Abstract
The non-protein amino acid homocysteine (Hcy), a metabolite of the essential amino acid methionine, is implicated in the pathology of human cardiovascular and neurodegenerative diseases. In addition to its elimination by the remethylation and transsulfuration pathways, Hcy is also metabolized to the thioester Hcy-thiolactone in an error-editing reaction in protein biosynthesis when Hcy is mistakenly selected in place of methionine by methionyl-tRNA synthetase. In humans, the accumulation of Hcy-thiolactone can be detrimental because of its intrinsic ability to modify proteins by forming ^-Hcy-protein adducts, in which a carboxyl group of Hcy is ^-linked to e-amino group of a protein lysine residue. ^-linked Hcy occurs in each protein examined and constitutes a significant pool of Hcy in human blood. N-Hcy proteins are recognized as neo-self antigens and induce an auto-immune response. As a result, IgG and IgM anti-N-Hcy-protein auto-antibodies, are produced in humans. Serum levels of anti-N-Hcy-protein IgG auto-antibodies are positively correlated with plasma total Hcy, but not with plasma cysteine or methionine levels, which is consistent with the etiology of these autoantibodies. In a group of male patients with stroke, the levels of anti-N-Hcy-protein IgG auto-antibodies and total Hcy are significantly higher than in a group of healthy subjects. In a group of male patients with angiographically documented coronary artery disease, seropositivity for anti-N-Hcy-protein IgG auto-antibodies occurs 5-times more frequently than in controls and is an independent predictor of coronary artery disease. These findings show that an auto-immune response against ^-Hcy-proteins is a general feature of atherosclerosis and provide support for a hypothesis that ^-Hcy-protein is a neo-self antigen, which contributes to immune activation, an important modulator of atherogenesis. Plasma Hcy lowering by folic acid administration leads to significant decreases in anti-^-Hcy-protein IgG auto-antibody levels in control subjects, but not in coronary artery disease patients. The results of these Hcy-lowering treatments suggest that, while primary Hcy-lowering intervention is beneficial, secondary Hcy-lowering intervention in coronary artery disease patients may be ineffective in reducing the advanced damage caused by Hcy, and may explain at least in part the failure of vitamin therapy to lower cardiovascular events in recent Hcy-lowering trials. Chronic activation of immune responses towards ^-Hcy-protein associated with hyperhomocysteinemia over many years would lead to vascular disease.
Keywords: autoantibodies; atherosclerosis; coronary artery disease; Hyperhomo-cysteinemia; homocysteine thiolactone hypothesis; protein N-homocysteinylation; stroke
Introduction
Cardiovascular disease is a major cause of morbidity and mortality in industrial nations. Despite advances in our understanding of cardiovascular disease, traditional risk factors such as hyperlipidemia, hypertension, smoking, and diabetes do not accurately predict cardiovascular events and over half of all coronary events occur in persons without overt hyperlipidemia [1; 2; 3; 4]. Thus, a search continues for new markers and strategies to guide the development of novel antiatherosclerotic therapies beyond low-density lipoprotein (LDL) cholesterol reduction. Although atherosclerosis has been viewed as a lipid storage disease [5], the growing body of evidence suggests that inflammation participates in all stages of atherosclerosis from the initial lesion to the end-stage thrombotic complications [2; 6; 7; 8; 9; 10; 11]. The principal culprits responsible for the initiation of inflammation appear to be proteins modified by products of lipid peroxidation or by glucose, particularly oxidized or glycated LDL. Modified LDL induces both innate and adaptive immune responses, and autoantibodies against modified LDL are present in atherosclerotic plaques and in circulation [12; 13].
