Comparison of modification sites formed on human serum albumin at various stages of glycation

Abstract

Background

Many of the complications encountered during diabetes can be linked to the non-enzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation.

Methods

Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed.

Results

It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation.

Conclusions

The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied.

Introduction

The glycation of various proteins in the body is accelerated during diabetes [1]. This process, known as the Maillard reaction, involves the addition of reducing sugars and/or their degradation products to free primary and secondary amine groups on proteins [2], [3]. The first step of this reaction involves the nucleophilic addition of a reducing sugar to a primary amine group (e.g., as found on a lysine or at the N-terminus of a protein). In this stage a reversible Schiff base is formed which can undergo a slow irreversible rearrangement to form more stable Amadori products that accumulate over time. The total amount of accumulation of these products is known to be dependent on the type of sugar that is causing the glycation [4], the incubation time and sugar concentration [5], and the type protein that is being modified [6], [7], [8].

The Maillard reaction is fairly well-characterized for proteins with long lifetimes in the body (e.g., collagen [9], [10], [11], [12] and lens crystalline [13], [14], [15]) and for some proteins with shorter half-lives (e.g., hemoglobin [16], [17], [18]). However, until recently there has been little information on this process for human serum albumin (HSA) [8], which is the major carrier agent for many drugs and small endogenous solutes in blood [2], [19]. HSA has 2 major binding regions for drugs. These sites are known as Sudlow sites I and II and are located in subdomains IIA (within residues 150–292) and IIIA (within residues 384–489) of HSA, respectively [2], [20]. Previous studies have found that glycation can alter the interactions of HSA with certain solutes [21], [22], [23] and may, under certain conditions, affect the overall structure of HSA [24], [25]. It is expected from these observations that characterization of the modifications that occur on HSA would be valuable in helping determine why such changes in binding can occur during glycation.

Previous studies with glycated HSA prepared under specific conditions have indicated that the most significant sites for early glycation are the N-terminus and lysines 199, 281, 439, and 525 [2], [26], [27]. Similar work looking at the modification of HSA with methylglyoxal has identified certain regions that are prone to advanced glycation end product (AGE) modification [28], including arginine 410 as a major region of modification and arginines 114, 186, 218, and 428 as regions with smaller amounts of modification. These findings are consistent with recent qualitative [29] and quantitative studies [30] that have been conducted by our group using minimally-glycated HSA. However, no past studies have looked at how these glycation patterns are altered when comparing samples of HSA with different levels of glycation.

In this study, HSA with various levels of glycation will be prepared in vitro under solution conditions and reaction times that are similar to those found for HSA in serum during diabetes [31], [32]. The modifications that occur on the resulting samples of glycated HSA will be examined by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [33]. A comparison will then be made between the results obtained for the HSA made with different extents of glycation and with previous results reported for minimally glycated HSA [29], [30]. The modifications that are found will also be compared to the known locations of the major drug binding sites on HSA. This data should provide a more complete picture of how the structure of HSA and its function as a carrier agent for drugs are affected by various stages of glycation during diabetes.