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PubMed1982

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.

Animals↗
PubMed2006

The interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1.

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.

Animals↗
PubMed1985

Procollagen secretion meets the minimum requirements for the rate-controlling step in the ascorbate induction of procollagen synthesis.

Ascorbate addition to primary avian tendon cells has been shown previously to cause a approximately 6-fold increase in procollagen translation that is first observable after 4 h and reaches a maximum level after 48 h. Similarly, procollagen mRNA has been shown to increase after ascorbate addition by approximately 6-fold starting at 12 h and reaching a maximum level by 72 h. The rate constant for procollagen secretion is now shown to also react to ascorbate by a 6-fold change. This results in a drop in the half-life of procollagen within the cell from 120 to 20 min. In sharp contrast to the other steps in the procollagen pathway, the change in the secretion rate constant is extremely fast occurring in less than 30 min. Moreover, after ascorbate addition, greater than 80% of the internal procollagen can be secreted at the fast rate. Since this change results from an increase in hydroxylation of proline residues and since the hydroxylation reaction has been localized to the endoplasmic reticulum, this evidence strongly supports the model that the slow step in the secretion pathway is transport out of the endoplasmic reticulum. Further support for this comes from electron microscope autoradiography of [3H]proline-labeled cells where the labeled procollagen pool within the cells was highly localized to the endoplasmic reticulum.

2,2'-Dipyridyl↗
PubMed1981

Procollagen production and procollagen messenger RNA levels and activity in human lung fibroblasts during periods of rapid and stationary growth.

The production of procollagen molecules by human diploid fetal lung fibroblasts (HFL-1 cells) remains constant in both rapid and stationary growth phases. However, log phase cells degrade 3-fold more newly synthesized collagen inside the cell prior to secretion than do stationary phase cells. Procollagen mRNA levels, measured by hybridization with a type I procollagen mRNA-specific complementary DNA, are approximately 2-fold higher in confluent cells than in log phase cells. There are no significant differences in the ability of either log phase or confluent HFL-1 cell procollagen mRNA to be translated in an in vitro cell-free translation system. Therefore, the ability of HFL-1 cells to maintain constant collagen production irrespective of the growth status of the cells results from the combined action of a number of regulatory mechanisms, including changes in procollagen mRNA levels, the utilization of procollagen mRNA, and intracellular procollagen degradation.

Cell Division↗
PubMed2002

Inhibition of type I procollagen production in photodamage: correlation between presence of high molecular weight collagen fragments and reduced procollagen synthesis.

Three-dimensional lattices of reconstituted, polymerized type I collagen were subjected to partial hydrolysis by organ culture fluid from human skin or by various matrix metalloproteinases, including matrix metalloproteinase-1 (interstitial collagenase), -2 (72 kDa gelatinase A), -8 (neutrophil collagenase), -9 (92 kDa gelatinase B), or -13 (collagenase 3). Following partial digestion, human dermal fibroblasts were incubated on the enzyme-treated or control lattices and examined for ability to contract the collagen lattice and synthesize type I procollagen. Collagen lattices partially degraded by organ culture fluid were contracted by fibroblasts under conditions in which control collagen lattices were not. On the partially degraded collagen, fibroblasts synthesized reduced amounts of type I procollagen (approximately 70% reduction). Purified matrix metalloproteinases with collagenolytic activity duplicated the effects of the human skin organ culture fluid, although matrix metalloproteinases 8 and 13 were less efficient than matrix metalloproteinase-1 (65% vs 40% and 18% reduction in type I procollagen production for matrix metalloproteinases 1, 8, and 13, respectively). Matrix metalloproteinases 2 and 9 were without effect on intact collagen; however, when collagen lattices were subjected to digestion by a combination of matrix metalloproteinases 1 and 9, fragments produced by matrix metalloproteinase-1 were further degraded by the gelatinase. Collagen contraction and inhibition of procollagen synthesis were both reduced. Matrix metalloproteinase-2 was less effective than matrix metalloproteinase-9 in clearing matrix metalloproteinase-1-generated fragments. Matrix metalloproteinase-2 was also less effective in preventing contraction and inhibiting the downregulation of type I procollagen synthesis. These observations suggest that in the presence of high molecular weight fragments of type I collagen, type I procollagen synthesis is inhibited. As these fragments are processed further, there is less inhibition of type I procollagen production.

