Isolation and characterization of a human collagen alpha 1(I)-like gene from a cosmid library

We have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends through most of the cloned DNA and must, therefore, be at least 35kb in length.     

Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene.

Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens.     

Regional chromosome mapping of human collagen genes alpha 2(I) and alpha 1(I) (COLIA2 and COLIA1)

Summary For the assignment of the genes for the pro-2(I) (COLIA2) and the pro-1(I) (COLIA1) collagens, cDNA and genomic DNA probes were used in in situ hybridization experiments on human prometaphase chromosomes. An improved staining method is reported for the simultaneous identification of chromosomes and the autoradiographic grains after the hybridization procedures. With this procedure more cells with higher resolution could be used for the assignment of genes by in situ hybridization. Statistical analysis of the grains located on respectively 660 and 302 metaphases using pro-2(I) and pro1(I) DNA probes, confirmed the assignment of these genes to human chromosomes 7 and 17. Analysis of the grain distribution on prometaphase chromosomes showed that the location of the pro2(I) collagen gene is in the region 7q21.3–22.1. The location of the pro-1(I) collagen gene was found to be in band 17q21.31–2005.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.     

Isolation and characterization of genomic DNA coding for alpha 2 type I collagen.

We have isolated and characterized a segment of the chick alpha 2 collagen gene by screening a library of chick genomic fragments using as hybridization probe an alpha 2 collagen cDNA clone. Several clones were isolated and one of them, lambda gCOL 204, was used for further studies. The DNA of lambda gCOL 204 hybridizes to a unique species of mRNA the size of alpha 2 collagen mRNA. This mRNA can be translated into a unique polypeptide which comigrates in SDS-gel electrophoresis with pro-alpha 2 collagen. Electron microscopic analysis by R-loop technique indicates that lambda gCOL 204 contains 7Kb of the alpha 2 collagen gene. This 7 Kb piece constitutes the 3' end of the gene. The same clone also contains 9 Kb of DNA that is immediately adjacent to the 3' end of the alpha 2 collagen gene. The cloned segment of the alpha 2 collagen gene is interrupted by 8 intervening sequences of various lengths. The coding sequences for collagen in this clone add up to approximately 1,800 bp, which correspond to about 1/3 of alpha 2 collagen mRNA. DNA sequence analysis of a small coding segment of lambda g COL 204 reveals a characteristic collagen type sequence which encodes for an amino acid sequence identical to a sequence found in calf alpha 2 collagen. The sequence of this region of the protein has not yet been determined for the chick alpha 2 collagen.     

Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule

Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.     

Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.

Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.     

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins of the periodontium. Identification of collagens with the [alpha1(I)]2alpha2 and [alpha1(III)]3 structures in bovine periodontal ligament.

Insoluble collagen was prepared from bovine periodontal ligament. Isolation and characterization of CNBr peptides originating from the alpha1(I), alpha2, and alpha1(III) chains showed that the tissue contained both type I and type III collagens. Further evidence for the presence of type III collagen was obtained by the isolation of alpha1(III) chains from pepsin-treated ligament collagen, with properties similar to those of human alpha1(III) chains. Estimates based on the amounts of certain CNBr peptides indicated that about one-fifth of the collagen of periodontal ligament is type III, the remainder being type I collagen.     

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.     

The characterization of type I and type III collagens from human peripheral nerve.

The normal chemical features of peripheral nerve collagens were determined on postmortem, histologically normal adult human femoral nerve. 1. Genetically distinct type I, [alpha1(I)2]alpha2, and type III, [alpha1(III)]3, were isolated by differential salt precipitation and the component subunit chains, alphal(I), alpha2 and alphal(III) were obtained by ion-exchange chromatography and gel filtration. 2. The molecular weight of alphal(I) and alpha2 of type I collagen was 95 000 and that for type III was 280 000. Reduction of type III with dithiothreitol yielded expected alpha1(III) chains of 95 000 molecular weight. 3. The amino acid composition of the three collagen chains, alpha1(I), alpha2, and alpha1(III), was the same as previously reported values for the corresponding chains from human skin except for slightly elevated hydroxylysine content. 4. Peripheral nerve collagen was found to contain 81% type I collagen and 19% type III. These results indicate that peripheral nerve collagen characteristics closely simulate that of human skin and differ from that of human aorta and other parenchymal organs. These data will permit a chemical analysis for possible abnormalities of peripheral nerve collagen in various neurogenic disorders.     

Cloning and characterization of five overlapping cDNAs specific for the human pro alpha 1(I) collagen chain.

We report the cloning of five overlapping cDNAs bearing sequences specific for the human pro alpha 1(I) collagen chain. Poly-A RNA enriched for collagen sequences was purified from normal human fibroblasts and used as template to synthesize double stranded cDNA. The cDNA was inserted into the Eco RI site of pBR 322 by blunt-ending and dG:dC tailing. The clones were screened by colony hybridization using the original RNA population and the resulting five positive clones subjected to restriction endonuclease mapping analysis and DNA sequencing. These overlapping clones cover from residue 247 in the alpha chain to part of the 3' end untranslated region of the pro alpha 1(I) mRNA for a total of 3400 nucleotides.