Differently cross-linked and uncross-linked carboxy-terminal telopeptides of type I collagen in human mineralised bone.
In bone matrix, type I collagen is stabilised by covalent cross-links formed between adjacent collagen molecules; the majority of which is believed to be immature, divalent bonds. For studying these immature forms in detail, we have developed an immunoassay for a synthetic peptide SP 4 that is analogous with and detects a linear epitope within the C-telopeptide of alpha1-chain of type I collagen. The SP 4 assay, together with the ICTP assay, which is specific for the trivalently cross-linked C-telopeptide, was used for the isolation of the differently cross-linked C-telopeptide structures of the alpha1-chain of type I collagen present in mineralised human bone. Amino acid analysis, peptide sequencing and MALDI-TOF mass spectrometry were used to identify and characterise each of the isolated structures. The cross-link content of each isolated peptide was identified. In the trivalent ICTP peptide, only 40% was cross-linked with pyridinoline, the remainder of the cross-links being currently uncharacterized. The divalent peptides contained only previously characterised cross-linking structures. Most of the divalent cross-links were dihydroxylysinonorleucine (DHLNL), with minor amounts of hydroxylysinonorleucine (HLNL). The relative proportion of the HLNL cross-link was slightly higher in the divalent alpha1Calpha2H peptide. A substantial amount of uncross-linked telopeptide structures was also found. Previous studies, where direct chemical cross-link analyses have been used to assess the maturity of cross-linking, have inferred that bone contains more divalently than trivalently cross-linked C-telopeptides. The immunochemical peptide approach used in this study may help to detect presently uncharacterized, trivalent cross-links, the presence of which is strongly suggested in this study. It also provides additional information regarding the extent and maturity of tissue type I collagen cross-linking in health and disease.
Eriksen
Heidi A
HA
Department of Clinical Chemistry, University of Oulu, Finland. heidi.eriksen@oulu.fi
Sharp
Christopher A
CA
Robins
Simon P
SP
Sassi
Mirja-Liisa
ML
Risteli
Leila
L
Risteli
Juha
J
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D002113:Calcification, Physiologic / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D024042:Collagen Type I; D010455:Peptides; C052929:collagen type I trimeric cross-linked peptide; D003094:Collagen