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PubMed1995

Type III procollagen peptide in the adult respiratory distress syndrome. Association of increased peptide levels in bronchoalveolar lavage fluid with increased risk for death.

OBJECTIVE: To determine whether bronchoalveolar lavage fluid levels of the N-terminal peptide of type III procollagen (procollagen III) are increased in patients with the adult respiratory distress syndrome and, if so, whether increased procollagen III levels in lavage fluid are associated with increased fatality rates. DESIGN: Prospective cohort study. SETTING: Intensive care units of a tertiary care hospital affiliated with a medical school. PATIENTS: 117 consecutive patients with the adult respiratory distress syndrome prospectively identified on admission; 6 healthy volunteers served as controls. MEASUREMENTS: Bronchoalveolar lavage fluid procollagen III levels in 117 patients at 3, 7, and 14 days after onset of the adult respiratory distress syndrome (total of 196 lavage samples). RESULTS: The median procollagen III level was 1.75 U/mL (range, 0 to 13.4 U/mL) in lavage fluid obtained from patients with the adult respiratory distress syndrome. We detected procollagen III levels in lavage fluid from 80% of patients (94 of 117) but not in 6 normal volunteers. The overall fatality rate was 41% (48 of 117 patients). In a univariate analysis, the relative risk (RR) for death was increased in patients with procollagen III levels of 1.75 U/mL or more obtained on day 3 (RR, 2.4; 95% CI, 1.3 to 4.3), day 7 (RR, 2.7; CI, 1.4 to 5.4), and day 14 (RR, 2.7; CI, 1.1 to 6.3). Inclusion of other variables in a multivariate model only minimally decreased the risk associated with increased procollagen III levels. CONCLUSION: Increased levels of type III procollagen in bronchoalveolar lavage fluid are frequently detected in patients with the adult respiratory distress syndrome and are strongly associated with increased risk for fatal outcome independent of other variables related to fatality in patients with the syndrome.

Adolescent↗
PubMed1991

Propeptide-mediated regulation of procollagen synthesis in IMR-90 human lung fibroblast cell cultures. Evidence for transcriptional control.

We have demonstrated previously that the carboxyl- and amino-terminal propeptides of type I procollagen can inhibit procollagen synthesis by specifically decreasing procollagen mRNA levels. The objective of the present experiments was to determine the mechanism by which propeptides cause these pretranslational effects. IMR-90 fibroblasts were exposed to medium containing carboxyl-terminal propeptide of type I procollagen, and nuclear run-off assays were performed by hybridization to a specific alpha 1 chain type I procollagen cDNA probe. Specific type I procollagen transcription rates were found to be decreased by 50% in the presence of 75 nM carboxyl-terminal propeptide compared with control (untreated) cells. Total cellular transcription rates as well as beta-actin mRNA rates were not affected significantly by any concentration of carboxyl-terminal propeptide. Propeptide radiolabeled with 125I was found to be taken up by cultured cells. Furthermore, exogenous carboxyl-terminal propeptide levels increased in the cytosolic compartment and eventually reached a steady-state level of 18 +/- 2 pmol/g cell protein by 30 min. Of particular interest was the finding that levels of radiolabeled carboxyl-terminal propeptide were also detected in the nuclear fraction and increased with time, reaching a plateau after 60 min of incubation. Incubation of nuclei from IMR-90 cells in medium containing varying concentrations of carboxyl-terminal propeptide resulted in nuclear transcription rates that were decreased by 40% compared with untreated controls. beta-Actin nuclear message levels remained unchanged under identical conditions. We conclude that carboxyl-terminal propeptide of type I procollagen can be internalized and become associated with the nuclear compartment. This suggests a feedback regulatory role on procollagen synthesis by a direct effect on procollagen gene transcription.

Animals↗
PubMed1989

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV.

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.

Adult↗
PubMed1987

A hepatic fibrogenic factor stimulates the synthesis of types I, III, and V procollagens in cultured cells.

