The covalent structure of calf skin type III collagen. III. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB4 (positions 403--551).

The cyanogen bromide peptide alpha 1(III)CB4 comprises the sequence region from position 403 to 551 of the alpha 1(III) chain. Almost the entire sequence of this region was elucidated using two hydroxylamine- and one chymotrypsin-derived fragments for automated Edman degradation. The sequence analysis of alpha 1(III)CB4 was completed with the help of trypsin and one protease V8-derived peptide. Comparison with the corresponding region of the alpha 1(I) chain revealed a striking homology between the two chains in this region which is higher than for the entire alpha chains.     

The covalent structure of calf skin type III collagen. V. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB9A (position 789 to 927).

The cyanogen bromide peptide alpha 1(III)CB9A is 139 amino acid residues in length and occupies positions 789--927 along the alpha 1(III) chain. Peptides necessary for the complete sequence analysis were obtained after fragmentation of alpha 1(III)CB9B with trypsin, protease from Staphylococcus aureus V8, hydroxylamine and chymotrypsin. They were separated mainly by chromatography on Sephadex G-50 and phosphocellulose and subsequently sequenced using the automated Edman degradation procedure.     

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.     

The covalent structure of calf skin type III collagen. VI. The amino acid sequence of the carboxyterminal cyanogen bromide peptide alpha 1(III)CB9B (position 928--1028).

The C-terminal cyanogen bromide peptide alpha 1(III)CB9B is 101 amino acid residues in length and occupies position 928--1028 along the alpha 1(III) chain. For sequence analysis, alpha 1(III)CB9B was fragmented with trypsin and chymotrypsin. The peptides obtained were separated using molecular sieve and ion exchange chromatography and sequenced using the automated Edman degradation procedure.     

The covalent structure of calf skin type III collagen. II. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB1,8,10,2(Positions 223--402).

The cyanogen bromide peptide alpha 1-(III)CB1,8,10,2 is 180 amino acid residues in length and occupies position 223 to 402 along the alpha 1(III) chain. In order to elucidate its amino acid sequence, alpha 1(III)CB1,8,10,2 was fragmented with hydroxylamine, protease from Staphylococcus aureus V8 and trypsin. Peptides necessary for sequence analysis with the automated Edman degradation were separated using molecular and ion exchange chromatography. Edman degradation of the hydroxylamine-derived fragments resulted in the elucidation of 80% of the entire sequence. The rest was completely established by sequence analysis of some protease V8 and trypsin-derived peptides.     

Platelet-reactive sites in collagens type I and type III. Evidence for separate adhesion and aggregatory sites.

The adhesion of human and rabbit platelets to collagens and collagen-derived fragments immobilized on plastic was investigated. Adhesion appeared to be independent of collagen conformation, since similar attachment occurred to collagen (type I) in monomeric form, as fibres or in denatured state. The adhesion of human platelets was stimulated to a variable degree by Mg2+, but rabbit platelet adhesion showed little if any dependence on this cation. Collagens type I, III, V and VI were all able to support adhesion, although that to collagen type V (native) was lower than that to the other collagens. Adhesion to a series of peptides derived from collagens I and III was measured. Attachment did not require the presence of peptides in triple-helical configuration. The extent of adhesion ranged from relatively high, as good as to the intact parent collagen molecule, to little if any adhesive activity beyond the non-specific (background) level. The existence of very different degrees of activity suggests that platelet adhesion is associated with specific structural sites in the collagen molecule. Adhesion in many instances was essentially in accord with the known platelet-aggregatory activity of individual peptides. However, two peptides, alpha 1(I)CB3 and alpha 1(III)CB1,8,10,2, exhibited good adhesive activity although possessing little if any aggregatory activity. Of particular interest, despite its near-total lack of aggregatory activity, adhesion to peptide alpha 1(I)CB3 was as good as that to the structurally homologous peptide alpha 1(III)CB4, in which is located a highly reactive aggregatory site. This implies that platelet adhesion to collagen may involve sites in the collagen molecule distinct from those more directly associated with aggregation.     

Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type III collagen.

Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the 'amino donor' residues cross-linked to non-helical 'aldehyde donor' residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.     

Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis.

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific 'maps' were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific 'maps' were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.     

Quantitation of type I and III collagens using electrophoresis of alpha chains and cyanogen bromide peptides

The methods of quantitating the relative amounts of type I and III collagens in samples containing crosslinked collagen chains were evaluated using electrophoresis of alpha chains and cyanogen bromide peptides. The densitometry areas of the alpha I(I) chains from type I collagen and the alpha I(III) chains from type III collagen were reduced because of the failure of the crosslinked chains to dissociate. However, the ratios of the unit densitometry areas of these chains (area of chain/micrograms type I or III collagen loaded) were constant for type I and III collagens prepared from the same samples of tissue. A calibration factor, which was the same for dermis and mitral valve, was derived to convert the densitometry area ratios to the weight ratios of type I to III collagens. In contrast, the densitometry areas of the alpha I(I) CB8 (type I collagen marker) and the alpha I(III) CB5 (type III collagen marker) were not reduced by crosslinked collagen chains. A calibration factor was also derived to convert the ratios of the densitometry areas of the marker peptides to weight ratios of type I to type III collagens. Almost identical results were obtained when electrophoresis of alpha chains and of cyanogen bromide peptides was used with these calibration factors to quantitate the relative amounts of type I and III collagens in tissue extracts which contained different amounts of crosslinked chains.     

Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.     

Covalent structure of collagen: amino acid sequence of alpha 1 (III)-CB5 from type III collagen of human liver

Type III collagen was prepared from human liver by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Ten distinct peptides were obtained by cyanogen bromide digestion. The peptide alpha 1 (III)-CB5 was further purified by carboxymethylcellulose chromatography, and its amino acid sequence was determined. Automatic Edman degradation of intact alpha 1 (III)-CB5, tryptic and thermolytic peptides, and hydroxylamine-derived fragments was used to establish the total sequence. The mammalian collagenase site contained in the alpha 1 (III)-CB5 sequence was ascertained by digestion of native type III collagen with purified rheumatoid synovial collagenase. Collagenase cleavage occurred at a single Gly--Ile bond, one triplet before the corresponding specific cleavage site of type I collagen. The present work brings the known sequence of human liver type III collagen to include alpha 1 (III)-CB3-7-6-1-8-10-2-4-5. These correspond to the homologous region of alpha 1 (I)-CB0-1-2-4-5-8-3-7 residues 11--804.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids.

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 x 10(6)-5 x 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.     

Isolation and characterization of CNBr peptides of human (alpha 1 (III) )3 collagen and tissue distribution of (alpha 1 (I) )2 alpha 2 and (alpha 1 (III) )3 collagens.

[Alpha 1(III)]3 collagen was solubilized by pepsin digestion of normal human placental membranes and was purified by differential salt precipitation and carboxymethylcellulose chromatography. This collagen was digested with CNBr, and the resultant nine peptides were isolated and characterized. The chains are cross-linked by cysteinyl residues in the COOH-terminal peptide. Isolation of peptides derived from CNBr digestion of insoluble tissues was used as an assay for the presence of [alpha 1(I)]2alpha 2 and [alpha 1(III)]3 collagens. Both types are present in human skin, intestine, liver, spleen, kidney, lung, aorta, umbilical cord, placental membranes, and myocardium. Bone and tendon contain [alpha 1(I)]2alpha 2 collagen but, unlike the other tissues, lack [alpha 1(III)]3 collagen. Both [alpha 1(I)]2alpha 2 and[alpha 1(III)]3 collagens are present in scars of human skin, myocardium, tendon, and liver and of rabbit skin. The degree of hydroxylation of proline was 4 to 5% lower in the same peptides in skin, bone, and tendon than in the other tissues. The degree of hydroxylation of lysine in the same peptides derived from different tissues varied more widely.     

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.     

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.     

Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.     

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.