Quantification of extracellular matrix turnover by isotope-labelled proteomics: Variation across tissues and age.

Abstract

Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix, assembled during development, stays with us for life. However, recent work suggests that type-I collagen in mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives in the tendon. Furthermore, the properties of these pools are altered during ageing and in other tissues. Tail tendon, heart and brain tissue were harvested from C57BL/6J mice (aged 8, 22, 52 or 78 weeks) fed for fixed periods on a heavy-lysine diet. Protein was extracted for analysis using a sequential two-step protocol: fraction 1 (F1), in sodium laurate/deoxycholate; and fraction 2 (F2), in sodium dodecyl sulphate with ultra-sonication. In adult tendon, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 +/- 2 days in F1, compared to 700 +/- 100 days in F2; matrix protein half-lives could be placed in a hierarchy of collagens > glycoproteins and proteoglycans > matrix-associated proteins. This distinction and the significantly different turnover of F1 and F2 were lost in 52 and 78 week-old animals. Mechanically comparable tissue like the heart showed the presence of 2 distinct pools of matrix half-lives. This difference in turnover is lost, however, in the brain, used as an example of a mechanically different tissue. This work further evidences the faster turnover/permanent model of mechanically active, stiff matrices. It also suggests decreasing turnover as a characteristic of the ageing matrix.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Karl Kadler (Supervisor), Qing-Jun Meng (Supervisor) & Joe Swift (Supervisor)