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PubMed1999

Increased tropoelastin and procollagen expression in the lung of nitrofen-induced diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: Collagen and elastin, the predominant components of the lung connective tissue network, have been suggested to have an important influence on lung compliance and maximal expansion. Decrease in lung compliance and distensibility often is seen in human congenital diaphragmatic hernia (CDH) lung as well as in experimentally produced CDH lung. The aim of this study was to investigate mRNA levels of tropoelastin and alpha1 (I) procollagen, the precursors of elastin, and type I collagen, respectively, in CDH lung and to determine whether antenatal dexamethasone treatment has any effect on the production of these extracellular matrix proteins. METHODS: CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (0.25 mg/kg) was given on day 18.5 and 19.5. Cesarean section was performed on day 21. The fetuses were divided into three groups: group I, normal controls; group II, nitrofen-induced CDH; and group III, nitrofen-induced CDH with antenatal dexamethasone treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate relative amounts of tropoelastin and alpha1 (I) procollagen mRNA. RESULTS: Levels of both tropoelastin and alpha1 (I) procollagen mRNA were significantly increased in group II compared with group I (P< .05). Neither tropoelastin nor alpha1 (I) procollagen mRNA levels were significantly different between group II and III. CONCLUSIONS: The increased local synthesis of tropoelastin and type I procollagen in CDH lung may be responsible for the increased rigidity and decreased compliance observed in the CDH hypoplastic lung. Glucocorticoids have no effect on pulmonary tropoelastin and alpha1 (I) procollagen gene expression in CDH lungs.

Animals↗
PubMed2001

Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha1(I) and TIMP-1.

BACKGROUND/AIMS: Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism. METHODS: Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen alpha1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) beta1 were determined by multi-probe ribonuclease protection assay. RESULTS: After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen alpha1(I), TIMP-1 and TGFbeta1 (P < 0.01). Silymarin downregulated elevated procollagen alpha1(I), TIMP-1 and TGFbeta1 mRNA levels by 40-60% (P < 0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III. CONCLUSIONS: Silymarin suppresses expression of profibrogenic procollagen alpha1(I) and TIMP-1 most likely via downregulation of TGFbeta1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

Animals↗
PubMed1993

The effect of interferon alpha and prednisolone on the serum concentrations of the aminoterminal domain of procollagen type III(PIIIP) and type IV (7-S collagen) in patients with chronic hepatitis B.

Since interferons are reported to inhibit collagen synthesis in vivo and in vitro, they are possible anti-fibrogenic drugs. However, little is known about the effect of interferons on connective tissue metabolism in liver disease. To investigate the effect, serum concentrations of the aminoterminal domain of procollagen type III and type IV (7-S collagen) were measured in 16 patients with chronic hepatitis B during alpha interferon treatment. The levels were also determined during prednisolone withdrawal treatment to investigate the effect on hepatic collagen turnover. In patients with chronic hepatitis B, the serum levels of both markers were significantly elevated before any treatment (procollagen type III: 1.10 +/- 0.39 U/ml, 7-S collagen: 6.69 +/- 3.71 ng/ml) compared with levels in 41 healthy volunteers (pro-collagen type III: 0.53 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.72 +/- 0.57 ng/ml, P < 0.001). Both levels were even more elevated during interferon therapy (procollagen type III: 1.36 +/- 0.58 U/ml, P < 0.05; 7-S collagen: 8.77 +/- 4.68 ng/ml, P < 0.01). On the other hand, serum procollagen type III and 7-S collagen levels were both decreased during prednisolone treatment (procollagen type III: 0.62 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.66 +/- 1.69 ng/ml, P < 0.01). These preliminary data suggest that interferon alpha enhanced, but prednisolone inhibited the turnover of procollagen type III and IV in the liver. Further study is required to interpret this observation in relation to anti-fibrotic therapy.

Adolescent↗
PubMed2002

Photoprotection prevents TPN-induced lung procollagen mRNA in newborn guinea pigs.

