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Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent α chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three α chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its α or β subunits or with the individual α or β subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with α subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.

Type XII and type XIV collagens are closely related homotrimeric and disulfide-bonded extracellular matrix constituents that contain two short triple helical domains near the C -terminus and a very large modular non-triple helical domain at the N -terminus. This domain contains multiple fibronectin type III repeats, von Willebrand factor A-like modules and a module homologous to the N-terminal region of thrombospondin. When visualized by rotary shadowing electron microscopy they have a cross shape with a thin collagenous tail, 75 nm in length, connected to a central globule from which three arms are sticking out (from 60 to 90 nm depending on the collagen type and the splicing variant). Collagens XII and XIV have been localized around collagen I-containing fibrils in dense connective tissues. Despite the presence in their primary structure of modules and sequences characteristic of adhesive glycoproteins, these molecules do not appear to have cell adhesion activity. They appear rather to play a role in modulating the biomechanical properties of collagen-containing tissues by mediating deformability of fibril bundles. Collagen XIV also serves as a ligand to procollagen N -proteinase and may contribute in this way to sequester this activity around the collagen fibrils.

Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.

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. SI 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 chpin 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 splce donor site in intron 27 was changed to an A in one allcle of the patient's type III procollagen gene. Using a cDNA genomic DNA hybrid probe in Si 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 absenci: of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient'; fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism. © 1995 Wiley-Liss, Inc.

The picture emerging from recent discoveries about collagens, other matrix macromolecules and their modes of organization is one of a complex modular system operating at all levels, molecular, supramolecular and whole tissue. This complexity provides multiple opportunities for interactions between structurally similar and dissimilar components. The analysis of these interactions will eventually help us to understand the regulatory principles underlying the morphogenesis of extracellular matrices.

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.

Collagen XI is found mainly as a component of cartilage fibrils. Among the different transcripts identified by RT-PCR for the alpha1 (XI) chain, the major tissue form has been reported to be the splicing product of exons I, III and V. In this study, two other splice isoforms of the alpha1(XI) chain were identified using N-terminal sequencing. Like the major alpha1(XI) chain, the fully processed isoforms begin at Gln254 within the N-terminal domain encoded by exon I. This sequence is followed by sequences encoded by exon IIA or III. An anti-peptide antibody allowed the identification of the exon IV encoded sequence within both isoforms. Therefore, these isoforms of the alpha1(XI) chain correspond to the splicing of exons I, IIA, III, IV and V or of exons I, III, IV and V, thus presenting larger acidic sequences than the major form. They could mediate strong ionic interactions within the cartilage matrix.

International audience; Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.

International audience; Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

International audience; We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

This study has examined the frequency and distribution of polymorphisms in the type I collagen coding sequences. RNA from a group of human skin fibroblast cell lines was analyzed by the chemical cleavage mismatch detection method using hydroxylamine, a reagent specific for C-base mismatches, and overlapping cDNA probes covering the entire prepro alpha 1(I) and prepro alpha 2(I) coding regions. Mismatches were detected at only two nucleotide positions, one in each of the type I collagen sequences, suggesting that polymorphisms are relatively rare within these cDNAs. cDNA sequence analysis demonstrated that the prepro alpha 1(I) mismatch, detected in only one cell line, was due to a sequence polymorphism involving the wobble position of the codon for arginine residue 59 within the amino-propeptide globular subdomain of the pro alpha 1(I) chain and not resulting in a change in the polypeptide primary structure. In contrast, the prepro alpha 2(I) mismatch, detected in 6 of the 16 cell lines, was shown to arise from a sequence polymorphism affecting the identity of Y-position residue 459 of the alpha 2(I) triple helical domain, resulting in an alanine/proline dimorphism at this position. This study is the first to identify a type I collagen coding sequence polymorphism resulting in an alteration at the level of the amino acid sequence. The data suggest that at least some alpha 1(I) and alpha 2(I) helix Y positions may be tolerant of sequence variation, particularly if the replacing amino acid is proline, a residue involved in stabilizing the collagen triple helix.

We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5′ junction exon and the two last 3′ exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.

International audience; Type XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions.Type XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions.

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12–q14. Interestingly, within this region of human chromosome 6 resides the α1(IX) collagen gene (COL9A1), a member of the FACIT group.

cDNA from embryonic chick skin has been isolated and characterized which encodes a novel member of the FACIT (fibril-associated collagen with interrupted triple helices) group whose other known members are collagen types IX and XII. Nucleotide sequence analysis of the cDNA, combined with characterization of a pepsin-resistant fragment of the protein from embryonic chick skin, demonstrates that the collagen chain is more closely related to the chain of type XII collagen than to those of type IX. It is most similar to a collagen, type XIV, recently identified in bovine skin. It is possible, therefore, that the cDNA codes for a chain of chicken type XIV collagen. From the additional data on molecular structure obtained by sequencing the cDNA, the FACIT family appears to consist of at least two classes of molecules: one of which contains the three chains of type IX collagen, and a second which includes the chains of collagen types XII and XIV.