Severe hyperhomocysteinemia secondary to mutations in the CBS, MTHFR, or MS gene causes pathologies in multiple organs, including the cardiovascular system and the brain, and leads to premature death due to vascular complications [16; 17; 18]. McCully observed advanced arterial lesions in children with inborn errors in Hcy metabolism and proposed that Hcy causes vascular disease [19]. Although severe hyperhomocysteinemia is rare, mild hyperhomocysteinemia is quite prevalent in the general population and is associated with an increased risk of vascular [20] and neurological complications [21; 22], and predicts mortality in heart disease patients [23]. The strongest evidence that Hcy plays a causal role in atherothrombosis comes from the studies of severe genetic hyperhomocysteinemia in humans and the finding that Hcy-lowering by vitamin-B supplementation greatly improves vascularoutcomes in CBS deficient patients [16; 17; 18]. For example, untreated CBS-deficient patients suffer 1 vascular event per 25 patient-years [16] while vitamin-B-treated CBS-deficient patients suffer only 1 vasular event per 263 patient-years (relative risk 0.091, p<0.001) [18]. Hcy-lowering therapy started early in life also prevents brain disease from severe MTHFR deficiency [24]. Furthermore, studies of genetic and nutritional hyperhomocysteinemia in animal models also provide a strong support for a causative role of Hcy [14; 25; 26]. In humans, lowering plasma Hcy by vitamin-B supplementation improves cognitive function in the general population [27] and leads to a 21-24% reduction of vascular outcomes in high risk stroke patients [28; 29], but not in myocardial infarction (MI) patients [29; 30]. Hcy-lowering trials are currently ongoing, and the results of these trials are required before making recommendations on the use of vitamins for prevention of vascular disease [31].
Atherosclerosis, a disease of the vascular wall, is initiated by endothelial damage. Endothelial dysfunction, immune activation, and thrombosis, characteristic features of vascular disease [8], are all observed in hyperhomocysteinemia in humans [16] and experimental animals [26]. The degree of impairment in endothelial function during hyperhomocysteinemia is similar to that observed with hypercholesterolemia. Multiple mechanisms, such as protein homocysteinylation, unfolded protein response, decreased bioavailability of nitric oxide, oxidative stress, altered cellular methylation and epigenetic regulation, and the induction of innate and adaptive immune responses appear to contribute to Hcy pathobiology in cardiovascular disease [14; 25; 26; 32; 33; 34; 35; 36; 37]. The Hcy-thiolactone hypothesis [36][37a, 37b] states that metabolic conversion of Hcy to Hcy-thiolactone, catalyzed by methionyl-tRNA synthetase (MetRS) (Eq. 1), followed by protein N-homocysteinylation by Hcy-thiolactone (Eq. 2), causes a variety of pathophysiological consequences including protein [38] and cell damage [39; 40; 41], enhanced thrombosis [42; 43], and induction of auto-immune responses [14; 35; 36]. In this topic, I will discus the mechanism of formation of N-Hcy-proteins, new neo-self antigens derived from Hcy, summarize evidence for their presence in the human body, and describe their antigenic properties and emerging evidence for an important role of anti-N-Hcy-protein autoantibodies in vascular disease.
Overview of Homocysteine Metabolism
Homocysteine (Hcy) is a sulfur-containing amino acid that is found as an intermediary metabolite in all living organisms. In mammals Hcy is formed from dietary methionine (Met) as a result of cellular methylation reactions [16]. In this pathway, dietary Met is taken up by cells and then activated by ATP to yield S-adenosylmethionine (AdoMet), a universal methyl donor (Figure 1). As a result of the transfer of its methyl group to an acceptor, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The reversible enzymatic hydrolysis of AdoHcy is the only known source of Hcy in the human body. Levels of Hcy are regulated by remethylation to Met, catalyzed by the enzyme Met synthase (MS), and transsulfuration to cysteine, the first step of which is catalyzed by the enzyme cystathionine P-synthase (CBS). The remethylation requires vitamin Bi2 and 5,10-methyl-tetrahydrofolate (CH3-THF),generated by 5,10-methylene-THF reductase (MTHFR). The transsulfuration requires vitamin B6.
