Glucosepane Is a Major Protein Cross-link of the Senescent Human Extracellular Matrix

Abstract

The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysinearginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n ‫؍‬ 110) and glomerular basement membrane (GBM, n ‫؍‬ 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p ‫؍‬ 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes.Reducing sugars react nonenzymatically with proteins to form adducts and cross-links referred to as advanced glycation end products (1). Their accumulation is particularly high in long-lived proteins, such as lens crystallins and collagen, and results in intra-and intermolecular cross-linking. The latter has been hypothesized to result in an age-and diabetes-related stiffening of collagenous tissues and is believed to play an important role in the etiology of atherosclerosis and cardiovascular disease (3) as well as the loss of elasticity in lungs, joints, and skin (4 -6). These age-related processes are markedly accelerated by diabetes and are associated with morbidity and mortality in diabetic individuals (7,8). In skin, protein crosslinking is associated with an age-related loss of elasticity, increased stiffening, and wrinkling (9). However, this process occurs ubiquitously with age, suggesting a fundamental underlying mechanism.Although most of the age-related changes in the extracellular matrix could be duplicated by incubating reducing sugars with proteins, Eble et al. (10) suggested that the major cross-links are not stable to conventional conditions of acid hydrolysis. Indeed, Biemel et al. (11) recently showed that glucose and sugar-derived dicarbonyl intermediates react with human serum albumin to form various acid...

Identification of Glucose-Derived Cross-Linking Sites in Ribonuclease A

“…In this aspect, our in vitro nonoxidative incubation results conform well with the predominant level of glucosepane observed during analysis of in vivo human tissue modifications. 11,12 Most established protocols and routine methods used to identify and confirm cross-links in vivo utilize complete acid hydrolysis or exhaustive enzymatic digestion of proteins to the constituent amino acids, and amino acids with modifications. 11,22 The cross-links identified by these protocols will include both intra-and intermolecular cross-links.…”

“…Our laboratory provided additional data supporting the premise that glucosepane is the single major glycation derived cross-link known to date in aging human skin and glomerular basement collagen. 12 In addition, its levels are dramatically elevated in diabetes, 13 a condition that is associated with increased stiffness of all collagenrich tissues. Glucosepane levels increased up to ~2 nmol/mg collagen in old nondiabetic controls, and further increased to ~4.3 nmol/mg in diabetic patients.…”

Advanced glycation end-products: Mechanics of aged collagen from molecule to tissue

“…1A) [13]. Recently, it has been found that among all different AGEs in human tissues the most abundant is glucosepane, a lysine-arginine crosslinking product [14,15].…”

Bacterial enzymes that can deglycate glucose- and fructose-modified lysine

“…Fructoselysine is a major protein adduct in extracellular matrix proteins, haemoglobin, lens proteins and other cells in which glucose permeates freely. It is the precursor to glucosepane, a lysine-arginine protein cross-link that is increased severalfold in diabetes [1], as well as several glycoxidation products [2]. For this reason, considerable efforts are being deployed to find ways to block or reverse its formation.…”