Isolated photosynthetic reaction center from the bacterium Rhodospirillum rubrum was extracted with acetone-methanol. Its main pigments were identified as bacteriochlorophyll, bacteriopheophytin, and spirilloxanthin. The extinction coefficients of these pigments in acetone-methanol were determined. Quantitative spectroscopic analysis of the dry acetone-methanol extracts indicated a bacteriochlorophyll to bacteriopheophytin mole ratio of 2 and led to an extinction coefficient at 868 nm of 71.3, 142.6, or 214 mm-1 cm-1, depending on whether the photosynthetic center was assumed to contain 2, 4, or 6 bacteriochlorophyll molecules. The extinction coefficient of P870 was determined by mixing photo-oxidized photosynthetic center with ferrocytochrome c in a stopped flow spectrophotometer. Complete reaction was observed when the reactants were mixed in equimolar ratio calculated on the basis of an extinction coefficient of 143 mm-1 cm-1 at 868 nm. This photosynthetic center is proposed to contain 4 moles of bacteriochlorophyll, 2 moles of bacteriopheophytin, and 1 mole of spirilloxanthin per equivalent of P870.

Rhodospirillum rubrum chromatophores were solubilized by various detergent treatments and fractionated. The main fractions were a particle enriched in P870 photochemical activity, a purified photochemical reaction center, and AUT “photoreceptor” particles. These fractions and others were compared to the chromatophore preparation by immunodiffusion in the presence of deoxycholate, in order to solubilize the antigenic components of the membrane. They were compared also by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. These techniques show that each particle has different components. The purified reaction center preparation contains two antigens and three electrophoretic components with apparent molecular weights of 28,000, 25,500, and 21,000. These three components are present in all the fractions studied, although in small amounts in the pellet obtained after solubilization with dodecyl dimethylamine N-oxide. This fraction contains, as a major component, a polypeptide of about 10,000 apparent molecular weight which is thought to be associated with light-harvesting pigments. This polypeptide is virtually absent from purified photochemical reaction center. The reaction center protein is estimated to represent 15% of the chromatophore preparation on a protein basis.

The bacteriochlorophyll ( P -800 and P -870) of the carotenoidless photoreaction center isolated from Rhodospirillum rubrum (strain G9) is bleached irreversibly when the preparations are exposed to intense near infrared light in the presence of oxygen. This effect is much smaller in preparations, extracted from the wild type, which contain, as shown earlier, 1 mol of spirilloxanthin per mol of P -870. This photodynamic effec is shown to be due to singlet O 2 . The oxidation of adrenaline in the presence of superoxide dismutase and the oxidation of 1,3-diphenylisobenzofuran are used as reporter reactions. Singlet oxygen is presumably generated by the triplet-triplet energy transfer 3 bacteriochlorophyll → O 2 ( 3 Σ ). Four purified bacterial carotenoids, spirilloxanthin, sphaeroidene, sphaeroidenone and chloroxanthin were attached onto the carotenoidless photoreaction center from strain G9 in nearly 1 : 1 mol ratios with respect to P -870. Once fixed, these carotenoids confer protection against the photodynamic bleaching of bacteriochlorophyll. The relative photoprotection efficiency was 1.0 for spirilloxanthin and sphaeroidene, 0.4 for chloroxanthin and 0.2 for sphaeroidenone. The fixed carotenoids display optical activity and their molar ellipticity appears to be correlated with their relative photoprotection efficiency. The efficiency of energy transfer to P -870 is 0.90 for sphaeroidene, 0.35 for sphaeroidenone, 0.30 for chloroxanthin and 0.20 for spirilloxanthin. The energy transfer efficiency from the carotenoids to bacteriochlorophyll is suggested to be governed by the rate of the internal conversion processes of the excited singlet state of the carotenoids. A study of the difference absorption and CD spectra of the reconstituted minus carotenoidless preparations leads to the interpretation that the fixed carotenoids are in a central monocis conformation.

1. 1. The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. 2. 2. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. 3. 3. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. 4. 4. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1α, 2α, 3α collagen in human cartilage.

We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino-terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us.

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.

The existence of discordant results regarding the effects of intracerebroventricular (icv) administration of GRF on GH secretion prompted a reexamination of the central actions of GRF and a detailed chemical characterization of the peptide designated as hpGRF-44-NH2. The icv injection of 10 micrograms rat (r) GRF to freely-moving rats caused an acute stimulation of GH release, whereas 10 micrograms of the putative hpGRF peptide icv continued to suppress spontaneous GH secretion. Through a series of biochemical and immunologic studies we demonstrate that peptide hpGRF-44-NH2, code number 92-81-5G-41-47, is predominantly ovine (o) CRF and also contains a small amount (3-5%) human (h) GRF-44-NH2. We conclude that the major effect of icv administered GRF, at high doses, is to stimulate GH release and that the central actions previously attributed to the hpGRF peptide are, in fact, due to the oCRF component of this compound.

P870 was isolated from wild type Rhodospirillum rubrum by solubilization with dodecyldimethylamine oxide followed by ammonium sulfate precipitation. The particle contains some carotenoids, 0.3 mole of ubiquinone per mole of P870 and no cytochrome. The apparent particle weight is 140 000. Electrophoresis in dissociating media containing 0.1% sodium dodecyl sulfate shows the existence of at least two and more likely three protein bands. The complex is calculated to have a minimal molecular weight of 70 600 ± 3200 and is supposed to be formed of three protomers.

Collagen XIV is known to bind to the dermatan sulfate chain of decorin and to the heparan sulfate chain of perlecan. To study its possible interaction with glycosaminoglycans, the NC1 domain of chicken collagen XIV was overproduced in E. coli. Purified NC1∗ (6–119)∗ appears poorly organized (the asterisks indicate the presence of extension sequences), but V8-protease generated fragments containing the 84–108 basic sequence tend to fold into α-helix. These fragments interact specifically with heparin, which induces an α-helical fold with a maximum effect for equimolar heparin/peptide ratio. These data demonstrate the existence of a glycosaminoglycan binding site in NC1.