Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D

Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned. Three recombinant plasmids containing type I collagen specific sequences were characterized: p alpha 1R1 is 1600 bp and spans approximately 500 amino acid residues within the triple helical region of alpha 1(I) and p alpha 1R2 is 900 bp in size and covers the entire 3' noncoding and about half of the C-terminal propeptide region of alpha 1(I) collagen mRNA. The third recombinant p alpha 2R2 is 1500 bp and contains alpha 2(I) sequences specific for the entire 3' noncoding and C-terminal propeptide region. Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse greater than human greater than chick. Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed two distinctly different molecular weight patterns characteristic of alpha 1(I) (4.7 and 5.7 kb) and alpha 2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe. Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA. The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the polymerase chain reaction to clone and sequence a cDNA encoding the bovine alpha 3 chain of type IV collagen

A novel type IV collagen, alpha 3(IV), has previously been isolated from a collagenase digest of bovine and human glomerular and lens basement membranes. The cloning and sequencing of a cDNA encoding the alpha 3(IV) chain is described here. Using the polymerase chain reaction, with primers derived from the known 27-residue bovine alpha 3(IV) amino acid sequence, a 68-base pair bovine genomic fragment (KEM68) which encodes the known peptide sequence, was synthesized. KEM68 was then used to screen a bovine lens cDNA library and a 1.5-kilobase partial cDNA clone obtained, encoding 471 residues of the bovine alpha 3(IV) chain: 238 residues from the triple helical collagenous domain and all 233 residues of the noncollagenous domain. The collagenous repeat sequence has three interruptions, coinciding with those in the alpha 1(IV) chain. The noncollagenous domain has 12 cysteine residues in identical positions to those of other type IV collagens and 71, 61, and 70% overall similarity with the human alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains. The noncollagenous domain of alpha 3(IV) is of particular interest as it appears to be the component of glomerular basement membrane that reacts maximally with the Goodpasture antibody. Furthermore, such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients with Alport syndrome. The alpha 3(IV) cDNA clone described here now permits study of the molecular pathology of COL4A3 in Alport syndrome.     

The complete primary structure of the alpha 2 chain of human type IV collagen and comparison with the alpha 1(IV) chain

The complete primary structure of the human type IV collagen alpha 2(IV) chain has been determined by nucleotide sequencing of cDNA clones. The overlapping cDNA clones cover 6,257 base pairs with a 5'-untranslated region of 283 base pairs, the 5,136-base pair open reading frame, and the 3'-untranslated region of 838 base pairs. The predicted amino acid sequence demonstrates that the complete translation product consists of 1,712 residues corresponding in molecular weight to 167,560. The translated polypeptide has a signal peptide of 36 amino acids, an amino-terminal noncollagenous part of 21 residues, a 1,428-residue collagenous domain with 23 interruptions, and a carboxyl-terminal noncollagenous (NC) domain of 227 residues. The calculated molecular mass of the mature human alpha 2(IV) chain is 163,774 Da.     

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.     

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

Structural and functional features of the alpha 3 chain indicate a bridging role for chicken collagen VI in connective tissues

Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.     

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

The complete primary structure of a nematode alpha 2(IV) collagen and the partial structural organization of its gene.

We have isolated and characterized cDNA and genomic DNA clones which encode an alpha 2(IV) collagen chain from the parasitic nematode Ascaris suum. In addition we have determined, by nucleic acid sequence analysis, the structural organization of approximately two-thirds of the gene. This analysis has shown that the gene contains at least 15 introns, and those that have been characterized range in size from 141 to 854 base pairs. The derived protein sequence contains 1763 amino acids and includes a putative 26-amino acid signal sequence. The collagenous triple-helical region contains 17 interruptions, many of which occur in the same positions as those in the human alpha 1(IV) and alpha 2(IV) chains. Comparison of the genomic DNA sequence with the cDNA sequence has revealed the presence of a sequence within the gene which appears to be an intact and normal exon that is not represented in our cDNA sequence. The presence of this putative exon raises the possibility that the A. suum alpha 2(IV) collagen gene may undergo alternative splicing.     

Complete primary structure of the alpha 1-chain of human basement membrane (type IV) collagen

We have determined the primary structure of the alpha 1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the alpha 1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human alpha 1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The alpha 1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Isolation and characterization of a cDNA clone for the amino-terminal portion of the pro-alpha 1(II) chain of cartilage collagen

We have isolated a cDNA clone (pRcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(II) chain mRNA. A synthetic oligonucleotide was used both as a primer for cDNA synthesis and as a probe for screening a cDNA library. The probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(II) chain. This primer was chosen so that the resulting cDNA would contain the sequence of the 5' end of the mRNA. The nucleotide sequences of the cDNA were determined and compared with that of three other interstitial procollagen chain mRNAs (pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) chain mRNA). pRcol 2 contains a 521-base pair (bp) insert, including 153 bp of the 5' untranslated region plus 368 bp coding for the signal peptide, the amino-terminal propeptide, and a part of the telopeptide. The signal peptide of the type II collagen chain is composed of about 20 amino acids. There is little homology between the amino acid sequence of the signal peptide in the pro-alpha 1(II) chain and that of three other interstitial procollagen chains. The NH2-terminal propeptide is deduced to contain short nonhelical sequences at its amino and carboxyl ends and an internal helical collagenous domain comprising 25 repeats of Gly-X-Y with one interruption. There is a strong conservation of the amino acid sequence of the carboxyl-terminal part of the NH2-terminal propeptide in the pro-alpha 1(II), pro-alpha 1(I), and pro-alpha 2(I) chains. Type II collagen mRNA does not contain a sequence corresponding to a uniquely conserved nucleotide sequence around the translation initiation site which occurs in mRNA for other procollagen chains.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

The alpha 1 chain of type XIII collagen consists of three collagenous and four noncollagenous domains, and its primary transcript undergoes complex alternative splicing.

We have recently reported a characterization of cDNA clones that encode an apparently novel human collagen that undergoes alternative splicing. These cDNAs covered one-third of the corresponding 2.5-2.8-kilobase mRNAs. We have now determined the complete primary structure of the protein encoded by several overlapping cDNAs isolated from a human endothelial cell library. Since the deduced translation product of the cDNAs is different in structure from all other collagen types, we have given the collagen chain encoded by the cDNAs the designation alpha 1 (XIII). The deduced polypeptide consists of three collagenous domains and four noncollagenous domains, two of them separating the collagenous domains and two located at the N-terminal and C-terminal ends of the polypeptide. Cysteine residues are found in three of the noncollagenous domains and also in the extreme N-terminal collagenous domain. Surprisingly, comparison of the nucleotide sequences encoded by the overlapping cDNA clones demonstrates that there are several alpha 1 (XIII) collagen mRNAs in HT-1080 human fibrosarcoma cells and human endothelial cells which differ in coding potential. Nuclease S1 mapping experiments suggest that these different mRNAs arise through alternative splicing of the precursor RNA at five locations within the coding region. This property makes type XIII collagen unique among all the collagen types studied so far. Its polypeptide length, therefore, may vary between 614 and 526 amino acids, depending on what internal splicing has taken place.