Unravelling the molecular contributions to collagen higher order structure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand

Thumbnail Image
Open Access Location
Journal Title
Journal ISSN
Volume Title
Massey University
The Author
Abnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types of cross-links in different species. Furthermore, no research has been done to compare the relative distribution of diastereomers of hydroxylysine even within the same species. In order to make a valid comparison, collagen needs to be purified from skin to a high degree and separated into different collagen types and sub- structures as much as possible. To achieve this, the extraction and purification of collagen from the skins of four different mammalian species displaying different skin tensile strengths has been optimised. Different extraction methods were used to prevent the loss of specific features of the collagens that were characterised that may otherwise be lost. Amino acid analysis revealed that while the ratios of the two hydroxylysine diastereomers differed between different animals and extraction methods, the differences were not significant. Mass spectral analysis of cross-links showed that goat skin differed from the other three animals in its cross-link profile. Amino acid analysis combined with mass spectral analysis revealed that on average 70% of proline residues were hydroxylated, a figure much higher than previously thought. Mass spectral analysis also revealed that there are some differences between the glycosylation pattern of different animals, and the ratios of the different types of collagen which are extracted from each animal. While these findings need to be confirmed, they challenge some long held beliefs about the collagen molecule and provide a firm foundation for future work.
Table 2 and Figure 3 are re-used with the respective publisher's permission.
Collagen, Structure, Synthesis, Proteins, Crosslinking