A hepatic fibrogenic factor (HFF) isolated from fibrotic rat livers has previously been shown to stimulate the transcription of type I procollagen genes in cultured fibroblasts (Raghow, R., Gossage, D., Seyer, J. M., and Kang, A.H. (1984) J. Biol. Chem. 259, 12718-12723). To test if the expression of other collagen genes was similarly affected by the fibrogenic factor, we measured the rates of types I, III, and V procollagen synthesis in two different cell lines after treatment with HFF. The effect of fibrogenic factor on types I and III procollagens was tested in rat fibroblasts, while a human rhabdomyosarcoma cell line was used to evaluate the effect of HFF on type V procollagen synthesis. Incubation with rat fibroblasts resulted in a 3-4-fold stimulation of the synthesis of both types I and III procollagens in a time-dependent manner. The stimulated rates of types I and III procollagen synthesis accompanied an increase in the steady-state levels of their corresponding mRNAs. When A204 cells, which are derived from a rhabdomyosarcoma and exclusively synthesize type V procollagen, were incubated with the fibrogenic factor, a 3-4-fold stimulation of the synthesis of both pro-alpha 1(V) and pro-alpha 2(V) chains was seen. Using a cDNA probe for pro-alpha 2(V), we also observed that there was a 2-3-fold increase in the steady-state level of pro-alpha 2(V) mRNA in A204 cells after treatment with the fibrogenic factor. In both rat fibroblasts and A204 cells the steady-state levels of beta-actin mRNA were minimally affected by fibrogenic factor, suggesting that the procollagen genes were preferentially affected. Since types I, III, and V collagens are present in the normal liver and accumulate aberrantly in the fibrotic liver, we suggest that fibrogenic factor may play an important role in determining the altered collagen composition of the fibrotic liver. Based on these data, we also speculate that the regulation of the biosynthesis of a variety of procollagens in diverse cell types by HFF possibly occurs by a common mechanism.

Animals↗
PubMed1984

Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase.

The specific mammalian collagenase isolated from cultures of metastatic mouse PMT sarcoma cells cleaves murine procollagen IV into two segments, of approximate mass ratio 3:1. These fragments were separated by velocity sedimentation, visualized by electron microscopy, and analyzed. The longer COOH-terminal procollagen segment has a 270-nm collagenous portion with a knob at one end. This knob consists of the three previously identified, noncollagenous carboxyl propeptides, of approximately 30,000 daltons each. These carboxyl propeptides are chain-specific, and the three chains of each segment have the same amino to carboxyl orientation. The collagenase cuts through all three chains at one site, and the three-component chains of both the longer COOH-terminal procollagen segment and the shorter NH2-terminal procollagen segment are linked by interchain disulfide bridges. The enzyme cuts off the same COOH-terminal procollagen segment from procollagen IV monomers and tetramers, and the flexibility of this segment is similar to that of interstitial collagen helices. The amino ends of the NH2-terminal procollagen segments derived from tetramers remain joined as the 32-nm long "7 S collagen" junctional complex, and the remaining 89-nm long projecting threads are significantly more flexible than the COOH-terminal procollagen segment. The electrophoretic mobilities of the enzyme cleavage products are consistent with a heterotrimeric composition of this procollagen IV.

Amino Acid Sequence↗
PubMed1980

Ascorbate-induced fibroblast cell matrix: reaction of antibodies to procollagen I and III and fibronectin in an axial periodic fashion.

Fibronectin and procollagen types I and III are constituents of the extracellular matrix of human fibroblasts. Ultrastructural immunocytochemistry using the peroxidase anti-peroxidase method showed fibronectin and procollagen antibodies reacting in continuous fashion on 10 nm diameter extracellular fibrils on human fibroblasts. Intracellular localization showed an intense accumulation of procollagen within cells cultured under routine conditions. This accumulation appeared almost as if there were a blockade in secretion of procollagen under routine culture conditions. Cells treated with ascorbic acid do not have the dense intracellular accumulation of procollagens seen with the apparent blockade of secretion in cells cultured under routine conditions. Ascorbate treated cells also have a more pronounced extracellular accumulation of matrix fibronectin and procollagen constituents. At the electromicroscopic level a new 40 nm diameter fibril is formed after ascorbic acid treatment of human fibroblasts. Antibody to fibronectin and procollagen I and III are seen binding to the 40 nm diameter fibrils in a periodic or stuttered appearance. The fibronectin and procollagen antibodies react with a 70 nm axial repeat along these 40 nm fibrils formed after ascorbate treatment. These studies suggest that under routine culture conditions "precursor" fibrils of fibronectin and procollagen are formed. Ascorbic acid treatment leads to enhanced matrix formation. Ultrastructural studies clearly show antibodies to fibronectin bind to fibronectin on native collagen fibrils formed by human fibroblasts cultured with ascrobic acid. Lastly there is an asymmetric or 70 nm axial periodic distribution of fibronectin along these definitive or mature collagen fibrils formed after ascorbic acid treatment.

