Destino final de unidades

Certain lysine residues of nascent collagen -chains are post-translationally hydroxylated by a family of enzymes called lysyl hydroxylases, of which three isoforms have thus far been identified (Myllyla et al., 2007). As in the case of the prolyl 4- hydroxylases, these enzymes are ascorbate-dependant (Myllyla et al., 1984). The resulting hydroxylysine (Hyl) residues are important for the later formation of specifically-located extracellular collagen cross-links.

Following the assembly of the collagen molecules into fibrils, intermolecular cross- linking takes place. This process serves to further stabilise the structure, increasing fibrillar strength and preventing excessive slippage between collagen molecules (Sasaki and Odajima, 1996b; Avery and Bailey, 2005). This is a very specific mechanism of cross-linking and is initiated by lysyl oxidase enzymes, which oxidatively deaminates the -amino group of a single telopeptide lysine or hydroxylysine residue to form the corresponding aldehyde (Bailey, Paul and Knott, 1998). Lysyl oxidase acts on lysines in the telopeptide regions only when it is bound to a conserved sequence, i.e. - Hyl-Gly-His-Arg- in the triple-helical region of an adjacent collagen molecule (Reiser, McCormick and Rucker, 1992a; Bailey et al., 1998). Following this, cross-link formation is a spontaneous condensation reaction brought about by the reaction of telopeptide lysyl or hydroxylysyl aldehydes with a lysine or hydroxylysine in the triple helix of a neighbouring molecule to form a dimeric Schiff base double bond. Four such species can be formed (Figure 1.4): telopeptide lysyl aldehydes react with triple-helix lysine or hydroxylysine residues to form the aldimines dehydro-lysinonorleucine (deH-LNL) and dehydro-hydroxylysinonorleucine (deH-HLNL); hydroxylysine aldehydes can react with triple-helix lysine or hydroxylysine residues to form the ketoimines lysino- ketonorleucine (LKNL) and hydroxylysino-ketonorleucine (HLKNL) after a rearrangement – the Amadori rearrangement – which gives rise to these stable cross- links (Bailey et al., 1998; Bailey, 2001).

With maturation, these divalent cross-links spontaneously react further with species on other molecules, to give more reduced, stable trivalent cross-links (Figure 1.5). The other species involved are histidine residues, and other lysyl- and hydroxylysyl- aldehydes. Histidine reacts with deH-HLNL to form histidino-hydroxylysinonorleucine (HHL). The reaction of HLKNL or LKNL with a further telopeptide hydroxylysyl- aldehyde residue results in the formation of a hydroxylysyl-pyridinoline (HL-Pyr) or lysyl-pyridinoline (LysPyr) cross-links, respectively (Willett et al., 2007). HL-Pyr and

LysPyr (sometimes referred to as pyridinium cross-links) are more stable and non- reducible than their immature, divalent predecessors (Fujimoto and Moriguchi, 1978; Avery, Sims and Bailey, 2009).

Figure 1.4 The telopeptide lysine/hydroxylysine aldehyde reactions with

lysine/hydroxylysines in the collagen triple helix of a neighbouring molecule in a fibril, to form divalent immature cross-links which are stable under physiological conditions. Reprinted from Bailey et al. (1998), © 1998, used with permission from Elsevier.

These immature and pyridinium cross-links occur in specific locations, linking molecular ends (non-helical telopeptides) to helical loci. These are highly conserved sites between species. They form between the lysyl/hydroxylysyl aldehydes in the 1- and 2-chain N-telopeptides and lysines/hydroxylysines at helix positions 930 in the

1-chain or 933 in the 2-chain, and between the lysyl/hydroxylysyl aldehydes in the

1-chain C-telopeptides and lysines/hydroxylysines in and lysines/hydroxylysines in position 87 in triple helix on the 1- or 2-chain (Reiser et al., 1992a; Hanson and Eyre, 1996). The HHL forms between the deH-HLNL formed between a C-terminal telopeptide lysyl aldehyde and a hydroxylysine at position 87 in the triple helix region of an 1-chain, and histidine 92 in the 2-chain (Mechanic et al., 1987; Yamauchi et al., 1987; Reiser et al., 1992a).

Figure 1.5 Formation of the characterised mature cross-links: (a) HHL derived from

the divalent Schiff base HLNL and triple helical histidine and (b) the pyridinolines derived from the divalent keto-imines and the telopeptide hydroxylysyl aldehyde. Reprinted from Bailey et al. (1998), © 1998, used with permission from Elsevier.

The types of cross-links found are more reliant on tissue type, location and maturity than on species. Before birth and in the early months or years of life, the immature cross-links predominate before reacting to form the trivalent, mature cross-links. The nature of the cross-links formed and their rate of formation is heavily influenced by lysyl hydroxylase and lysyl oxidase activities in different tissues (Bailey, 2001; Avery and Bailey, 2005).

In tendon there is variability in hydroxylation depending on the particular tendon being considered. Varying proportions of deH-HLNL and HLKNL are formed, leading to both HHL and pyridinoline cross-links predominating with increasing age. Therefore tendon’s mechanical strength and thermal stability tends to increase with age (Avery and Bailey, 2005; Willett et al., 2010). Curiously, in rat tail tendon (RTT) tissue hydroxylation is very low and so deH-HLNL is initially formed. However, though this decreases with age and the tendon continues to strengthen, it does not react with

histidine to form HHL. Therefore it is possible that a cross-link that has yet to be identified is formed (Bailey, 2001; Avery and Bailey, 2005). In comparison with a load- bearing tissue such as human patellar tendon, tail tendon from young rats has different mechanical properties showing it to be less mechanically resilient to applied stress and more prone to mechanical rupture. This is associated with a markedly lower level of mature cross-links and higher level of immature cross-links in the RTT as compared with the human tissue (Svensson et al., 2013). Therefore clearly, it can be considered that the pattern and profile of enzymic cross-linking of tissue is attuned to the requirements of that tissue.