Mechanical Stretching Changes Cross-Linking and Glycation Levels in the Collagen of Mouse Tail Tendon

J Biol Chem. 2020 Jun 16;jbc.RA119.012067. doi: 10.1074/jbc.RA119.012067. Online ahead of print.

Abstract

Collagen I is a major tendon protein whose polypeptide chains are linked by covalent cross-links. It is unknown how the cross-linking contributes to the mechanical properties of tendon or whether cross-linking changes in response to stretching or relaxation. Since their discovery, imine bonds within collagen have been recognized as being important in both cross-link formation and collagen structure. They are often described as acidic or thermally labile, but no evidence is available from direct measurements of cross-link levels whether these bonds contribute to the mechanical properties of collagen. Here, we used MS to analyze these imine bonds after reduction with sodium borohydride while under tension and found that their levels are altered in stretched tendon. We studied the changes in cross-link bonding in tail tendon from 11-week-old C57Bl/6 mice at 4% physical strain, at 10% strain, and at breaking point. The cross-links hydroxy-lysino-norleucine (HLNL), dihydroxy-lysino-norleucine (DHLNL), and lysino-norleucine (LNL) increased or decreased depending on the specific cross-link and amount of mechanical strain. We also noted a decrease in glycated lysine residues in collagen, indicating that the imine formed between circulating glucose and lysine is also stress-labile. We also carried out mechanical testing, including cyclic testing at 4% strain, stress relaxation tests, and stress-strain profiles taken at breaking point, both with and without sodium borohydride reduction. The results from both the MS studies and mechanical testing provide insights into the chemical changes during tendon stretching and directly link these chemical changes to functional collagen properties.

Keywords: chemistry; collagen; connective tissue; glycation; imine bond; mechanical stress; physical strain; protein cross-linking; stress; tendon.