (114 amino acids) were secreted poorly from Chinese hamster lung cells. In contrast, the WT and mutant proteins were secreted equally well by Mov-13 and COS-7 cells. Because Chinese hamster lung cells are epithelial-like, whereas Mov-13 and COS-7 cells are fibroblast-like, the failure of the former to secrete a collagen lacking N-propeptides may be unrelated to the structure of the protein. In all cases, triple helical assembly of the transfected gene products occurred normally. Similar experiments indicated that murine proα1 chains (marked by a substitution of Ile for Met-822) with a deletion of exon 2 were secreted normally and without post-translational overmodification by 3T6 cells (9Fenton S.P. Protein Engineering to Study Collagen Structure and Junction. University of Melbourne, 1996Google Scholar). Exon 2 encodes 65 of the 129 amino acids in the murine α1 N-propeptide, which comprise the “globular” domain of the propeptide, and include all 10 of the conserved cysteines. A great deal of evidence points to a correlation between synthesis of the molecular chaperone, HSP47, and type I collagen (10Nagata K. Trends Biochem. Sci. 1996; 21: 23-26Abstract Full Text PDF Scopus (249) Google Scholar, 11Nagata K. Matrix Biol. 1998; 16: 379-386Crossref PubMed Scopus (160) Google Scholar). HSP47 has been postulated to promote triple helix formation in the endoplasmic reticulum by binding and stabilizing partially folded triple helical intermediates of procollagen, thus inhibiting the intracellular aggregation and degradation of the precursor and facilitating its transcellular transport and secretion (12Koide T. Asada S. Nagata K. J. Biol. Chem. 1999; 274: 34523-34526Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 13Koide T. Aso A. Yorihuzi T. Nagata K. J. Biol. Chem. 2000; 275: 27957-27963Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). On the other hand, interactions between HSP47 and peptide sequences in the N-propeptide have also been demonstrated (14Hu G. Gura T. Sabsay B. Sauk J. Dixit S.N. Veis A. J. Cell. Biochem. 1995; 59: 350-367Crossref PubMed Scopus (37) Google Scholar). The latter observations are supported by studies in which Mov-13 fibroblasts were stably transfected with a control or a mutant Col1a1 gene that lacked exon 2. Coimmunoprecipitation experiments indicated that binding of HSP47 to the mutant collagen was reduced compared with the control, thus implicating the N-propeptide indirectly in binding to HSP47 (9Fenton S.P. Protein Engineering to Study Collagen Structure and Junction. University of Melbourne, 1996Google Scholar). Because failure to remove the N-propeptide from procollagen results in formation of abnormal collagen fibrils, both in humans with type VII Ehlers-Danlos syndrome (15Smith L.T. Wertelecki W. Milstone L.M. Petty E.M. Seashore M.R. Braverman I.M. Jenkins T.G. Byers P.H. Am. J. Hum. Genet. 1992; 51: 235-244PubMed Google Scholar) and in animals with dermatosparaxis (16Lenaers A. Ansay M. Nusgens B. Lapière C.M. J. Biochem. 1971; 23: 533-543Google Scholar), consideration has been given to the role of the N-propeptide in regulating both NH2-terminal proteolysis and fibrillogenesis. There is good evidence that the triple helical conformation of collagen is required for efficient proteolysis by procollagen N-proteinase (PNP; 2), but the role of the N-propeptide in the proteolytic event is uncertain. An indication that the N-propeptide is not essential for proteolysis is provided by the demonstration that a recombinant homotrimeric protein, composed of three proα2(I) chains with shortened triple helices, was at least partially cleaved by exogenous PNP (17John D.C.A. Watson R. Kind A.J. Scott A.R. Kadler K.E. Bulleid N.J. Nat. Biotechnol. 1999; 17: 385-389Crossref PubMed Scopus (75) Google Scholar). Proα2(I) chains naturally lack the sequence encoded by exon 2 in proα1(I). However, these results leave open the possibility that the presence or conformation of the N-propeptide could modulate this proteolytic event. A feedback regulatory role for the N-propeptide, after its release from procollagen by PNP, was originally suggested by the observation that bovine dermatosparactic fibroblasts, which have a defect in PNP activity, synthesize higher amounts of collagen than control cells (18Wiestner M. Krieg T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with a feedback a of the N-propeptide reduced collagen synthesis it was to bovine or human fibroblasts (18Wiestner M. Krieg T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). it was that this peptide the of I and in a J. PubMed Scopus Google Scholar, J. S.H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and that the of bovine cells with a the N-propeptide reduced collagen synthesis by these cells J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The for these are not The have also been implicated in feedback regulation of procollagen synthesis C.