Figure 1. Neo-self antigen, protein N-linked Hcy (N-Hcy-protein), is a byproduct of Hcy metabolism in humans.
A fraction of Hcy is also metabolized by MetRS to a thioester, Hcy-thiolactone (Figure 1), in an error-editing reaction in protein biosynthesis when Hcy is mistakenly selected in place of Met [44; 45; 46; 47; 48]. The flow through the Hcy-thiolactone pathway is increased by a high-Met diet [49], inadequate supply of CH3-THF [49; 50; 51], or impairment of re-methylation or trans-sulfuration reactions by genetic alterations of enzymes, such as CBS [49; 50; 52; 53], MS [52; 53], and MTHFR [49]. Because of its exceptionally low pKa value (Table 1), Hcy-thiolactone is neutral at physiological pH and thus can diffuse out of the cell (Figure 1) and accumulate in the extracellular fluids [49; 50; 51; 54; 55]. Hcy-thiolactone is hydrolyzed to Hcy by intracellular [56] and extracellular Hcy-thiolactonases [57; 58; 59; 60], previously known as bleomycin hydrolase (BLH) and paraoxonase 1 (PON1), respectively. Because of the oxidative environment in the blood, extracellular Hcy forms disulfides, mostly with serum proteins [38; 57] such as albumin [34] and globulins [61], and only ~1% of plasma total Hcy exists in a free reduced form in humans [62]. Furthermore, as discussed in a greater detail in the following sections of this topic, Hcy-thiolactone reacts spontaneously with proteins, forming #-Hcy-proteins (Figure 1), which are recognized as neo-self antigens by the immune system.
The Mechanism of Protein N-Homocysteinylation
Fundamental physical-chemical properties of Hcy (Table 1) underlie its ability to undergo metabolic conversion to Hcy-thiolactone. During protein biosynthesis Hcy is often mistakenly selected in place of Met by MetRS and metabolized to Hcy-thiolactone in an error-editing reaction according to Equation (1) [44; 55; 66].
Table 1. Physical-chemical properties of L-Hcy-thiolactone and L-Hcy [46]
|
Property |
 |
 |
|
Chemical character |
Aminoacyl-thioester |
Mercaptoamino acid |
|
UV spectrum |
 |
 |
|
Stability at 37°C, to.5 phosphate-saline human serum |
 |
 |
 |
 |
 |
|
Chemical reactivity |
Acylates amino groups of protein lysine residues c Reacts with aldehydes to afford tetrahydrothiazines a Resistant to oxidation Base-hydrolyzed to Hcy |
Condenses to Hcy-thiolactone Reacts with aldehydes to afford tetrahydrothiazines a Oxidized to disulfides Reacts with nitric oxide to afford S-nitroso-Hcy d |
It should be noted that the high energy of the anhydrate bond of ATP is conserved in the thioester bond of Hcy-thiolactone, which is responsible for the chemical reactivity of Hcy-thiolactone (Table 1). Thus, Hcy-thiolactone spontaneously modifies proteins by forming N-Hcy-protein adducts, in which Hcy is N-linked to the s-amino group of protein lysine residues as shown in Equation (2) [38; 46; 50].
These two reactions, studied extensively in vitro in model systems and ex vivo in cultured cells [38; 46; 47; 48; 50], are relevant in vivo, as demonstrated in humans and mice [49; 53; 54; 61; 67]. Protein N-homocysteinylation is a novel example of protein modification reaction that expands the biological repertoire of known protein modifications by other metabolites, such as glucose, products of lipid peroxidation, or certain drugs, such as penicillin or aspirin [38]. These protein modification reactions have two common aspects: a) each involves protein lysine residues as sites of modifications, and b) are linked to human pathological conditions, including diabetes, vascular disease, Alzheimer’s disease, or drug allergy or intolerance [38]. The primary focus of this topic is on the mechanism of generation of protein N-linked Hcy epitopes in humans and their immunogenic properties.
