Changes in the structure and organisation of collagen due to glycation

Various techniques have been employed in the study of structural changes and effects brought about by glycation of collagen, at the molecular, fibrillar and super-fibrillar level.

At the molecular level, using circular dichroism Reigle et al. (2008) found that following

in vitro glycation of RTT, glycated collagen molecules solubilised into acid showed no

significant change in triple helical content as compared with non-glycated collagen. However binding experiments with heparin – a structural analogue to the heparin sulphate GAG chains found on some PGs, which have a specific binding site along the collagen molecule (Di Lullo et al., 2002; Sweeney et al., 2008) – resulted in a significantly lower triple helical content in the glycated collagen than the non-glycated

protein (Reigle et al., 2008). Therefore glycation appears to destabilise the collagenous molecular structure.

In the same study, using spectrophotometry to monitor progress, Reigle et al. (2008) also demonstrated that the kinetics of in vitro fibrillogenesis were altered by glycation, the process taking place more slowly for the glycated collagen than for the unglycated protein. However using negative staining and TEM, they found that the control and glycated collagens both formed fibrils that retained a 67 nm D-periodic length and showed no significant differences in the ranges of diameters of the fibrils formed (Reigle et al., 2008).

Also employing TEM work in this area Bai et al. (1992) found that on incubation of RTT with 0.2M ribose in phosphate buffered saline (PBS), the cross-sectional appearance of the fibrils changed markedly. Even after a few days, they became larger and deviated from their initial (and control) circular appearance, becoming much more irregularly-shaped. After two weeks, all the fibrils showed a distorted cross-section, and many of them were in contact or fused with others. Interestingly, despite these marked cross-sectional changes, no differences were discernable in the axial structure and the 67 nm D-periodicity remained the same as in the fibrillogenesis experiments carried out by Reigle et al. (2008). Using X-ray diffraction to study RTT glycated in vitro Tanaka et al. (1988a) produced data, which also showed an unchanged D-period length following the glycation treatments they studied and that the molecular packing expanded across the fibril and became less ordered. They found that the arrangement of the molecules along the fibril remained tightly ordered, but produced evidence that indicated a change in the conformation or electron densities of the molecular terminal telopeptide regions (Tanaka et al., 1988a). This latter finding was also reported for human tendon tissue in diabetic patients as compared with control (James et al., 1991). Work using neutron diffraction suggested that hydroxylysine residues associated with the N- and C- telopeptides for cross-linking were also particularly susceptible to glycation during in

vivo studies of diabetes in rats (Wess et al., 1993).

Previous TEM work has concentrated on the positive staining banding pattern described in section 1.1.4.1 (see also, Figure 1.3). This staining method relies on binding of PTA stain to the basic amino acid residues lysine and arginine (Tzaphlidou et al., 1982b; Chapman et al., 1990; Hadley et al., 1998). As glycation and AGE formation involve these positively charged residues, it is reasonable to presume that post-glycation reductions in positive staining banding pattern intensities can yield information about glycation along fibrils. Hadley et al. (1998) used this to investigate

the glycation of human scleral collagen by fructose and found the glycation not to be a uniform event along the fibril, but rather to occur preferentially in four of the positive staining bands of the D-period (Hadley et al., 1998). Previously, using biochemical techniques, Reiser et al. (1992b) had also identified four specific lysine residues along type I collagen molecules, within fibrils, that are preferentially glycated in vitro and suggested that this was due in part to primary amino acid sequence. Reiser et al. (1992b) also suggested that the proximity of other groups, especially acidic amino acids might catalyse the Amadori reaction, and other work has provided evidence for this in other proteins (Shapiro et al., 1980; Venkatraman, Aggarwal and Balaram, 2001; Johansen, Kiemer and Brunak, 2006). However this did not explain all of the preferential sites of glycation found and they also suggested that higher order structure might be an important factor and that with increasing age and so more disordered tissue resultant from glycation-related damage, site-specificity of glycation might diminish (Reiser et al., 1992b). However, Reiser et al.’s (1992b) data did not support this as being a major determining factor of site specificity.

Other studies were in agreement that there are preferentially glycated sites. Wess et al. (1990; 1993) using neutron diffraction to study tendon AGE cross-linking sites in vivo and Mikulíková et al. (2007) using electrophoretic and HPLC separation techniques coupled to mass spectrometry to study in vitro-glycated tendon, both identified preferentially-glycated lysines. However, there is not complete consensus between these studies. There is consistency between some of the identified sites, i.e. Wess et al. (1990) and Mikulíková et al. (2007) agreed on one site of preferential glycation (lysine 855), and Reiser et al. (1992b) agreed with one of Wess et al.’s (1990) suggested sites (lysine 434) but not with the other (lysine 855). Reiser et al. (1992b) and Hadley et al. (1998) also agreed on another specific locus (lysine 479). However the other sites identified by Reiser et al. (1992b) and Mikulíková et al. (2007) appear to be uncorroborated by other studies.

Clearly different approaches can generate different specific data regarding preferred sites of glycation. Even within the same study, Le Pape et al.’s (1984) investigation of preferred glycation sites has yielded discrepant results between an in vivo diabetes model and an in vitro glycation approach. In addition, not all studies have concurred that there are preferred sites (see Brennan (1989b)). Therefore although there is general agreement inasmuch as the breadth of effects that glycation has on structure and other aspects of normal collagen functioning, study of more specific aspects of the origins and mechanisms of these effects might be difficult to accomplish until the

nature and locations of the glycation reactions can be determined with greater consensus.