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. R. PubMed Scopus Google Scholar), but of this has been In of the role of the N-propeptide in the biogenesis and regulation of type I collagen synthesis, we a targeted deletion of exon 2 in the Col1a1 collagen gene and generated mice that lacked the globular of the N-propeptide encoded by this exon were in this by the that in the of exon of and the of the protein. the that the protein encoded by a proα1(I) cDNA gene that lacked exon 2 was at least by some that the of the mutant not be embryonic on the of to secrete type I Mouse lines were established on both a mixed 129 and C57BL/6 background and on a pure 129 OlaHsd/Sv background. in this mixed background mice that lack the amino sequence encoded by exon 2 of the proα1(I) chain are normal in appearance and are fertile. To the extent that these mice have been studied, it that none of the functions to the N-propeptide has been by its to synthesis of type I collagen or to its function as a protein. However, homozygous mutant 129 background mice were generated at only 64% of the by The N-propeptide may some essential function in embryonic which is to the of 129 of the Col1a1 were provided by and were to a and the exon from are in 2. Exon 2 was by of of Col1a1 at and and of the a that in of the A by was in the to generate a with of and of with the The exon also a exon which a but not the amino sequence of the protein. The and of the have been R. R. Cell. Biol. 1998; PubMed Google Scholar, Matrix Biol. 1999; PubMed Scopus Google Scholar). stem cells from 129 mice from T. were on cells in The was with 2 amino acids and of cells the exon was as L.T. Li M. J. Biol. 1998; PubMed Scopus Google Scholar). 2 cells were with of exon and were to in were and were by for of the to the The for be from which also the of the and the used in the of and 2. Because we a correlation between and J. S. C.M. R. 1997; PubMed Scopus Google Scholar), we the of targeted and with a normal of for mice, generated after of were to heterozygous exon These mice were with 129 mice the the control of the provided by to homozygous and heterozygous exon were by both and and a of from the WT and a from the exon was of with cells were in with bovine 2 and amino acids cells were transfected with of were in which was after the studies in of the WT and exon were into the The is to the and the is in exon The exon also the in exon The was to from of the by the were of was used for synthesis of the cDNA 10 of and were used for of the The of is a sequence used in other the of the is from the Col1a1 cDNA sequence and is to the sequence that the exon was from of WT and exon mice the was the of and of were at 65 for 10 by on for 2 and of reverse were and the was at for The was by at for was with the in exon and the reverse in exon of were at of lung, and were from mice at of were in in and with and for was as L.T. Li M. J. Biol. 1998; PubMed Scopus Google Scholar). the was in in and in the was with and with were with and and with a was from mice with of were into and with animals was from the and A was for of The was and in of at with was by at for and in of was at with An of collagen was to by at for in The was with and was by the of to a of and at for The was by the of to with and in a at The of collagen in was by with a fibroblasts were from and WT and exon mice as Biol. Cell. 2000; PubMed Scopus Google Scholar). were to with and at for in with and In some experiments the was also with The was was and the was into two was for 2 at with of The was in the of were by in by and in of into proteins secreted into the was by and the proteins were by and mice were to as J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). were with on WT and exon mice mice of were at and and the were with a of of were in to in in and were with and or with The for the of a Col1a1 that exon 2 is and in 2. Because with the of other genes indicated that the of a kinase into the and with at a in targeted we with this the exon were by of in mutant a of and WT a of with of of from targeted with 2 provided that the structure of the Col1a1 gene was as targeted a of and WT a of not were on targeted to a normal and two with a in and were into These were into the of were from and these were with both C57BL/6 and 129 and were used to of the However, of these heterozygous animals to homozygous exon failure of by the transcription Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), which in embryonic of homozygous mutant heterozygous mutant mice were with mice and for the presence or of and by In all mice were of the by not of these mice homozygous exon mice, as by both and not of mixed background heterozygous mice and homozygous exon animals in the expected of However, breeding of pure 129 OlaHsd/Sv background heterozygous mice than homozygous mutant was equally for mutant mice