The Molecular Mechanism of Hcy-Thiolactone Synthesis
In living organisms, the formation of Hcy-thiolactone is a consequence of error-editing reactions of aminoacyl-tRNA synthetases [44; 45; 47; 48; 68; 69; 70; 71]. Because of its similarity to protein amino acids Met, leucine, and isoleucine, the non-protein amino acid Hcy poses a selectivity problem in protein biosynthesis. Indeed, Hcy enters the first step of protein biosynthesis and forms Hcy-AMP with methionyl-, leucyl-, and isoleucyl-tRNA synthetases [72] [72a]. However, misactivated Hcy is not transferred to tRNA [65], and thus cannot enter the genetic code. Instead, Hcy-AMP is destroyed by editing activities of these aminoacyl-tRNA synthetases [44; 66], as shown in Equation (1). Hcy editing is universal, occurs in all organisms investigated, including bacteria [53; 72; 73; 74] [72a, yeast [52; 53; 75], plants [76], mice [36; 49], and humans [36; 49; 53; 54; 67], and prevents direct access of Hcy to the genetic code [44; 45; 46; 47; 48].
Although studied in several systems [77], molecular mechanism of Hcy editing is best understood for E. coli MetRS [78; 79; 80]. The Hcy editing reaction occurs in the synthetic/editing active site [78]], whose major function is to carry out the synthesis of Met-tRNA [80]. Whether an amino acid completes the synthetic or editing pathway is determined by the partitioning of its side chain between the specificity and thiol-binding sub-sites of the synthetic/editing active site [79]. A sub-site that binds carboxyl and a-amino groups of cognate or non-cognate substrates does not appear to contribute to specificity [78].
Methionine completes the synthetic pathway because its side chain is firmly bound by the hydrophobic and hydrogen bonding interactions with the specificity sub-site (Figure 2). Crystal structure of MetRS-Met complex [80] reveals that hydrophobic interactions involve side chains of Tyr15, Trp253, Pro257, and Tyr260; Trp305 closes the bottom of the hydrophobic pocket, but is not in the contact with the methyl group of the substrate methionine. The sulfur of the substrate methionine makes two hydrogen bonds: one with the hydroxyl of Tyr260 and the other with the backbone amide of Leu13.
Figure 2. The aminoacylation of tRNA with Met catalyzed by MetRS.
The non-cognate substrate Hcy, missing the methyl group of methionine, cannot interact with the specificity sub-site as effectively as cognate methionine does. This allows the side chain of Hcy to move to the thiol-binding sub-site, which promotes the synthesis of the thioester bond during editing (Figure 3). Mutations of Tyr15 and Trp305 affect Hcy/Met discrimination by the enzyme [78]. Asp52, which forms a hydrogen bond with the a-amino group of the substrate methionine, deduced from the crystal structure of MetRS-Met complex [80], is involved in the catalysis of both synthetic and editing reactions, but does not contribute to substrate specificity of the enzyme. The substitution Asp52Ala inactivates the synthetic and editing functions of MetRS [65; 78; 79].
Figure 3. The formation of Hcy-thiolactone during Hcy editing catalyzed by MetRS.
Futrhermore, the thiol-binding sub-site also supports the ability of MetRS to edit in trans, i.e., to catalyze thioester bond formation between a thiol and the cognate methionine (Figure 4). With CoA-SH or cysteine as a thiol substrate, MetRS catalyzes the formation of Met-S-CoA thioesters [81] and Met-Cys di-peptides [79], respectively. The formation of Met-Cys di-peptide proceeds via a Met-S-Cys thioester intermediate, which spontaneously rearranges to the Met-Cys di-peptide. Remarkably, the formation of Met-Cys di-peptide as a result of editing in trans, is as fast as the formation of Hcy-thiolactone during Hcy editing.