Cell Size↗
PubMed1988

Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts.

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.

Animals↗
PubMed1985

Altered steady-state ratio of type I/III procollagen mRNAs correlates with selectively increased type I procollagen biosynthesis in cultured keloid fibroblasts.

Regulation of collagen gene expression was studied in fibroblast cultures established from patients with keloids, fibrotic lesions of the skin. In selected keloid fibroblast cultures, an overproduction of type I procollagen was observed. This increase was accompanied by a parallel increase in type I procollagen-specific mRNA levels, as detected by dot-blot and RNA transfer hybridizations, without concomitant change in type I procollagen gene copy number. At the same time, type III procollagen mRNA levels were unaltered, resulting in markedly elevated type I/III procollagen mRNA ratios. Thus, keloid fibroblasts offer a unique model to study the independent regulation of the gene expression of two genetically distinct procollagens, type I and type III.

Cells, Cultured↗
PubMed1994

Substitution of serine for glycine 883 in the triple helix of the pro alpha 1 (I) chain of type I procollagen produces osteogenesis imperfecta type IV and introduces a structural change in the triple helix that does not alter cleavage of the molecule by procollagen N-proteinase.

Type I procollagen secreted by dermal fibroblasts from an individual with osteogenesis imperfecta type IV was a mixture of normal molecules and molecules that were post-translationally overmodified. The individual was heterozygous for a G to A transition in the COL1A1 gene that resulted in the substitution of serine for glycine 883 in one or both of the pro alpha 1 (I) chains. The thermal stability of molecules containing overmodified chains was lower by 2 degrees C than that of normal molecules. However, following cleavage of the molecules with vertebrate collagenase, the temperature of denaturation of the overmodified A fragments (residues 1-775 of the helix did not contain the substitution) was 2 degrees C greater than that of A fragments from normal molecules. The rates of cleavage by procollogen N-proteinase (EC 3.4.214.14) (N-proteinase) of procollagen molecules in normal and osteogenesis imperfecta samples were not significantly different. The procollagen molecules in the osteogenesis imperfecta sample were also indistinguishable from those in control samples by rotary shadowing electron microscopy. The results suggest that this substitution of serine for glycine in the alpha 1 (I) chain of procollagen, like the substitution of aspartate for the same glycine previously described (Lightfoot, S. J., Holmes, D. F., Brass, A., Grant, M. E., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 25521-25528), can alter the structure of the triple helix N-terminal to the site of the substitution. However, in contrast to the aspartate for glycine substitution, the structural change is insufficient to delay the cleavage of the procollagen by N-proteinase and results in a mild rather than lethal phenotype.

Amino Acid Sequence↗
PubMed1998

Transcriptional activation of the alpha1(I) procollagen gene and up-regulation of alpha1(I) and alpha1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice.

OBJECTIVE: To investigate the levels of expression of type I and type III collagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mice and to examine the transcriptional regulation of the alpha1(I) procollagen gene (COL1A1) in these cells. METHODS: Dermal fibroblasts from Tsk2 mice and from normal age- and sex-matched control mice were studied. Steady-state levels of alpha1(I) and alpha1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot-blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of the COL1A1 promoter ligated to the chloramphenicol acetyltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift assays were performed with nuclear extracts prepared from normal and Tsk2 dermal fibroblasts. RESULTS: Synthesis of collagen was almost 100% higher in Tsk2 dermal fibroblasts than in control fibroblasts. Up-regulation of mRNA for 2 extracellular matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the alpha1(I) procollagen mRNA steady-state levels were 50% higher, and those of the alpha1(III) procollagen mRNA 100% higher, in Tsk2 cells. The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of alpha1(I) collagen mRNA in Tsk2 cells were largely due to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing a relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2 fibroblasts compared with normal fibroblasts. Competition experiments using consensus Spl and nuclear factor 1 (NF-1) oligonucleotides and supershift experiments using anti-Sp1 and anti-NF-1 antibodies indicated that at least 2 transcription factors, Sp1 and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts. CONCLUSION: Dermal fibroblasts from Tsk2 mice display increased collagen synthesis and up-regulation of alpha1(I) and alpha1(III) procollagen mRNA in vitro. The data also directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest that the transcription factors Sp1 and NF-1 or their homologs play an important role in the upregulated expression of this gene in Tsk2 fibroblasts. These findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study of the connective tissue alterations in this disease.