generated from and that were at of we and homozygous exon homozygous mutant mice were generated at only 64% of the expected frequency. There was indication that exon mice after To the of exon 2 in the Col1a1 gene the or function of type I collagen, it was to that the was of the sequence could to degradation of 2000; PubMed Scopus Google Scholar). transfected cells with a the normal sequence of in gene or with a lacking exon 2. of a reverse WT with and generate a whereas of a with a deletion of the exon 2 sequence generate a provided that of the occurred normally. in a of the by of exon to exon was with the mutant collagen whereas the proα1(I) sequence the expected of sequence of a with a in from the used to generate the deletion of exon 2 and a reverse in the formation of a a in of the in 2. The which is between the and 2 is by a from and a of results were from the sequences of from exon mice generated from either or The evidence for of the was by sequence of the of reverse from of mutant and control in to the WT the exon in a protein chain in which the amino sequence in exon is to that in exon The composed of from exon and 2 from exon to the amino as the from the normal of exon to exon 2. lacking the 65 amino acids encoded by exon 2 normal on and are fertile. The is not and is indication of of of the by a normal appearance and of collagen as by with and and not There were in the collagen of or in the of collagen that was with from the of mutant and WT mice These findings a indication that are normally in the of the protein sequence encoded by exon 2. the of of was normal in exon mice, and of the at and not These experiments and were with mixed background of collagen from was with at in in all and between and in are the in a Collagen was with at in in all and between and in are the studies of a in the of collagen and a for in in from exon and in The were in and were in the to the The of the 65 amino acids encoded by exon 2 N-propeptide of amino which be released by PNP the collagen α1(I) chain is to its normal NH2-terminal sequence of collagen by indicated that the of α1(I) chains from WT and exon type I was the The presence of of in the exon α1(I) chains have been as a reduced of this is of that the exon collagen in in chains compared with that from WT The of this observation is not However, the could a of the gene and a in formation of the N-propeptide, which is in amino sequence to the α1(I) is that the N-propeptide on exon α1(I) chains could have been released by limited proteolysis by during of the collagen from evidence that proteolysis not was provided by NH2-terminal sequence of exon α1(I) amino sequence of after from to indicated a terminus not of collagen chains by PNP normally at a and the NH2-terminal to which be by the that exon α1(I) chains NH2-terminal that is generated normally by feedback of collagen synthesis by released N-propeptides occurred in one that fibroblasts from exon in synthesis and secretion of type I procollagen and intermediates in However, as in was in the and of on of from WT and exon cells. The between chains by the two not be in this In a of of for for WT cells and for exon cells. that feedback of procollagen synthesis by the N-propeptide not in fibroblasts the in this However, we have not established that of N-propeptide were by WT fibroblasts during the of this and it is also that the N-propeptide, generated in the of exon 2 has some have used gene by of the gene with the to generate proα1(I) chains with a deletion of the exon 65 amino acids that comprise the NH2-terminal globular domain of the N-propeptide Because was a for or of the that from deletion of exon we that this was in a in which a the was by into cells. The of the expected was by sequence of both and cDNA from mutant Exon I procollagen was secreted normally as by the of of into proteins in the of fibroblasts and by the of these proteins on The N-propeptide was also cleaved normally by PNP, as suggested by the normal of α1 chains from mutant collagen on and by the expected of these of collagen by occurred as by of the of collagen with in mutant and WT Collagen to be normal by and were only slightly as by in exon The of was also normal in these was evidence that the of the N-propeptide to procollagen synthesis, it these was by the lack of its globular NH2-terminal The of studies of the structure and function of type I procollagen was by a failure to between the of the N- and a of the W. The Google Scholar). it that association, and facilitation of triple helix formation during molecular assembly were that be to the than to N-propeptides (5Doege K.J. Fessler J.H. J. Biol. Chem. 1986; 261: 8924-8935Abstract Full Text PDF PubMed Google Scholar, 7McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (97) Google Scholar). However, were that the N-propeptides function to intracellular fibrillogenesis and to in the aggregation and of collagen during the formation of extracellular These may have in observations that dermatosparactic which are in PNP A. Sieron A.L. Li S.W. U. Petty Wertelecki W. W. W. Byers P.H. Lapière C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and the N-propeptide on a of α1 in collagen formation (16Lenaers A. Ansay M. Nusgens B. Lapière C.M. J. Biochem. 