Identification of a New Cross-link and Unique Histidine Adduct from Bovine Serum Albumin Incubated with Malondialdehyde

Abstract

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1⑀-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mM malondialdehyde. This cross-link was identified as its partial degradation product N ⑀ -(2-carboxyl,2-aminoethane)-N ⑀ -methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/ arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4-and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.Glycated collagen promotes the oxidation of polyunsaturated fatty acids to a myriad of reactive aldehydes (1, 2). Many of these have only one functional group, leaving the most toxic major products as 4-hydroxynonenal and malondialdehyde (MDA), 1 the latter being the focus of this paper. These oxidation products, mainly derived from the fats carried by lipoprotein, are implicated in the progression of atheroma by reacting with the collagen arterial wall.Compared with many aldehydes, MDA is relatively nonreactive at neutral pH as it has a pK a value of 4.46, above which it favors the enolate salt form (3) stabilized by a conjugated ⌸-bond system. Resonance stabilization reduces the electrophilicity of MDA, decreasing its reactivity with proteinbased amine groups such as lysine and arginine, for example, in comparison with reaction with glutaraldehyde. Thus, MDA has a half-life in vitro with 10 mM lysine of approximately 2 days (3). However, free MDA is removed from the bloodstream much faster, having a half-life of ϳ2 h in rats (4), with 2-propenal adducts being detected in urine (2). Nonetheless, a significant proportion of MDA has a longer half-life in vivo because it binds to protein amine groups mainly as a temporary and unstable 3-iminoprop-1-en-1-ol adduct, ␤-lysylaminoacrolein (5). The presence of these labile MDA adducts is supported by various ways of measuring MDA, which indicate that at least 80% MDA in tissues is bound reversibly to protein in vivo (5).Incubation of collageneous rat tail tendon in a 10 mM MDA solution for 24 h at 37°C and...

Tendon injury and repair – A perspective on the basic mechanisms of tendon disease and future clinical therapy

“…The collagen fibril is an exquisite example of cellular mediated protein self-assembly [92,93] -with emergent mechanical properties that depend on the diameter of the collagen fibril [88], as regulated by small leucine-rich proteoglycans [60,[94][95][96][97][98][99][100]. The properties are also highly dependent on the extent of covalent collagen molecule cross-linking as regulated by the enzyme lysyl-oxidase [85,86,[101][102][103][104]. Consequently, depending on the degree of collagen packing and extent of collagen crosslinking, collagen fibrils have elastic moduli on the order of several GPa, and failure properties on the order of 0.5-1 GPa [90,105].…”

Carbonylation Induces Heterogeneity in Cardiac Ryanodine Receptor Function in Diabetes Mellitus

“…At low micromolar concentrations, RCS regulate cell proliferation, prevent aggregation of proteins, and tag proteins for degradation (Nagaraj et al, 2003;Barrera et al, 2004;Dalle-Donne et al, 2006;Wong et al, 2010;Segré and Chiocca, 2011). In diabetes, production of RCS, including lipid-derived malondialdehyde and 4-hydroxynonenal and glucose-derived glyoxal, deoxyglucosone, and methylglyoxal (MGO), increases (Slatter et al, 2004;Lapolla et al, 2005;Fosmark et al, 2009;Vicentini et al, 2011). These electrophiles react with susceptible basic amino acids on proteins to form RCS adducts.…”

“…It has been known for more than 20 years that RCS and RCS adduct levels are elevated in the serum of individuals with diabetes (Makita et al, 1992;Slatter et al, 2004;Lapolla et al, 2005;Fosmark et al, 2009;Vicentini et al, 2011) and scavengers of RCS can prevent diabetic complications (Brownlee et al, 1986). Although RCS adductbreaking strategies have been not been successful in clinical studies (Hartog et al, 2011), therapies involving agents that scavenge RCS and/or prevent RCS production have been successful (Bolton et al, 2004;Williams et al, 2007).…”

Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal Caco-2 cells

“…One consequence of hydroxyl radical attack is lipid peroxidation [1]. The reaction leads to damages in cell membrane structure and the major product, malondialdehyde (MDA), can further react with cellular protein and DNA [2,3]. Cytotoxicity and carcinogenesis were both reported as a result of MDA exposure [4,5].…”