Characterization of pepsin-solubilized bovine heart-valve collagen.

Collagens extracted from heart valves by using limited pepsin digestion were fractionated by differential salt precipitation. Collagen types were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, amino acid analysis and cleavage with CNBr. Heart-valve collagen was heterogeneous in nature, consisting of a mixture of type-I and type-III collagens. The identity of type-III collagen was established on the basis of (a) insolubility in 1.7 M-NaC1 at neutral pH, (b) behaviour of this collagen fraction on gel electrophoresis under reducing and non-reducing conditions, (c) amino acid analysis showing a hydroxyproline/proline ratio greater than 1, and (d) profile of CNBr peptides on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showing a peak characteristic for type-III collagen containing peptides alpha1(III)CB8 and alpha1(III)CB3. In addition to types-I and -III collagen, a collagen polypeptide not previously described in heart valves was identified. This polypeptide represented approx. 30% of the collagen fraction precipitated at 4.0 M-NaCl, it migrated between beta- and alpha1-collagen chains on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its electrophoretic behaviour was not affected by disulphide-bond reduction. All collagen fractions from the heart valves contained increased amounts of hydroxylysine when compared with type-I and -III collagens from other tissues. The presence of beta- and gamma-chains and higher aggregates in pepsin-solubilized collagen indicated that these collagens were highly cross-linked and suggested that some of these cross-links involved the triple-helical regions of the molecule. It is likely that the higher hydroxylysine content of heart-valve collagen is responsible for the high degree of intermolecular cross-linking and may be the result of an adaptive mechanism for the specialized function of these tissues.     

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.     

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.     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

Type II collagen arthritis: identification of arthritogenic epitopes using fractionated anticollagen IgG

Affinity-purified anticollagen IgG was fractionated on purified cyanogen bromide-derived collagen peptide Sepharose. The antibody fraction bound to the peptides was eluted and tested for its ability to induce passive arthritis in recipients. Anticollagen IgG bound to peptide 5 (alpha 1(II)-CB8-10 and alpha 1(II)CB11-8) and to peptide 6 (alpha 1(II)CB11) were active in inducing passive arthritis. Other peptide bound fractions were inactive. These observations suggest that the arthritogenic domain in Type II collagen is restricted to alpha 1(II)CB11.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

A structural mutation of the collagen alpha 1(I)CB7 peptide in lethal perinatal osteogenesis imperfecta

Structurally abnormal type I collagen was identified in the dermis, bone, and cultured fibroblasts obtained from a baby with lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I)-chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution. This heterozygous peptide defect was not detected in the collagens from either parent. The defect was localized to a 224-residue region at the NH2 terminus of the alpha 1(I)CB7 peptide by mammalian collagenase digestion. Analysis of unhydroxylated collagens produced in cell culture indicated that the mutant alpha 1(I)CB7 migrated faster on electrophoresis suggesting that the abnormality may be a small deletion or a mutation that alters sodium dodecyl sulfate binding. The post-translational hydroxylation of lysine residues was increased in the CB7 peptide and also in peptides CB3 and CB8 which are toward the NH2 terminus of the alpha 1(I)-chain. The COOH-terminal CB6 peptide was normally hydroxylated. These findings support the proposal that the lysine overhydroxylation resulted from a perturbation of helix propagation from the COOH to NH2 terminus of the collagen trimer caused by the structural defect in alpha 1(I)CB7.     

Changes in the cross-linking of collagen from rat tail tendons due to diabetes

The acid solubility of Type I collagen from rat tail tendons decreases due to diabetes. This finding has been taken as evidence that collagen from diabetics may be more cross-linked than normal. We compared CNBr peptide maps prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for [3H] NaBH4-reduced tail tendons from streptozotocin-diabetic rats with maps from age-matched control rats. At least through 30 weeks of diabetes, the distribution of mass of both cross-linked and uncross-linked CNBr peptides was identical in diabetic and control tendons. Therefore, the number of cross-linked peptides did not increase due to diabetes. We analyzed the 3H-cross-linking compounds present on the CNBr peptides and found that the 3H content of peptides cross-linked in control tendons through the bivalent, reduced cross-links hydroxylysinonorleucine and lysinonorleucine was diminished on corresponding peptides from diabetic tendons as a function of duration of diabetes. The cross-linked peptides, however, persisted. Therefore, we conclude that a larger fraction of these bivalent cross-links is found in an unknown, non-reducible form in tendons from diabetic compared with control rats. This resembles a phenomenon normally associated with maturation and/or aging where the non-reducible form of the cross-links is acid-stable. An increase in the fraction of the cross-links that is non-reducible and acid-stable would explain, at least in part, the decrease in acid solubility of the collagen. Non-enzymatic glycation (NEG) was not very specific, since most CNBr peptides bound some glucose. However, peptides from the alpha 2-chain seemed to be preferential targets for NEG. While NEG clearly increased due to diabetes, we found no evidence that increased NEG led to an increased number of cross-links in tail tendon collagen from streptozotocin diabetic rats.     

