Advances in collagen cross-link analysis

Abstract

The combined application of ion-trap mass spectrometry and peptide-specific antibodies for the isolation and structural analysis of collagen cross-linking domains is illustrated with examples of results from various types of collagen with the emphasis on bone and cartilage. We highlight the potential of such methods to advance knowledge on the importance of post-translational modifications (e.g., degrees of lysine hydroxylation and glycosylation) and preferred intermolecular binding partners for telopeptide and helical cross-linking domains in regulating cross-link type and placement.

Fig. 1

Hierarchical depiction of a heterotypic collagen fibril, emphasizing the internal axial relationships required for mature cross-link formation. Upper: Three-dimensional concept of the type II/IX/XI heterotypic fibril of developing cartilage matrix. Middle: Detail illustrating required nearest neighbor axial relationships for trifunctional intermolecular cross-links to form in collagens of cartilage, bone, and other high-tensile strength tissue matrices. The exact 3D spatial pattern of cross-linking bonds is still unclear for any tissue. Lower: Detail of the axial stagger of individual collagen molecules required for pyridinoline cross-linking.

Fig. 2

Chemical pathway of cross-linking interactions initiated by lysyl oxidase that predominates in skeletal tissue collagens. Bone collagen features both pyrrole and pyridinoline cross-links whereas mature cartilage contains only pyridinolines. Lysines of triple-helix and telopeptide origin are tracked by color to the mature cross-linking structures.

Fig. 3

Examples of cross-linked peptides recovered on in-gel trypsin digestion of type II collagen (CNBr-digested) from fetal and adult human cartilage. The collagens were extracted from tissue by pepsin digestion and purified by 0.9 M NaCl precipitation before CNBr digestion [17]. (a) Divalent cross-links are prominent in fetal cartilage and in this example the cross-link in the identified peptide is glucosyl galactosyl hydroxylysino-ketonorleucine derived from glcgalHyl at K87 in the triple-helix. (b) The trifunctional cross-link hydroxylysyl pyridinoline predominates in cross-linked peptides from adult cartilage. The structure of the peptide shown, with one telopeptide arm cleaved immediately after the cross-linking hydroxylysine (X), probably reflects cleavage by pepsin at this peptide bond. Note that from the MS results, it is not possible to determine the relative positions of these two telopeptide arms on the pyridinol ring. The lowest panel in each example (c and f) shows the principal fragment ions on MS/MS from the 3⁺ parent ion and their origins as b-ion (aminoterminal) and y-ion (carboxyterminal) cleavage products.

Fig. 4

RP-HPLC resolution and mass spectrometric structural identification of cross-linked peptides from human bone collagen digested by cathepsin K in vitro. (a) RP-HPLC and detection by fluorescence of trivalent pyridinoline-containing peptides from the cathepsin K digest. (b and c) LC/MS resolution and detection of a major pyridinoline-containing peptide from fraction 38. The HP and LP forms of the same peptide are resolved in the mass spectrum as ions that differ by 16 mass units (= oxygen atom), e.g., 905.7 and 901.7 for the 4⁺ charge state. (d and e) LC/MS resolution and detection of a major pyrrole-containing cross-linked peptide from fraction 39 of the HPLC profile. The predominant 2⁺ charge state of the pyrrole structure is a useful distinguishing feature from the predominant 3⁺ charge of the equivalent peptide in which the cross-link is a pyridinoline residue (latter not shown).

Fig. 5

Examples of cross-linked peptides recovered from a bacterial collagenase digest of human cartilage type II collagen. Both N-telopeptide and C-telopeptide forms, from each of the two intermolecular cross-linking sites (Fig. 1) can be identified.

Fig. 6

Summary of methods used to locate the sites of intermolecular cross-linking and the interacting sequences in cartilage type XI collagen polymers. The lower right and upper panels summarize published results on bovine type XI collagen [27]. Lower left panel shows Western blot results on human cartilage using two polyclonal antisera, one against the human α1(XI) cross-linking N-telopeptide sequence, the other against the human α2(XI) N-telopeptide.

Fig. 7

Collagen IX. Flow diagram (upper) showing the methods, results (middle), and molecular interpretation (lower) indicating that the α2(IX)NC1 domain of collagen IX (and, combined with previous results, all three NC1 domains) participates in covalent cross-linking to the surface of type II collagen fibrils.

Fig. 8

Cross-linked peptides isolated by immuno-affinity chromatography from adolescent human urine. (a) NTx-peptides, originating from bone resorption, affinity enriched on mAb 1H11. (b) CTx-II peptides, originating from mineralized growth plate resorption, affinity enriched on mAb 2B4.