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.     

Immuno-electron-microscopic localization of types III pN-collagen and IV collagen,laminin and tenascin in developing and adult human spleen

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.     

Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.     

Production of both interstitial and basement membrane procollagens by fibroblastic WI-38 cells from human embryonic lung

The synthesis and secretion of type IV procollagen, in addition to that of procollagen types I and III, was detected in cells derived from human embryonic lung (WI-38) by immunofluorescence, metabolic labeling, immunoprecipitation, collagenase digestion and the characteristic polypeptide sizes of both intact procollagen type IV chains and their initial pepsin-resistant fragments as determined by polyacrylamide gel electrophoresis. Locally obtained human embryonic lung cells secreted the same procollagens, but neither embryonic nor adult human skin fibroblasts were found to secrete type IV procollagen in amounts detectable by the same methods.     

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Isolation of cDNA clones for basal lamina components: type IV procollagen

We have isolated cDNA clones for mouse type IV procollagen from a library constructed from total poly A+RNA of 13.5 day mouse embryo parietal endoderm (PE) cells. In Northern analysis these clones hybridise to a 6.8 kb RNA which is abundant in embryonic PE cells and in differentiated F9 teratocarcinoma cells. Hybrid selection and in vitro translation of the cDNA specific mRNA produced a single polypeptide of Mr = 165 000. This polypeptide was specifically immunoprecipitated with mouse type IV procollagen antisera and comigrated on SDS-gel electrophoresis with one of the two in vitro synthesised chains of type IV procollagen. Undifferentiated F9 teratocarcinoma cells can be induced by retinoic acid and dibutyryl cAMP to differentiate in vitro into endoderm-like cells which resemble mouse PE cells in synthesising large amounts of basement membrane proteins, including type IV procollagen. Here we show, using one of the cDNA clones as a probe for type IV procollagen, that an increase in cellular concentration of type IV procollagen mRNA occurs within 24 to 48 hours of induction, reaching a constant high level by 72 hours.     

Demethylation of the type I procollagen genes in transformed fibroblasts treated with 5-azacytidine

Transformation of the human embryonic lung fibroblast line, WI-38, with simian virus 40 (SV40) results in inactivation of the type I procollagen genes. No type I collagen or procollagen mRNA is detected in these transformed cells, as determined by polyacrylamide gel electrophoresis. Analysis of the methylation patterns of these genes showed the type I procollagen genes to be hypermethylated at certain cytosine residues in the transformed cells. However, several of the cytosine residues were methylated in the normal cells where these genes are expressed. These methylation patterns can be altered by treatment of the cells with 5-azacytidine or 5-azadeoxycytidine, but without a resultant activation of the type I procollagen genes. These results show that demethylation alone is not sufficient for gene activation, but that other signals are also required.     

Biosynthesis of type IV collagen by cultured rat Schwann cells

We have obtained evidence that rat Schwann cells synthesize and secrete type IV procollagen. Metabolic labeling of primary cultures of Schwann cells plus neurons and analysis by SDS PAGE revealed the presence of a closely spaced pair of polypeptides in the medium of these cultures that (a) were susceptible to digestion by purified bacterial collagenase, (b) co-migrated with type IV procollagen secreted by rat parietal endoderm cells, and (c) were specifically immunoprecipitated by antibodies against mouse type IV collagen. Limited pepsin digestion of metabolically labeled medium or cell layers produced a pepsin- resistant fragment characteristic of pro-alpha 1(IV) chains. Removal of neuronal cell bodies from the cultures immediately before labeling did not reduce the amount of type IV procollagen detected in the medium. This indicated that Schwann cells, not neurons, were responsible for synthesis of type IV procollagen. We believe type IV procollagen is a major constituent of the Schwann-cell extracellular matrix based upon (a) its presence in a detergent-insoluble matrix preparation, (b) its presence in the cell layer of the cultures in a state in which it can be removed by brief treatment with bacterial collagenase or trypsin, and (c) positive immunofluorescence of Schwann cell-neuron cultures with anti-type-IV collagen antibodies. Secretion of type IV procollagen was substantially reduced when Schwann cells were maintained in the absence of neurons. This observation may account for the previously reported finding that Schwann cells assemble a basal lamina only when co-cultured with neurons (Bunge, M. B., A. K. Williams, and P. M. Wood, 1982, Dev. Biol., 92:449).     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

Mechanism for the regulation of post-translational modifications of procollagens synthesized by matrix-free cells from chick embryos

Three possible mechanisms are considered to account for the variations of post-translational modifications in different collagen types. 1) The cells have different amounts of post-translational modifying enzymes, 2) the rate of prolylhydroxylation of different procollagen types is varied, and 3) the rate of chain association of pro-alpha chains of different collagen types is modulated. In an attempt to examine the three possibilities, we have determined the activities of prolyl hydroxylase and lysyl hydroxylase, and we have examined the kinetics of the secretion of procollagens and the kinetics of pro-gamma chain formation of different procollagen types in matrix-free cells isolated from tissues of 17-day-old chick embryos. Type II collagen synthesized by cartilage cells contains more hydroxylysine than type I collagen synthesized by tendon and cornea cells. It was found, however, that cartilage cells contain significantly less lysyl hydroxylase than tendon and cornea cells. In contrast, we found only a small difference in the amount of prolyl hydroxylase in tendon, cornea, and cartilage cells. The secretion of type I procollagen by tendon and cornea cells can be described by two first order processes. In contrast, the secretion of type II procollagen by cartilage cells, type IV procollagen by lens cells, and type V procollagen by cornea cells can be described by single first order processes. Examination of the formation of pro-gamma components of procollagen types I and II revealed that it occurs via intermediate dimers of two pro-alpha chains. The formation or pro-gamma(I) chains in tendon and cornea cells is about three times faster than the formation of pro-gamma(II) chains in cartilage cells. These results are consistent with the hypothesis that the rate of association of pro-alpha chains regulates the synthesis of procollagens with different degrees of post-translational modifications.     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.