Cross-linking of structural proteins in ageing skin: an in situ assay for the detection of amine oxidase activity

Abstract

With increasing age, dynamic tissues such as lungs, blood vessels and skin lose their ability to both deform and recoil, culminating in tissue stiffening. This loss of tissue elasticity, which profoundly impacts tissue function and thus morbidity, may be due not only to changes in the relative abundance of key extracellular matrix proteins within tissues but also to their accumulation of post-translational modifications. Whilst to date attention has focussed primarily on the age-related non-enzymatic formation of advanced glycation end products, the accumulation of pathological enzyme-mediated cross-links may also lead to age-related tissue stiffening. The lysyl oxidase (LOX) family of enzymes are constitutively expressed in adult tissues and are known to drive the catalysis of cross-links in both fibrillar collagens and elastin. Although immunochemical approaches are commonly used to localise the inactive pro-enzyme of LOX, and biochemical methods are employed to quantify activity in homogenised tissue, they do not allow for the in situ localisation of the enzyme. Thus, we have developed a novel assay to both detect and localise LOX enzyme activity in situ. LOX family members are amine oxidases and this assay uses the principle that an amine substrate in the presence of this class of enzyme will be oxidised to an aldehyde and hydrogen peroxide (H2O2). In turn, H2O2, when combined with luminol and horseradish peroxidase, will produce a light-emitting reaction that can be detected by film autoradiography. The development of a technique to localise specific amine oxidase activity in tissue sections may provide crucial additional information on the exact role played by this class of enzymes in mediating age-related tissue stiffening.

Significance of the Lysyl Oxidase Members Lysyl Oxidase Like 1, 3, and 4 in Gastric Cancer

“…This result might be explained by several studies, which reported that the activity of LOX family was higher in the aged group than in the young group in skin [36,37].…”

Proteome analysis in the assessment of ageing

“…2). Collagen cross-linking can either be induced enzymatically via lysyl oxidase enzymes or chemically as a result of advanced glycation end products formation (Langton et al, 2013). The later modification was shown to trigger pathological age-related tissue stiffening (Sherratt, 2009).…”

Combining nano-physical and computational investigations to understand the nature of “aging” in dermal collagen

“…An agerelated stiffening of tissue was observed in tendon, vascular and myocardial tissue, cartilage and skin. 9,10,18,43 A stiffening of tissue was also observed in diabetic volunteers. A notable in vitro study reported an increase in stiffness of rabbit tendon incubated with glucose.…”

“…5 With elastin turnover approaching a lifespan, structural and physiochemical changes to the elastin network accumulate with age. 9,10 Decorin and versican undergo changes in their GAG chains, with studies showing a reduction in the molecular size of their polysaccharide chains. 6,11 HLA content in the dermis decreases as it is produced less by fibroblasts and disappears altogether in the epidermis as a function of age.…”

“…17 LOX activity is higher in skin than in other tissues, 18 showing an increase in expression in aged skin. 10 However, the proportion of LOX-derived cross-links compared to other covalent cross-links in the ECM reduces with age. 19 The proportion of nonenzymatic (glycation) cross-links, investigated in this study, increases, and therefore, they are associated with aging rather than the enzymatic LOX-derived cross-links.…”