1971; 23: 533-543Google Scholar). a role for the N-propeptide of type I procollagen in regulation of extracellular was from of the propeptide in association with in human R. R. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). intermediates either N- or and could also be demonstrated in both during and by and although was indication that these intermediates were involved in regulation of fibrillogenesis R. R. J. PubMed Scopus Google Scholar). studies of exon fibroblasts normal of secretion of type I for the globular domain of the N-propeptide in the of intracellular collagen However, the of abnormal collagen in the of exon mice the of and R. R. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. R. J. PubMed Scopus Google Scholar) that the N-propeptide a role in the of extracellular collagen fibrillogenesis. the of the N-propeptide has been its after its release by PNP, to function as a feedback of procollagen synthesis. for a function was provided by the observation that N-propeptide, from dermatosparactic collagen by reduced collagen synthesis by bovine fibroblasts in and that dermatosparactic bovine fibroblasts, which are in PNP activity, synthesized collagen in than control cells (18Wiestner M. Krieg T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Krieg T. R. EMBO J. PubMed Scopus Google Scholar). it was that bovine N-propeptide was of inhibiting the of in J. PubMed Scopus Google Scholar, J. S.H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), whereas N-propeptide reduced procollagen in human fibroblasts J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google Scholar). et al. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) transfected a N-propeptide into cells and a in type I collagen synthesis in these cells. Because the lacked a peptide the was in the and this was by with a to a encoded by the However, in to on these observations we have been to that human in and in was of inhibiting collagen synthesis by a of human and On the other hand, cells were stably transfected with a the N-propeptide, synthesis of the protein after only a transcription of the transfected as by the A of the latter which be in with the findings of et al. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), is that N-propeptide its synthesis. the results of in experiments the findings in this which are to the that for the feedback of the N-propeptide, not a role for the globular domain of the N-propeptide in the regulation of collagen synthesis. the globular domain of the N-propeptide not function in of the in collagen synthesis or fibrillogenesis, role it play in that a conserved domain of 65 amino acids be from type I procollagen, or other protein, without some has been proposed that the type collagen N-propeptide is involved in regulation of by with protein 2 α1 Y. A. J. Biol. 1999; PubMed Scopus Google Scholar). Exon 2 of the which also encodes the globular domain of the type N-propeptide, is in a that may be J. Biol. Chem. Full Text PDF PubMed Google Scholar, N. J. Biol. PubMed Scopus Google Scholar, A.M. 1994; PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google Scholar, B. T. PubMed Scopus Google Scholar). The of the 10 in the N-propeptide and the of other amino acids are conserved not only in the α1 N-propeptides of and but also in and protein, and its protein the on formation of in Genet. 1996; PubMed Scopus Google Scholar, Trends Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and the of protein J. B. S. E.M. 2000; PubMed Google Scholar). protein and play in the formation of the is of that type procollagen which the exon 2 has into and that the exon 2 sequence is required for this J. B. S. E.M. 2000; PubMed Google Scholar). that the type I collagen N-propeptide could play role in and in processes in other type I to the role proposed for the type collagen N-propeptide in In exon mice, this function be provided in by of the The failure of homozygous exon mice to be generated in the expected from the of heterozygous mutant 129 background mice a role for the N-propeptide in some during genes in this to the to type collagen synthesis, could a of exon mice to On the other hand, a sequence of the that are encoded by exon 2 of gene that the amino sequences in mice and from the sequences in other than sequences from as and not is not for a of the in type is that the globular domain of the α1(I) N-propeptide a function in for for of the for with NH2-terminal amino for with for with the and and of for and a of the
Börnstein et al. (Tue,) studied this question.