Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver

Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.     

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.     

The covalent structure of collagen. The amino-acid sequence of alpha2-CB4 from calf-skin collagen.

Sequencing of chymotrypsin, trypsin, collagenase- and hydroxylamine-derived peptides, using the automated Edman degradation procedure, yielded the complete amino acid sequence of alpha2-CB4 from calf skin collagen (321 residues). Together with the data from earlier work, an uninterrupted sequence in the helical region of the alpha2-chain from residues 1-393 is now known. Glycine is found in every third position of the peptide. Hydroxylation of proline and lysine occurs only in the Y-position of the triplet Gly-X-Y and is not complete in every position. Some residues, such as glutamic acid, leucine, phenylalanine and arginine, are distributed non-randomly between the X and Y-positions and this non-random distribution is different in the alpha1 and alpha2-chains. Comparison of the N-terminal 393 residues from the helical region of the alpha1 and alpha2-chains revealed a nearly identical distribution of charged polar residues arginine, lysine, glutamic and aspartic acids. The distribution of the triplet Gly-Pro-Hyp is simialr in both chains. The remaining residues in the alpha2-chain exhibit a high degree of substitutions when compared with those in the alpha1-chain. Approximately one in every two residues in both the X and Y-positions are substituted.     

The covalent structure of collagen. Amino acid sequence of alpha1-CB5 glycopeptide and alpha1-CB4 from chick skin collagen.

The amino acid sequences of chick skin alpha1-CB4 and alpha1-CB5 have been determined by automated Edman degradation of the intact peptides and of their tryptic and chymotryptic peptides. The two peptides contain 47 and 37 residues and comprise residues 56 to 102 and 103 to 139, respectively, of the alpha1(I) chain. In addition, alpha1-CB5 is the major hexose-containing peptide, previously reported to be active in mediating platelet aggregation. A comparison of the sequence with previously reported data on the homologous region of the rat skin alpha1(I) chain indicates that there are only three interspecies differences, or a sequence identity of 96%.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

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.     

Covalent structure of collagen: amino acid sequence of alpha 2-CB5 of chick skin collagen containing the animal collagenase cleavage site.

The amino acid sequence of the 112 residues from the amino terminus of alpha 2-CB5 from chick skin collagen was determined by automated sequential degradation of intact alpha 2-CB5 and several chymotryptic and tryptic peptides. This segment of the peptide includes the site of the action of animal collagenases. As compared to the sequence around the alpha 1 cleavage site, the alpha 2 sequence is notable for the remarkable constancy of the residues to the amino side and the relative abundance of hydrophobic residues to the carboxyl side of the cleavage site, suggesting that these features are important in the recognition by the enzyme. The sequence of this region of the alpha 2 chain is consistent with the Gly-X-Y triplet structure and the preference of certain residues for either the X or Y position in distribution. However, three of the six residues of leucine were found in the Y position rather than the X position. Leucine residues were found only once in the Y position in the alpha 1 (I) chain. This preference does not appear to hold in the alpha 2 chain.     

The amino acid sequence of chick skin collagen alpha1-CB7.

The amino acid sequence of chick skin collagen alpha1-CB7, the 268 CNBr peptide from the helical portion of the alpha1 chain, has been determined by automatic and manual degradation of tryptic and chymotryptic peptides, and of the COOH-terminal fragment produced by cleavage with animal collagenase. The resulting sequence shows 94% identity with that of the corresponding peptide from calf skin collage (Fietzek, P. P., Rexrodt, F. W., Hopper, K. E., and Kühn, K. (1973), Eur. J. Biochem. 38, 396). The bond cleaved by animal collagenase has been identified as Gly-Ile at residues 221-222 of alpha1-CB7.     

The order of cyanogen-bromide peptides and location of carbohydrate in the alpha2 chain of guinea-pig skin collagen.

The alpha2 chain of guinea pig skin collagen contains two additional methionyl residues in comparison with the alpha2 chain of other vertebrate species. The order of the three CNBr peptides unique to the alpha2 chain was established on the basis of the homology of their primary structures to sequences of previously ordered regions in the alpha1 and alpha2 chains of other colagens. The two larger peptides, 4A + 4B, were found to correspond to the region homologous to alpha2-CB4 of other species, while the smaller peptide, 3A, was homologous to the NH2-terminal portion of alpha2-CB3. Thus, the order of the peptides in the alpha2 chain of this collagen is 1-O-4A-4B-2-3A-3B-5. Periodate oxidation and alkaline or acid hydrolysis of the CNBr fragments showed that all of the hydroxlysine-linked carbohydrate in the alpha2 chain was present in alpha2-CB4B. Carbohydrate analyses were most consistent with the existence of single monosaccharide and disaccharide units in this region.     