BACKGROUND: Photo-exposed intravenous multivitamin solutions (MVP) carry a peroxide load. Peroxidation induces gene expression of procollagen. We hypothesized that photo exposure of the MVP solution might promote pulmonary fibrosis. The aim of the study was to assess the potential for MVP to increase procollagen mRNA. METHODS: Three day old guinea pigs were assigned to the following intravenous regimens, either: Control (C): 5% dextrose + 0.45% NaCl; C + 200 or 500 microM H(2)O(2); C + 500 microM H(2)O(2) + 10 microM GSSG; [C + 1% MVP +/- [amino acids + lipids]] +/- photoprotected. After 4 d, levels of pulmonary alpha1(I) procollagen mRNA and glutathione were determined. Results were compared by ANOVA. RESULTS: Photoprotection of MVP or TPN prevents light induction of procollagen mRNA. The effect of MVP + light was associated with a peroxide load coupled with a low glutathione level. This was also observed with the 500 microM H(2)O(2) group. The addition of GSSG prevented the increase of procollagen mRNA caused by H(2)O(2). CONCLUSION: An oxidant stress caused by the infusion of peroxides in an organism with a weak antiperoxide capacity induces the transcription of the gene encoding for procollagen alpha1(I). The results confirm the antiperoxide activity of lung glutathione. Parenteral nutrition could be a clinical condition favoring the initiation of lung fibrosis, especially in premature newborn infants who have low glutathione levels.

Amino Acid Sequence↗
PubMed1997

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

Amino Acid Sequence↗
PubMed1982

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos.

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.

Amino Acids↗
PubMed1977

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.

Animals↗
PubMed1999

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Amino Acids↗
PubMed1991

Cyclosporine induces elevated procollagen alpha 1 (I) mRNA levels in the rat renal cortex.

Chronic cyclosporine nephrotoxicity is a poorly understood drug side-effect characterized by renal cortical interstitial scarring. To evaluate procollagen mRNA levels as an early factor in the development of this form of renal fibrosis, we measured renal procollagen alpha 1 (I), alpha 1 (III), alpha 1 (IV) and beta-actin mRNA levels in rats treated with cyclosporine (CsA) or the olive oil vehicle (OO) for one or four weeks. Renal morphology was similar without atrophy or fibrosis in one week CsA and OO and four week OO rats. Four week CsA rats had focal cortical interstitial fibrosis and tubular atrophy. Cortical procollagen alpha 1 (I) mRNA levels were increased in CsA versus OO rats at one week (P less than 0.02) and four weeks (P less than 0.02). One week medullary procollagen alpha 1 (I) and all other one week medullary, and one and four week cortical procollagen and beta-actin mRNA levels were no different in CsA versus OO rats. The early increase in renal cortical procollagen alpha 1 (I) mRNA levels precedes renal morphologic abnormalities, and may represent an important step in the pathogenesis of cyclosporine-induced renal cortical fibrosis.

Actins↗
PubMed1989

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.

Basement Membrane↗
PubMed1990

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

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.

Adult↗
PubMed1991

Effect of dextran on synthesis, secretion and deposition of type III procollagen in cultured human fibroblasts.

When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase-sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms.

Blotting, Northern↗
PubMed2000

Involvement of collagen-binding heat shock protein 47 and procollagen type I synthesis in idiopathic pulmonary fibrosis: contribution of type II pneumocytes to fibrosis.

The most common pathologic form of idiopathic pulmonary fibrosis is usual interstitial pneumonia, which is characterized by patchy fibrotic areas, marked increase in the number of fibroblasts and type II pneumocytes, and excessive deposition of extracellular matrix proteins, especially collagen. Heat shock protein 47 is a collagen-binding stress protein and has a specific role in intracellular processing of procollagen molecules as a collagen-specific molecular chaperone. However, its role in the causation of fibrosis in usual interstitial pneumonia is unknown. In this study, we examined the expression of heat shock protein 47 and type I procollagen in 12 patients with usual interstitial pneumonia by immunohistochemistry on sequential sections. Heat shock protein 47 was localized predominantly in alpha-smooth muscle actin-positive myofibroblasts and surfactant protein-A-positive type II pneumocytes in active fibrotic areas of usual interstitial pneumonia. Type I procollagen was also expressed in those cells. In contrast, heat shock protein 47 and type I procollagen were weakly or not at all expressed in myofibroblasts and type II pneumocytes in bronchiolitis obliterans organizing pneumonia and normal lung tissue samples obtained from excised lung cancer tissues. The numbers of heat shock protein 47- and type I procollagen-positive cells to type II pneumocytes or myofibroblasts were significantly higher in usual interstitial pneumonia than in bronchiolitis obliterans organizing pneumonia and normal lung tissue specimens. Our results suggest that myofibroblasts and type II pneumocytes play an important role in the progression of fibrosis through the induction of heat shock protein 47, which regulates the synthesis/assembly of type I procollagen in usual interstitial pneumonia. HUM PATHOL 31:1498-1505.

Actins↗
PubMed2000

Procollagen synthesis by fresh and cryopreserved rat pulmonary valve grafts.