Animals↗
PubMed1997

Molecular cloning of the bovine alpha 1(IV) procollagen gene (COL4A1) and its use in investigating the regulation of expression of type IV procollagen by retinoic acid in bovine lens epithelial cells.

This report is the first to describe the isolation of a 400 base pair cDNA clone encoding part of the bovine alpha 1(IV) procollagen. Using the polymerase chain reaction (PCR), we have amplified a sequence of approximately 400 bp from this gene within the recombinant phage DNA. The cloned sequence encodes 94 amino acids that form part of the protein's helical region. The sequence contains one interruption in the Gly-Xaa-Yaa repeat unit. The third base of the codon for glycine at several sites differs from those seen in murine and human genes, as does the third base of proline codons. The bovine cDNA also contains fewer thymine residues. Northern blot hybridization has shown that the mRNA for bovine procollagen to be 6.2 kb in size. We have used the cDNA clone to investigate the effect of all-trans retinoic acid (RA) on the gene expression of alpha 1(IV) procollagen in cultured bovine lens epithelial (LE) cells. We have also observed that RA decreases total protein production and concomitantly increases type IV procollagen in a concentration dependent manner. An increase in alpha 1(IV)mRNA as well as increase in type IV procollagen suggest that the regulation of alpha 1(IV) gene by RA in the LE cells is at the transcriptional level. Further, our results support the hypothesis that RA inhibition of lens epithelium transformation to fibroblast-like cells may be due to the ability of RA to stimulate the production of basement membrane components by epithelia.

Amino Acid Sequence↗
PubMed1982

Partial purification of a procollagen C-proteinase. Inhibition by synthetic peptides and sequential cleavage of type I procollagen.

A procollagen C-proteinase which cleaves the C-propeptides from type I procollagen was purified about 125-fold from membranous bones of chick embryos. As estimated by gel filtration, the enzyme was about 80 000 daltons. When a reaction with modified procollagen was carried out, the enzyme preferentially cleaved the C-propeptides from the pro alpha chains in the order pro alpha 1, pro alpha 1, and then pro alpha 2. The enzyme was inhibited by several metal chelators and high concentrations of dithiothreitol. It was also inhibited by 5% fetal calf serum. A series of inhibitors of serine proteinases and sulfhydryl-containing proteinases were not inhibitory. Four oligopeptides were synthesized with amino acid sequences similar to the amino acid sequences around the sites at which the C-propeptides are cleaved during the conversion of procollagen top collagen in vivo. The peptide Tyr-Tyr-Arg-Ala-Asp-Asp-Ala inhibited the enzyme 35--60% in concentrations of 6--12 mM. Shorter peptides containing the Ala-Asp bond cleaved by the enzyme were less effective. The partially purified enzyme was also found to cleave the C-propeptides from type II and type III procollagens.

Animals↗
PubMed1995

Determination of various cytokines and type III procollagen aminopeptide levels in bronchoalveolar lavage fluid of the patients with pulmonary fibrosis: inverse correlation between type III procollagen aminopeptide and interferon-gamma in progressive patients.