The covalent structure of collagen: amino-acid sequence of the cyanogen-bromide peptides alpha1-CB2, alpha1-CB4 and alpha1-CB5 from calf-skin collagen.

The amino acid sequence of 120 residues in the N-terminal region of the alpha1-chain of calf skin collagen (comprising the cyanogen-bromide-derived peptides alpha1-CB2, alpha1-CB4 and alpha1-CB5) has been determined by automated Edman degradation. The lysyl residue in position 87 is completely hydroxylated, while those in positions 99 and 108 partially hydroxylated. Two substitutions are found with respect to the homologous region of the alpha1-chain from rat skin collagen. Positions 101 and 102 of calf skin collagen are occupied by Asp-Ala, in rat skin collagen by Asn-Thr. The extensive homology in this region is remarkable and is not found in other regions of the alpha1 and alpha2-chain.     

Chymotryptic and tryptic peptides of fragment alpha 1-CB3 from bovine corneal collagen. Pinpointing the sites of hexose attachment.

Tryptic peptides of citraconylated fragment alpha1-CB3 and chymotryptic peptides of fragment alpha1-CB3 of bovine corneal collagen were prepared, isolated and characterized. Their amino acid compositions were consistent with the amino acid sequence of fragment alpha1-CB3 from calf skin collagen. Two glycoside sites were identified in bovine corneal fragment alpha1-CB3, one of them being the first located in the overlap region of collagen. The results are related to the uniformly narrow collagen fibres found in cornea and essential for its transparency.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

Cross-linking of collagen. Location of pyridinoline in bovine articular cartilage at two sites of the molecule.

The location of pyridinoline in 18-month-old bovine articular cartilage was investigated by fractionation of CNBr-derived peptides by ion-exchange chromatography and gel filtration. Two peptides, PCP1 and PCP2, were isolated and were shown to contain stoichiometric amounts of pyridinoline. From its amino acid composition and sequence studies, peptide PCP1 was shown to comprise two C-terminal non-helical chains (CB14) linked through pyridinoline to the alpha 1(II)-CB12 portion of the helix. The CB14 chains appeared to be labile at their C-terminal ends, resulting in lower-than-expected amounts of homoserine, and only the N-terminal portion of the peptide was sequenced. Similar studies of peptide PCP2 showed that it contained two N-terminal non-helical chains (CB4) linked to the alpha 1(II)-CB9,7 portion of the helix. The isolated peptides therefore confirmed the function of pyridinoline in stabilizing the 4D stagger of adjacent molecules. The possibility that the cross-link could act both as an intra- and an inter-microfibrillar cross-link was considered. A mechanism of formation of pyridinoline was postulated that, together with other evidence, appears to support the view that, in cartilage, pyridinoline acts primarily as an intramicrofibrillar cross-link and does not contribute to increased stability during maturation through lateral aggregation and bonding of filaments.     

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.     

Engineering new peptidic inhibitors from a natural chymotrypsin inhibitor.

Three model peptides of different sizes (17-24 amino acid residues) mimicking the chymotrypsin inhibitor SCGI (a peptide of 35 amino acid residues) isolated from Schistocerca gregaria were designed and prepared by convergent peptide synthesis. Selective formation of disulphide bridges in the closing step was achieved without selective protection of cysteine residues. The natural pattern of the two disulphide bridges was determined by 2D homonuclear 1H NMR techniques. All three model peptides were characterized by amino acid analysis. MS and CD spectra. Preliminary results revealed that the two smaller model peptides exhibit no Inhibitory activity, whereas the larger one shows limited inhibition of chymotrypsin.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

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.     

The covalent structure of calf skin type III collagen. IV. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB5 (positions 552--788).

The cyanogen bromide peptide alpha 1(III)CB5 is 237 amino acid residues in length and occupies position 552--788 along the alpha 1(III) chain. For sequence analysis alpha 1(III)CB5 was fragmented with hydroxylamine, protease from Staphylococcus aureus V8, trypsin and the arginine-specific enzyme from mouse submaxillary gland. The peptides obtained were separated using molecular and ion exchange chromatography and sequenced with the automated Edman degradation procedure.     

Luteinizing hormone of the sperm whale. Isolation, separation into subunits and study of the amino acid sequence of the alpha-subunit

The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine.