Collagens II, IX and XI resist extraction with denaturants or serial digestion with streptomyces hyaluronidase, chondroitinase ABC, and trypsin at 37°C. Such serial digestion leaves little else in the cartilage but these three collagens as cross-linked polymers [12
]. The exact spatial relationships, manner and temporal order of assembly of these different collagen types into heteromeric fibrils are not well understood. Their interaction and existence as subunits of the same fibril network have been shown by immunoelectron microscopy [13
] and the isolation and structural identification of cross-linked heterotypic peptides [14
]. The basic structure of the fibrils seen by TEM is a four-dimensional (4D)-staggered polymer of collagen type II molecules heavily cross-linked head-to-tail by hydroxylysyl pyridinoline residues at the two telopeptide-to-helix sites.
Collagen IX molecules can decorate fibril surfaces, particularly those of thin fibrils in the pericellular basket [16
]. Cross-linking studies have identified at least six sites of cross-linking within the collagen IX molecule where covalent bonds form with either collagen II molecules or with other collagen IX molecules [14
] (Eyre D, Wu J, Weis M, unpublished observations, 2001; Fig. ). The cross-linking residues are either trivalent pyridinolines or divalent borohydride-reducible intermediates formed by the same lysyl oxidase-mediated mechanism as occurs in the major fibril-forming collagens.
The collagen II:IX:XI heterofibril. A molecular model of the collagen type IX fold and interaction site with a collagen II microfibril that can account for all known cross-linking sites between collagen II and IX molecules.
Each of the three collagen IX chains, α1(IX), α2(IX), and α3(IX), has one to three cross-linking sites, all of which are occupied in the matrix pool of type IX collagen, as judged from peptide mapping studies [17
]. The role of collagen IX in the matrix apparently requires the molecules to be covalently linked to the surface of type II collagen fibrils, which suggests a mechanical restraint of some kind. It is tempting to speculate from the biochemical evidence that collagen IX can also form a covalent bridge between fibrils, increasing network mechanical integrity and providing a restraint for entrapped proteoglycan osmotic swelling pressure. Interfibrillar cross-linking has not been proven, however, and it could be that covalently anchored molecular projections from fibril surfaces (the COL3 domain and terminal NC4 globular domain of α1(IX) project from the fibril surfaces) could restrict shear strains between fibrils in a mesh of thin fibrils embedded in a proteoglycan gel, without a need for direct covalent bonds between fibrils. Figure shows how collagen IX molecules can be accommodated on a fibril surface and can satisfy all the covalent interactions so far identified. In this model proposed by Miles et al.
], the COL1/NC1 domain docks in the hole region, oriented as shown in Figure , and the molecule hinges back on itself at the NC2 domain.
Collagen XI is found in developing cartilage as a heterotrimeric molecule of two novel collagen gene products (α1(XI) and α2(XI)) and a third chain (α3(XI)) identical in primary sequence to α1(II)B, the common form of splicing variant of the type II collagen gene [6
]. From mature articular cartilage, the isolated collagen XI fraction contains α1(V) and α1(XI) in roughly equal amounts [6
]. The α1(V) chain appears to occur in hybrid molecules together with α1(XI) and/or α2(XI) rather than in typical type V collagen molecules found in non-cartilaginous tissues. The biological significance of this is unknown.
The N-propeptide domains of all these chains are retained in the matrix and alternatively spliced variants can be expressed [20
]. Selective binding interactions with other matrix macromolecules can be expected as part of the distinctive function of these molecules. Immunolocalization studies [13
] and analyses of cross-linked peptides [15
] have shown that the collagen XI pool is intimately copolymerized with type II collagen. The type XI N-propeptide domains are thought to poke out of the hole domains of the collagen 4D-staggered lattice, perhaps acting to limit growth in fibril diameters [20
]. Collagen XI is most concentrated in the pericellular network of thin fibrils, and recent work has shown high-affinity binding sites for heparan and heparin sulfate in the triple helical domains [9
Cross-linked peptide analyses have shown that collagen XI molecules are cross-linked to each other through their N-telopeptide-to-helix interaction sites [15
]. They lack a cross-linking lysine in the C
-telopeptide except in the α3(XI) (αI(II)) chain. Interestingly, the N-telopeptide cross-linking lysines are located external to candidate metalloproteinase cleavage sites, in α1(XI), α1(V) and α2(XI), implying that any such cleavages could selectively depolymerize collagen XI [15
]. The N-terminal helical cross-linking site of collagen XI molecules was occupied (in α1(XI)) by the α1(II)C-telopeptide. By analogy to findings with the type I/V collagen heteromer of bone [21
], this is consistent with the formation of lateral cross-links between collagen II and XI molecules at this locus. Together, these findings can be interpreted as collagen XI initially forming a head-to-tail self-cross-linked filament that becomes integrated and cross-linked laterally onto or within the body of collagen II fibrils. Collagen XI could conceivably form an interconnecting, secondary filamentous network that provides links between fibrils as well as running within fibrils, not inconsistent with the current concept that collagen XI restricts the lateral growth of collagen II fibrils [22
]. Clearly, the majority of the covalent links of collagen XI are type XI to type XI [15
] and this fact needs to be accommodated in any workable model of fibril assembly.
Proteolytic and mechanical damage to the fibrillar network is believed to be a key, perhaps irreversible, stage in the destruction of joint cartilages in arthritis. Defining and being able to monitor the structure, assembly and biological mechanisms of degradation of the cartilage collagen heterotypic polymer are therefore important for the development and validation of rational therapeutic targets for treating and preventing joint disease.