Antibodies↗
PubMed1981

Procollagen messenger RNA levels and activity and collagen synthesis during the fetal development of sheep lung, tendon, and skin.

The rates of type I collagen synthesis in sheep lung, tendon, and skin were evaluated during the latter half of fetal development and compared with the levels of type I procollagen mRNA, quantified by molecular hybridization with a type I procollagen specific complementary DNA, and with the activity of total procollagen mRNA measured by in vitro cell-free translation. In the lung and tendon, the levels of type I procollagen mRNA and activity of total procollagen mRNA parallel collagen synthesis during development. In the skin, however, type I collagen synthesis declines sharply during fetal development, but both type I procollagen mRNA levels and total procollagen mRNA activity remain at the high levels of early development. These observations suggest that in developing lung and tendon, type I procollagen mRNA levels are likely the major determinants of the levels of type I collagen synthesis. In contrast, the dichotomy between type I procollagen mRNA levels and rates of type I collagen synthesis in the developing sheep skin suggest the skin utilizes mechanisms in addition to mRNA levels to modulate expression of the type I collagen gene.

Animals↗
PubMed1994

Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen.

Hsp47, an endoplasmic reticulum resident protein, has gelatin-binding and procollagen-binding properties and has been hypothesized to function as a molecular chaperone in regulating procollagen folding and/or assembly. In this report, we further investigate the interaction of Hsp47 with polysome-associated alpha 1(I) procollagen chains following antisense treatment of 3T6 cells. For these studies, we employed phosphorothioate oligodeoxynucleotides directed to the first five codons of Hsp47 that straddle the predicted translation initiation site of mouse Hsp47. Cells depleted of Hsp47 in this manner were observed to produce diminished amounts of fully elongated nascent alpha 1(I) procollagen while accumulating shorter procollagen peptides associated with peptidyl-tRNA. Pulse-labeling of cells with [35S]methionine followed by treatment with puromycin and immunoprecipitation with anti-Hsp47 and anti-procollagen antibodies revealed that Hsp47 is associated with alpha 1(I) procollagen at a very early point during translocation of the nascent procollagen chains. Although Hsp47 appears to possess properties similar to grp78/BiP, Hsp47 binding early during translocation favors a more specialized specific function relative to chain selection or completion of stable folding in type I procollagen.

Animals↗
PubMed1993

Progressive ovarian carcinoma induces synthesis of type I and type III procollagens in the tumor tissue and peritoneal cavity.

Increased serum concentrations of aminoterminal propeptide of type III procollagen occur in advanced ovarian cancer. To study their origin, we compared the expressions of type I and type III procollagens in ovarian tumor tissue and the peritoneal cavity with immunoassays for the propeptide domains of these procollagens. Samples of tumor cyst fluid, peritoneal ascitic fluid, tumor vein blood, and peripheral blood were obtained at operation from 50 women with malignant ovarian neoplasms and 61 women with benign neoplasms. The ascitic fluid concentrations of both type I and type III procollagen antigens were significantly higher in the malignant tumors than in the benign ones, but this difference was evident only for type I procollagen in the tumor cysts. The aminoterminal propeptide of type III procollagen concentration in the peripheral blood was higher in the patients with malignant tumors, whereas the concentrations were similar in the tumor veins. The enhanced type I procollagen synthesis in the malignant tumors did not affect the corresponding antigen in the blood. The findings suggest that progressive ovarian carcinoma invariably induces a fibroproliferative response, characterized by active expression of type I and type III procollagens. The increased circulating aminoterminal propeptide of type III procollagen is derived from the peritoneal cavity rather than from the tumor tissue via the ovarian vein.

Adult↗
PubMed1993

Procollagen binding to sphingomyelin.

The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.

Animals↗
PubMed1993

Expression of type I and type III procollagen by lens epithelial cells.