We measured levels of cytokines and type III procollagen aminopeptides (procollagen III peptides) in bronchoalveolar lavage fluid obtained from 20 patients with stable pulmonary fibrosis (PF) and seven patients with progressive PF, and nine control subjects to determine the role of cytokines in the development of PF. Procollagen III peptide levels were markedly increased in progressive PF patients. Tumour necrosis factor-alpha, interleukin-6, transforming growth factor-beta and interferon-gamma (IFN-gamma) levels were elevated in both PF patients as compared with controls, with a tendency of higher levels in progressive patients, whereas interleukin-1 beta (IL-1 beta) level was decreased in both PF patients. When the correlation between procollagen III peptide and various cytokine levels was analysed the only significant correlation was inversely between procollagen III peptide and IFN-gamma in progressive PF patients. These results indicated that although multiple cytokines may be involved in the development of PF, the negative role of IFN-gamma in active collagen synthesis could be also important.

Adult↗
PubMed1988

Low production of procollagen III by skin fibroblasts from patients with Ehlers-Danlos syndrome type IV is not caused by decreased levels of procollagen III mRNA.

The Ehlers-Danlos syndrome type IV represents a heterogeneous group of diseases, the molecular defect of which seems to reside in a defective synthesis and processing of collagen III. Here we present data concerning both protein and mRNA levels of collagens I and III in cell cultures established from skin of nine patients and six controls. All patients investigated were characterized by a reduced amount of procollagen III in fibroblast cultures. However, the levels of collagen III propeptides, measured by a radioimmunoassay in culture medium of fibroblasts from the patients, were either decreased or in the range of control levels. Using hybridization with cDNA probes specific for pro alpha 1 (I) and pro alpha 1 (III) collagen chains we determined the ratios of procollagens I and III mRNA. Although synthesis of procollagen III was markedly reduced in fibroblasts of all patients studied, no parallel decrease of procollagen III specific mRNA was found. However, two distinctive groups of patients were identified, one with an unaltered ratio of procollagen I/III mRNA and the other which had an even higher proportion of collagen III mRNA.

Cells, Cultured↗
PubMed1997

Serum levels of carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen and laminin P1 in Duchenne muscular dystrophy.

The striking proliferation of connective tissue in Duchenne muscular dystrophy is attributed, besides other components of the extracellular matrix, to an increase of endomysial and perimysial type III and type I collagen. We investigated if muscle fibrosis correlates to an increased serum concentration of procollagen I or III. Therefore, we measured the serum levels of carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen and laminin P1 in 20 boys with progressive muscular dystrophy (16 definite Duchenne muscular dystrophy, 2 suspected of Duchenne muscular dystrophy, 2 Becker muscular dystrophy). In contrast to collagen I and III the expression of laminin in the basement membrane is known to be normal in Duchenne muscular dystrophy. There was no significant alteration of serum concentration of procollagen III N-peptide, procollagen I C-peptide and laminin P1 in boys with Duchenne muscular dystrophy. Measuring these parameters is not useful for investigating the extent of muscle fibrosis or for monitoring the effect of therapeutic trials such as steroid treatment.

Adolescent↗
PubMed1979

Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method.

Frozen sections of the growing end of the rat incisor tooth were exposed to antisera or affinity prepared antibodies against partially purified type I, II, or IV procollagen in the hope of detecting the location of the corresponding antigens by the peroxidase-anti-peroxidase technique. The distribution of immunostaining was similar with antisera as with purified antibodies of a given type, but differed for each type; that is, predentin, odontoblasts, pulp and periodontal tissue were the sites of type I; blood vessel walls, pulp and periodontal tissue, of type III; and basement membranes, of type IV antigenicity. It was demonstrated, at least in cases of type I and III, that immunostaining detected the corresponding procollagens and related substances, but not the corresponding collagens. The interpretation of these observations is that: 1) odontoblasts elaborate procollagen I for release to predentin and subsequent transformation to dentinal collagen I; 2) pulp and periodontal cells produce procollagens I and III which presumably become collagens I and III respectively, while the adventitial cells of blood vessels give rise to collagen III; and 3) procollagen IV is associated with basement membranes and, occasionally, adjacent cells.

Animals↗
PubMed1979

Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content.

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.

Animals↗
PubMed1997

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Animals↗
PubMed1992

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.

Adult↗