OBJECTIVE: Allograft valves are frequently used in the repair of congenital cardiac anomalies. The failure rate may differ depending on the type of allograft used. Previous studies have shown that rat aortic valve grafts exhibit synthesis of procollagen, suggesting a capacity for repair and regeneration after implantation. No studies of pulmonary valve grafts in the heterotopic rat implant model have thus far been reported. This study was designed to investigate whether pulmonary valve grafts maintain in vivo viability, as demonstrated by procollagen synthesis, and whether cryopreservation, histocompatibility, or both affect this property. METHODS: Cryopreserved and fresh rat pulmonary valves were implanted into the abdominal aorta of syngeneic and allogeneic recipients. The grafts and native valves were excised 3 to 21 days after implantation. Valves were sectioned and immunohistochemically stained for procollagen. Computerized morphometry was used to calculate changes in intima, media, and adventitia as a percentage of cross-sectional area of the graft. Procollagen content was graded by semiquantitative methods. RESULTS: Pulmonary valve grafts had significantly greater collagen density in the intima and adventitia compared with native aortic and pulmonary tissues, but collagen density in the media was similar in all groups. The grafts demonstrated appreciably greater procollagen than the corresponding native valves. These findings were consistent in all grafts (ie, both fresh and cryopreserved, both syngeneic and allogeneic), irrespective of duration of implantation. CONCLUSIONS: Procollagen synthesis occurs in pulmonary valve grafts early after implantation, indicating viability of these tissues. This model of pulmonary valve implantation may have wide applicability to questions of allograft biology.

Animals↗
PubMed1983

On the state of aggregation of newly secreted procollagen.

Procollagen and partially processed procollagen from cultures of primary chicken embryo tendon cells appeared as segment-long-spacing (SLS)-like aggregates when drops of medium were negatively stained and examined by electron microscopy. Similar aggregates were obtained after negative staining of medium partially purified by gel filtration and also after staining thin sections of fixed, dehydrated, and embedded pellets formed by prolonged ultracentrifugation of whole culture medium. In contrast to results from electron microscopy, analysis by velocity density gradient sedimentation or sedimentation equilibrium indicated the exclusive presence of procollagen or partially processed procollagen monomers in solution. These contradictory data can be reconciled if procollagen exists in monomeric form when greatly diluted (as in culture medium), and in specific aggregated form (SLS) at high concentration. We believe that cells in vivo secrete procollagen in high, local concentration packaged in the SLS form. We propose that such zero-D arrayed packages are the precursors of native collagen fibrils.

Animals↗
PubMed1995

Characterization and partial amino acid sequencing of a 107-kDa procollagen I N-proteinase purified by affinity chromatography on immobilized type XIV collagen.

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (plH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 degrees C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, Km and Vmax values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH2-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the alpha 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance.

Amino Acid Sequence↗
PubMed2001

Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer.

The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the alpha1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proalpha1(V) homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.

Amino Acid Sequence↗
PubMed2001

Processing of type II procollagen amino propeptide by matrix metalloproteinases.

In many embryonic tissues, type IIA procollagen is synthesized and deposited into the extracellular matrix containing the NH(2)-propeptide, the cysteine-rich domain of which binds to bone morphogenic proteins. To investigate whether matrix metalloproteinases (MMPs) synthesized during development and disease can cleave the NH(2) terminus of type II procollagens, we tested eight types of enzymes. Recombinant trimeric type IIA collagen NH(2)-propeptide encoded by exons 1-8 fused to the lectin domain of rat surfactant protein D was used as a substrate. The latter allowed trimerization of the propeptide domain and permitted isolation by saccharide affinity chromatography. Although MMPs 1, 2, and 8 did not show cleavage, MMPs 3, 7, 9, 13, and 14 cleaved the recombinant protein both at the telopeptide region and at the procollagen N-proteinase cleavage site. MMPs 7 and 13 demonstrated other cleavage sites in the type II collagen-specific region of the N-propeptide; MMP-7 had another cleavage site close to the COOH terminus of the cysteine-rich domain. To prove that an MMP can cleave the native type IIA procollagen in situ, we demonstrated that MMP-7 removes the NH(2)-propeptide from collagen fibrils in the extracellular matrix of fetal cartilage and identified the cleavage products. Because the N-proteinase and telopeptidase cleavage sites are present in both type IIA and type IIB procollagens and the telopeptide cleavage site is retained in the mature collagen fibril, this processing could be important to type IIB procollagen and to mature collagen fibrils as well.

Amino Acid Sequence↗