Protein aggregation as a consequence of non-enzymatic glycation: Therapeutic intervention using aspartic acid and arginine

Abstract

Non-enzymatic glycation tempted AGEs of proteins are currently at the heart of a number of pathological conditions. Production of chemically stable AGEs can permanently alter the protein structure and function, concomitantly leading to dilapidated situations. Keeping in perspective, present study aims to report the glycation induced structural and functional modification of a cystatin type isolated from rai mustard seeds, using RSC-glucose and RSC-ribose as model system. Among the sugars studied, ribose was found to be most potent glycating agent as evident from different biophysical assays. During the course of incubation, RSC was observed to pass through a series of structural intermediates as revealed by circular dichroism, altered intrinsic fluorescence and high ANS binding. RSC incubation with ribose post day 36 revealed the possible buildup of β structures as observed in CD spectral analysis, hinting towards the generation of aggregated structures in RSC. High thioflavin T fluorescence and increased Congo red absorbance together with enhanced turbidity of the modified form confirmed the aggregation of RSC. The study further revealed anti-glycation and anti-aggregation potential of amino acids; aspartic acid and arginine as they prevented and/or slowed down the process of AGEs and β structure buildup in a concentration dependent manner with arginine proving to be the most effective one.

Introduction

Reducing sugars react with proteins via a multistep chemical process known as non-enzymatic glycation. The process follows a sequential multi-step course starting with the condensation between N-terminal group of a protein or free amino acid side-chain and carbonyl group of reducing sugar through a nucleophilic addition leading to the rapid formation of a reversible and unstable acid labile Schiff's base which undergoes rearrangement to form a more stable early glycation product known as Amadori product [1,2]. The stable Amadori-adduct thus formed undergoes oxidation, dehydration and other chemical reactions generating reactive dicarbonyl compounds which in turn lead to the formation of irreversible cross-linked structures known as advanced glycation end-product (AGE's) [3,4]. AGE's formation is a natural metabolic course but when their formation is overdone, AGE's reach the tissues and the circulation thereby proving fatal [[1], [2], [3]]. AGE's when formed in excess tend to promote oxidative stress which could lead to the alteration in structure and function of proteins thence rendering them functionless, thereby leading to variety of pathological situations [[5], [6], [7]].

Glycation of proteins as such is known to promote the generation of protein crosslink's with the resultant development of insoluble and protease-resistant aggregates and/or amyloid forms [8,9]. A number of human neurological and several other related disorders have been found to be associated with the formation of toxic protein aggregates and/or amyloid fibrils [10,11]. AGEs tailored amyloids have been found to be associated with the brain tissues of patients suffering from Alzheimer's, transmissible spongiform encephalopathy and the islets of Langerhans of diabetic patients [12]. Further proteins like amyloid β, tau, prions and α-synuclein, implicated in various neurodegenerative conditions have often been found to be glycated suggestive of an association between protein glycation and amyloidosis [5,11,13]. Moreover reports available demonstrate an increased accumulation of AGEs in the Alzheimer's disease (AD) brain thereby implicating AGEs in the pathogenesis of neuro degeneration [8,13].

Glycation as such not only affects the proteins of animal origin but also affect the proteins of plant origin. Until very recently the subject of plant protein glycation has gained much attention and is being studied intensively. Recently, presence of AGEs was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants [14]. Reports available indicate that environmental alterations irrespective of their nature result in the development of oxidative stress and other metabolic adjustments in plant tissues, together resulting in the glycation of plant proteome [15]. Besides acting as marker for ageing, senescence and tag for protein degradation in plants [16], plant derived AGEs if consumed show toxicity via activation of several signaling cascades leading to cell stress thereby contributing to cellular dysfunction and damage to the target organs [17]. Additionally if consumed, AGEs can show toxic and pro-inflammatory behavior leading to the development of inflammatory diseases such as atherosclerosis and type 2 diabetes mellitus [18] thereby proving fatal.

Proteins in general and proteases in particular have been recruited by nature to carry out varied and specialized physiological functions necessary for the sustenance and maintenance of life. Also recruited by nature are protease inhibitors which act as protease master regulators to avoid uncontrolled protease action, hence avoiding a variety of diseases [19]. One such protease inhibitor i.e. cystatin, is essentially the most widely represented class of cysteine protease inhibitors, ubiquitously distributed in plants and animals, therein serving important physiological functions. Deregulation in cystatin function can lead to a number of pathological states ranging from renal failure, rheumatoid arthritis to Alzheimer's disease, cerebral amyloid angiopathy and septic shock [11,19]. Like other proteins, cystatin also has the tendency for structural modification by glycation, eventually leading to the development of aggregated forms thereby creating a space for uncontrolled cysteine protease activity [11,20]. Further reports are available demonstrating the involvement and co-localization of cystatin forms with amyloid beta-protein (Aβ) in parenchymal and vascular amyloid deposits in the brain of AD patients, Down's syndrome, intracranial hemorrhage, cerebral infarction, and of elderly subjects without any neurological disorder [21].

In recent times much attention has been devoted to study the mechanism underlying generation, propagation and deposition of AGEs and aggregated protein forms and it has been observed that three major factors are responsible for the conversion of a functionally active protein form into inactive and aggregated one, which include high hydrophobicity, high tendency to switch from α-helical to β-sheet structure, and low net charge [5,11,22]. Keeping in view these factors, need has arised to identify a novel therapeutic approach so as to minimize the impact and to arrest and rectify the AGEs and aggregate related disorders. One such approach involves the use of naturally obtained chemical chaperones like amino acid, likes of which have received considerable attention as therapeutic agents [23] and have further been reported to show properties of anti-oxidant, anti-hypertensive, anti-atherosclerotic, immunomodulatory and anti-glycation activities [17,24]. Reports are also available demonstrating the potential of free amino acids to act as potent protein stabilizing [17] as well as glycation and AGEs inhibiting agents [[24], [25], [26]]. Moreover using amino acids as therapeutic entities could be of advantage because of their ease and scale-up of synthesis, familiarity to the body and low or negligible associated toxicity.

Keeping in view the deliberations made and for better understanding of the protein sugar interaction, a study was designed which focused on unraveling the behavior of a plant protease inhibitor purified from rai mustard seeds (Rai Seed Cystatin – RSC) in the presence of reducing sugars; glucose and ribose. RSC-glucose and RSC-ribose was used as a model system to mimic the in vivo protein-sugar milieu so as to unravel the conditions leading to glycation and hence aggregation of the proteins. Making use of different assays, we observed for the structural and functional alterations in the model protein, originated due to the protein-sugar interaction. The study further focused on examining the efficacy of amino acids; aspartic acid (Asp; D) and arginine (Arg; R) against the glycation and glycation induced generation, propagation and deposition of AGEs and aggregated protein forms.