Bovine type I collagen: A study of cross-linking in various mature tissues

The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000.     

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.     

Increased steady-state levels of laminin B1 mRNA in kidneys of long-term streptozotocin-diabetic rats. No effect of an aldose reductase inhibitor

The steady-state levels of mRNAs coding for two components of basement membranes, the alpha 1 chain of type IV collagen and the B1 chain of laminin, were measured in the kidneys of male CDF rats following the induction of diabetes with streptozotocin for periods of between 2 days and 28 weeks. The concentration of mRNA for the alpha 1 chain of type IV collagen/microgram of RNA decreased markedly with age in control and diabetic rats. The diabetic level was significantly lower than control after 2 and 11 weeks of diabetes. After 28 weeks, however, there was no significant difference from the levels in control animals. Treatment of control and diabetic rats with the aldose reductase inhibitor Statil (350 mg/kg diet) did not affect the levels of the mRNA for the alpha 1 chain of type IV collagen. In contrast to the continuous decline in the concentration of mRNA for the alpha 1 chain of type IV collagen, the level of mRNA for the B1 chain of laminin increased two-fold between 11 and 28 weeks after induction of diabetes. This increase occurred as aging of control rats reduced the level of laminin B1 mRNA by approximately 50%. Treatment with Statil had no effect on laminin B1 mRNA levels. In control rats there was no change in the ratio of the levels of mRNAs for laminin B1: alpha 1 (IV) collagen with age. The mean ratio was 0.97 +/- 0.10 at 19 weeks and 1.0 +/- 0.10 at 36 weeks of age. In diabetic rats there was a marked increase in the ratio from 0.85 +/- 0.11 at 19 weeks to 3.2 +/- 1.2 at 36 weeks of age. The increased abundance of mRNA for laminin B1 raises the possibility that increased synthesis of laminin contributes to the thickening and abnormal function of renal basement membranes in streptozotocin-diabetic rats.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

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.     

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.     

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.     

Isolation and purification of biopolymers using an affinity chromatography method. X. A biospecific approach to preparing collagen-like peptides containing segments binding fibronectin

A biospecific method for one-stage isolation of collagen peptides containing the fibronectin binding site is proposed. alpha 1CB7-peptide of the type I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45 kDa) of fibronectin. The method allows one to obtain, in a short time, highly purified preparation of alpha 1CB7-peptide necessary for biochemical studies.     

Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta.

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the 'slow' alpha (I)'- and alpha 2(I)'-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)'- and alpha 2(I)'-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)'-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)'- and alpha 2(I)'-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.     

Activation of human neutrophils by type I collagen. Requirement of two different sequences.

Contact between type I collagen purified from several species and human polymorphonuclear neutrophils (PMNs) triggers the production of O2.- by these cells. The activity of collagen is located in the alpha 1(I)-CB6 cyanogen bromide-cleaved (CB)-peptide, which is the C-terminal CB-peptide of the alpha 1(I) chain. Experiments based on the competitive inhibition of O2.- production by simultaneous incubation of PMNs with type I collagen and synthetic peptides identical to the conserved sequences of this collagen demonstrated that the binding of collagen to PMNs and the subsequent activation of these cells depend on the simultaneous presence of two sequences: Arg-Gly-Asp [residues 915, 916 and 917 of the complete alpha 1(I) chain, located in the helical part] Asp-Gly-Gly-Arg-Tyr-Tyr (residues 1034-1039, located in the C-terminal non-helical telopeptide).     

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.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).