PURPOSE: To identify the types of procollagen that are synthesized by cultured bovine lens epithelial cells and to determine what types of post-translational modifications are involved in procollagen biosynthesis and what patterns of procollagen gene expression occur in these cells. METHODS: The epithelial nature of the cultured cells was confirmed with transmission electron and phase contrast microscopy. To label collagen, primary or secondary monolayer cultures were pulsed with [3H] proline for various periods of time. Procollagens were identified by immunoprecipitation with specific antibodies, pepsin digestion, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procollagen messenger RNA was identified by Northern blot analysis using specific DNA probes. RESULTS: In the normal lens epithelium, cells are attached to each other by tight junctions punctuated with desmosomes. After the proteins were labeled, they were immunoprecipitated with antibodies specific to collagen precursors and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to further confirm their identification as types I and III procollagen or processed forms of these proteins. The secretion of procollagen was inhibited by treatment with 2,2'-dipyridyl and bands of underhydroxylated procollagen were observed in the cell layer after electrophoresis. The sizes of messenger RNA observed were 6.9 kb and 5.4 kb for type alpha 1(I), 5.0 kb for type alpha 2(I), and 5.3 kb for the type III transcripts. CONCLUSIONS: This is the first report that lens epithelial cells express fibrillar procollagen genes, making the lens capsule a unique ocular basement membrane.

Animals↗
PubMed2006

Plasminogen activator inhibitor-I-related regulation of procollagen I (alpha1 and alpha2) by antitransforming growth factor-beta1 treatment during radiation-impaired wound healing.

PURPOSE: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-beta1 (TGF-beta1)-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of alpha(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-beta1 treatment specifically inhibits alpha(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-beta1-associated influence on alpha(I)-procollagen synthesis. METHODS AND MATERIALS: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-beta1 treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-beta1, immunohistochemistry, and immunoblotting for PAI-1, alpha1/alpha2(I)-procollagen. RESULTS: Anti-TGF-beta1 significantly reduced TGF-beta1 mRNA (p < 0.05) and PAI-1 expression (p < 0.05). Anti-TGF-beta1 treatment in vivo significantly reduced alpha1(I)-procollagen protein (p < 0.05) and the number of expressing cells (p < 0.05) in contrast to significantly increased (p < 0.05) alpha2(I)-procollagen expression. CONCLUSION: These results emphasize anti-TGF-beta1 treatment to reduce radiation-induced fibrosis by decreasing alpha1(I)-procollagen synthesis in vivo. alpha1(I)-procollagen and alpha2(I)-procollagen might be differentially regulated by anti-TGF-beta1 treatment. Increased TGF-beta signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-beta1 treatment.

Animals↗
PubMed2002

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen.

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.

Animals↗
PubMed2000

Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate.

In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.

Animals↗
PubMed1998

Recombinant procollagen II: Deletion of D period segments identifies sequences that are required for helix stabilization and generates a temperature-sensitive N-proteinase cleavage site.

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 degreesC as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 degreesC lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 degreesC lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 degreesC that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the alpha1(II) chain.

Binding Sites↗
PubMed2001

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

ADAM Proteins↗
PubMed1981

Assembly and processing of procollagen type III in chick embryo blood vessels.

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.

Animals↗
PubMed1995

Aberrant splicing of the type III procollagen mRNA leads to intracellular degradation of the protein in a patient with Ehlers-Danlos type IV.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterized by fragile skin, blood vessels, and internal organs and associated with decreased production, secretion, or thermal stability of type III procollagen. Mutations in the gene for type III procollagen have been identified in patients exhibiting decreased secretion or thermal stability of the protein, but no defect has been elucidated to explain the decreased production of type III procollagen in some patients with EDS IV. We report on a patient with a moderate case of EDS IV who produced decreased amounts of type III procollagen despite normal levels of translatable type III procollagen mRNA. S1 nuclease analysis of the type III procollagen mRNA indicated a defect in the region encoding exon 27. Sequence analysis of cDNA clones and genomic fragments generated by polymerase chain reaction amplification revealed that sequences encoded by exon 27 were absent from 3 out of 5 cDNA clones and that a G at the +5 position of the splice donor site in intron 27 was changed to an A in one allele of the patient's type III procollagen gene. Using a cDNA-genomic DNA hybrid probe in S1 nuclease analysis, fragments consistent with mRNA species containing and lacking exon 27 were detected in a 1:1 ratio. Pulse label and chase experiments in the presence or absence of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient's fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism.

Adult↗