Amino-terminal Telopeptide Research Articles

Amino-terminal location of pyridinoline in dentin collagen.

Cross-linking is believed to be one of the major factors that characterize the calcifiability of dentin and bone collagens. Dehydro-dihydroxylysinonorleucine and pyridinoline which constitute the principal cross-links of dentin collagen have so far been located only in the carboxy terminal telopeptide of the molecules [alpha 1(I)-chain 87 x alpha 1(I)-chain 16C]. This situation suggested that the amino terminal telopeptide portion might be "open" without intermolecular cross-linking in hard tissue collagen fibrils. However, the present study provided evidence that pyridinoline is also located in amino-terminal telopeptides (alpha 1-chain 9N or alpha 2-chain 5N) and alpha 1-chain 930. Bovine dentin collagen was digested with trypsin followed by heating at 60 degrees C before and after the digestion. This method gave complete trypsin peptides of dentin collagen. Fluorescent pyridinoline peptides with a smaller molecular size were isolated by Sephadex G-50 superfine, DEAE-cellulose and reverse-phase HPLC. Automatic Edman analysis of several isolated peptides revealed the five-residue sequence, Gly-Ile-X-Gly-His-Arg, the only assignment of which was alpha 1-chain 928-933. The above evidence together with the amino acid compositions of the peptides led to the conclusion that pyridinoline is located not only in the carboxy-terminal but also in the amino-terminal telopeptide in dentin collagen.

The post-translational chemistry and molecular packing of mineralizing tendon collagens

Using cross-linking stereochemistry as indicators, the molecular environment of two collagens in the turkey leg Achilles tendon were compared. The tendon from one year old turkeys was dissected into nonmineralized, fully mineralized and transitionally mineralized portions. Amino acid composition and cyanogen bromide peptide mapping of these portions indicated that the collagens were essentially type I throughout. The fully mineralized compartment had a lysine hydroxylation level similar to turkey or mammalian bone collagen. The non- and transitionally mineralized collagens had a significantly higher lysine hydroxylation, typical of tendon or ligament. However, unlike mammalian tendon, the collagen cross-links were essentially derived from the carboxy-terminal ends of the molecules. The predominant cross-link in this portion was pyridinoline having a high content of 0.95 +/- 0.09 res/mole of collagen. The cross-links in the fully mineralized collagen were also essentially derived from carboxy-terminal aldehyde. However, here significant amounts of the lysyl analog of pyridinoline and lysine-involved bifunctional cross-links were present. The molecular loci of pyridinoline in nonmineralized collagen and the lysyl analog of pyridinoline in mineralized collagen were found to be identical. The total trifunctional cross-link level in the mineralized collagen, 0.55 +/- 0.05 res/mole of collagen, was virtually identical to that observed in old mammalian bone and dentin, and in long term in vitro incubation studies of predentin. We have tentatively concluded that the post-translational chemistry and molecular environments are different in these two turkey tendon fibrils. However, a relative paucity of amino-terminal based cross-links is a feature they have in common. The possible involvement of the amino-terminal telopeptides in collagen mineralization is discussed.

Structural and functional characterization of a splicing mutation in the pro-alpha 2(I) collagen gene of an Ehlers-Danlos type VII patient.

A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.

Amino-terminal propeptide of human pro-α2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule

Abstract We have determined the nucleotide sequence of a cDNA clone encoding the amino-terminal portion of human alpha 2(V) procollagen and found that the structure of the 186-residue amino-terminal propeptide closely resembles those of the fibril-forming procollagens. Juxtaposed to a 26-residue leader peptide, pro-alpha 2(V) exhibits a characteristic cysteine-rich globular region followed by 24 Gly-X-Y repeats which are interrupted by two short non-collagenous sequences. Upon closer examination, each of these two sequences was noted to display structural motifs characteristic of either pro-alpha 1(I) and pro-alpha 1(III) collagens or pro-alpha 1(II) collagen, respectively. Finally, within the amino-terminal telopeptide, a putative amino-terminal proteinase cleavage site, Ala-Gln, was identified. This latter finding strongly suggests that the alpha 2(V) amino-terminal propeptide can be potentially processed and thus leaves unresolved the issue pertaining to the nature of the collagenase-resistant sequence that is retained by mature type V collagen molecules.

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

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

Identification of hydroxypyridinium cross-linking sites in type II collagen of bovine articular cartilage.

In mature cartilage, collagen fibrils are strengthened by covalent intermolecular bonds provided by 3- hydroxypyridinium cross-linking residues. To determine the location of these trifunctional cross-links within the type II collagen molecule, CNBr peptides were analyzed from pepsin-soluble collagen and from guanidine hydrochloride insoluble collagen of bovine articular cartilage. The presence of hydroxypyridinium residues in collagen alpha chains and CNBr-derived peptides was detected by their characteristic natural fluorescence. Quantitatively, about one in three alpha chains from pepsin-soluble collagen was found to contain a hydroxypyridinium residue. Its distribution in the chains was limited to two CNBr peptides, which were purified by column chromatography on CM-cellulose and Bio-Gel P-30 followed by slab-gel electrophoresis in sodium dodecyl sulfate-polyacrylamide. The composition and properties of the two peptides indicated that the main component of one was alpha 1(II)- CB9 ,7 and of the other alpha 1(II) CB12 . It was suspected that two amino-terminal telopeptides were cross-linked by hydroxylysylpyridinoline to alpha 1(II) CB9 ,7 and two carboxy-terminal telopeptides to alpha 1(II) CB12 . The properties of fluorescent CNBr peptides isolated from digests of insoluble cartilage collagen supported this conclusion. Cleavage of the 3- hydroxypyridinium ring by UV light was exploited to confirm the identity of the cross-linked peptides. On UV irradiation, one cross-linked peptide released alpha 1(II) CB9 ,7, and the other, alpha 1(II) CB12 . The findings indicate there are only two hydroxypyridinium cross-linking sites within the triple-helical region of the type II collagen molecule, probably placed symmetrically at opposite ends at residues 87 and 930, where telopeptide aldehydes are known to react to form the initial "head to tail" intermolecular bonds.

Age-related changes in aldehyde location on rat tail tendon collagen

Summary Rat tail tendons were dissolved in denaturants and the α-chains were separated by chromatography and reduced with sodium [3H] borohydride. The distribution of reduced allysine residues among the cyanogen bromide peptides from these chains was studied. In young tendons, 90% of the allysine residues are in the amino-terminal telopeptide region and 10% are elsewhere, probably mostly in the carboxy-terminal region. In older tendons these percentages change to approximately 60% amino-terminal and 30% elsewhere.