A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.     

Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome.

Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.     

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta- galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro(alpha)1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5' cysteine residue by a serine within a highly conserved sequence of the pro(alpha)1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro(alpha)1(V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen.     

Further evidence for the dispersion of the human fibrillar collagen genes.

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.     

Biosynthesis and regulation of type V collagen in diploid human fibroblasts

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.     

Existence of malfunctioning pro alpha2(I) collagen genes in a patient with a pro alpha 2(I)-chain-defective variant of Ehlers-Danlos syndrome

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one-half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the pro alpha 2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for pro alpha 2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the pro alpha 2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning pro alpha 2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize pro alpha 2(I) collagen chains.     

Presence of different types of procollagen messenger RNAs in human hepatoma cell lines

Human hepatoma cell lines were shown for the first time to contain various types of procollagen mRNAs. The amounts and types of procollagen mRNAs differed depending on the cell lines. Pro alpha 1 (III) and pro alpha 1 (IV) collagen mRNAs were present in PLC/PRF/5, a hepatocellular carcinoma cell line, whereas pro alpha 1 (I), pro alpha 2 (I), pro alpha 1 (IV) and pro alpha 2 (V) collagen genes contrast, HepG2 cells derived from hepatoblastoma contained little, if any, mRNAs for these types of procollagens we had examined.     

Assignment of the human pro alpha 2(I) collagen structural gene (COLIA2) to chromosome 7 by molecular hybridization.

A cDNA for the pro alpha 2 chain of human type I collagen has been recently cloned and amplified. We have used this specific probe to identify the human chromosome carrying the pro alpha 2(I) collagen gene. The DNA from 17 independent human/hamster and human/mouse somatic cell hybrids was digested by Eco RI and the restriction pattern analyzed in Southern blot experiments, using the 32P-labeled cDNA as a hybridization probe. The gene coding for the pro alpha 2 collagen subunit could be unambiguously assigned to human chromosome 7. All the other chromosomes, including chromosome 17, were excluded.     

Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides

Peptides prepared from the amino termini of pro alpha 1(I) and pro alpha 1(III) collagen chains inhibit the production of pro alpha 1(I) and pro alpha 2 by rat calvaria rna in a reticulocyte cell-free system. The synthesis of other proteins was not altered, suggesting a specific effect on collagen production. Various peptides from the helical region of the alpha 1(I) chain did not alter translation. These studies, taken together with earlier studies showing inhibition of collagen synthesis by cells in culture receiving the amino-terminal peptides, are consistent with a regulatory function in collagen synthesis for the amino-terminal peptides from procollagen.     

Complete primary structure of human collagen alpha 1 (V) chain

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.     

Physical mapping by PFGE localizes the COL3A1 and COL5A2 genes to a 35-kb region on human chromosome 2

The genes encoding the alpha 1 chain of Type III collagen (COL3A1) and the alpha 2 chain of Type V (COL5A2) collagen have been mapped to the long arm of human chromosome 2. Linkage analysis in CEPH families indicated that these two genes are close to each other, with no recombination in 37 informative meioses. In the present study, DNA probes from the 3' ends of each gene have been physically mapped by pulsed-field gel electrophoresis. The probes recognized 11 macrorestriction fragments in common, ranging from greater than 1000 kb MluI and NotI fragments to a 35-kb SfiI fragment. Therefore, the COL3A1 and COL5A2 genes appear to exist as a gene cluster on chromosome 2. This is the third example of a collagen gene cluster. Other examples include the COL4A1-COL4A2 genes on chromosome 13q and the COL6A1-COL6A2 genes on chromosome 21q. The physical proximity of these genes may indicate common evolution and/or regulation.     

Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro alpha 1(I) collagen gene (COL1A1)

Dysfunctions of the genes coding for the two chains of the human type-I procollagen result in genetic disorders that affect the integrity of bone, ligaments, tendons, and other connective tissues. While the primary amino acid (aa) sequence of one of the two type-I subunits, pro alpha 2(I), has been derived in its entirety from the analysis of overlapping cDNAs, the sequence of the first 247 aa residues of the helical domain of the other polypeptide, pro alpha 1(I), had yet to be determined. To this end, we have sequenced nearly 4 kb of the human pro alpha 1(I) collagen gene and identified twelve open reading frames whose conceptual amino acid translation exhibits 95% homology to the first 247 aa of rat alpha 1(I) chain. Furthermore, with these and other data, some of which previously unpublished, we have derived the complete sequence of the first 7618 bp of the gene. This region comprises the 25 exons encoding the N-terminal pre-propeptide and five of the eight cyanogen-bromide-derived peptides. This information therefore represents a most useful reference for the characterization of molecular defects in individuals affected by various connective tissue disorders.