Albumin Thiolate Anion Is an Intermediate in the Formation of Albumin-S–S-Homocysteine

Sengupta, Shantanu; Chen, Hong; Togawa, Tadayasu; DiBello, Patricia M.; Majors, Alana K.; Büdy, Beatrix; Ketterer, Michael E.; Jacobsen, Donald W.

doi:10.1074/jbc.m104324200

Cited by 158 publications

(177 citation statements)

References 39 publications

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“…Consistent with this suggestion are the observations that S-Hcy-albumin and NHcy-albumin account for most (Ͼ90%) of S-linked and N-linked Hcy, respectively, present in human plasma protein and for Ͼ90% of total plasma Hcy (22,26,28). In addition to S-Hcyprotein (26,31,32) and N-Hcy-protein (12, 13, 26 -28), Hcy exists in circulation as free reduced Hcy (31), Hcy-thiolactone (24,25), and disulfide forms with low molecular weight thiols (31). Of these, Hcy-thiolactone (10, 17, 21-23, 26, 28, 29) and free reduced Hcy (37), which comprise 1.4 (24,25) and 2% (31) of plasma total Hcy, respectively, have been suggested to be the deleterious forms of Hcy.…”

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confidence: 72%

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Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation

Głowacki

Jakubowski

2004

Journal of Biological Chemistry

125

119

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Since the 1960s, it has been known that elevated levels of homocysteine (Hcy), 1 resulting from mutations in genes encoding Hcy-metabolizing enzymes, are harmful to humans (1, 2). During the past decade it has been established that even a mild increase in Hcy level is a risk factor for cardiovascular disease and stroke in humans (3, 4) and predicts mortality independently of traditional risk factors in patients with coronary artery disease (5). Plasma Hcy is also a risk factor for neurodegenerative disorders, such as dementia and Alzheimer's disease (6). In tissue cultures, Hcy does not support growth and induces apoptotic death in human endothelial cells (7). Animal and cell culture studies have shown that Hcy induces cell death and potentiates amyloid ␤-peptide toxicity in neurons (8).In humans, Hcy, formed from dietary methionine as a byproduct of cellular methylation reactions, is detoxified by folic acid-and vitamin B 12 -dependent re-methylation to methionine (2) or vitamin B 6 -dependent trans-sulfuration to cysteine (1). Whereas Hcy is formed in all human organs, most of its detoxification occurs in the liver and kidneys. Detoxification of Hcy in human vascular tissues and skin occurs only by re-methylation; enzymes of the trans-sulfuration pathway are not expressed in these tissues (9).Hcy is perhaps the most reactive amino acid in biological systems (1, 2). In addition to re-methylation to methionine or trans-sulfuration to cysteine (via cystathionine), Hcy is also metabolically converted to Hcy-thiolactone, S-nitroso-Hcy, AdoHcy, Hcy-containing disulfides, or homocysteic acid, each of which has been implicated in the pathology of hyperhomocystinemia (1, 10).Because of its similarity to the protein amino acid methionine, Hcy can exert its biological effects by interfering with protein biosynthesis (10 -18). For example, Hcy is metabolized to Hcy-thiolactone by methionyl-tRNA synthetase in a two-step reaction (19,20). In the first step (Reaction 1), methionyl-tRNA synthetase catalyzes activation of Hcy with ATP, which yields methionyl-tRNA synthetase (MetRS)-bound homocysteinyl adenylate.The second step (Reaction 2), in which the side chain thiolate of Hcy reacts with the activated carboxyl group of Hcy, yields Hcy-thiolactone.The energy of the anhydride bond in HcyϳAMP is conserved in the thioester bond in Hcy-thiolactone. Because of this, Hcythiolactone reacts with proteins, forming Hcy-containing adducts, in which the carboxyl group of Hcy is linked by an amide bond with ⑀-amino group of a protein lysine residue (21-23).Originally discovered in cultured cells (21-23), protein Nhomocysteinylation is now known to occur in humans (10,(12)(13)(14)(15)(16)(17)(18). Both Hcy-thiolactone (13,24,25) and N-linked proteinHcy (12,16,26,27) have been demonstrated in human blood. About 70% of circulating Hcy is N-linked to blood proteins, mostly hemoglobin and albumin (26). A protective mechanism against protein N-homocysteinylation appears to exist in humans (28,29).Two major forms of albumin exist in circulatio...

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confidence: 72%

“…3), the exchange with cysteine was Ͻ20% complete (not shown). Thiol-disulfide exchange reactions between albumin-Cys 34 -SH and cystine, homocystine, or Cys-S-S-Hcy disulfide are known to be slow, being about 20% or less complete in 4 h (32,36).…”

mentioning

confidence: 99%

Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation

Głowacki

Jakubowski

2004

Journal of Biological Chemistry

125

119

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“…[23][24][25] Furthermore, we recently demonstrated that when homocysteine and cysteine are simultaneously incubated with human albumin, Ϸ4-fold more homocysteine than cysteine becomes protein bound. 11 To identify the region(s) of fibronectin to which homocysteine binds, a limited trypsin digestion of fibronectin that had been labeled with radioactive homocysteine was carried out. Several of the resulting protein fragments that contained the radioactive homocysteine were sequenced by using LC/MS.…”

Section: Discussionmentioning

confidence: 99%

Homocysteine Binds to Human Plasma Fibronectin and Inhibits Its Interaction With Fibrin

Majors

Sengupta

Willard

et al. 2002

ATVB

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Objective-More than 70% of circulating homocysteine is disulfide-bonded to protein, but little is known about the specific proteins that bind homocysteine and their function as a consequence of homocysteine binding. Methods and Results-When human plasma was incubated with [35 S]L-homocysteine, most of the homocysteine bound to albumin. However, additional homocysteine-binding proteins were detected, and 1 of them comigrated with fibronectin. Treatment with 2-mercaptoethanol removed the bound homocysteine, demonstrating the involvement of disulfide bonding. In contrast, [35 S]L-cysteine did not bind to fibronectin. Purified fibronectin bound Ϸ5 homocysteine molecules per fibronectin dimer. SDS-PAGE of a limited trypsin digestion of homocysteinylated fibronectin showed that several tryptic fragments contained [35 S]homocysteine. Sequence analysis demonstrated that the fragments containing bound homocysteine had localized mainly to the C-terminal region, within and adjacent to the fibrin-binding domain. Homocysteinylation of fibronectin significantly inhibited its capacity to bind fibrin by 62% (PϽ0.005). In contrast, neither the binding of fibronectin to gelatin nor its capacity to serve as an attachment factor for aortic smooth muscle cells was affected. Key Words: homocysteine Ⅲ fibronectin Ⅲ fibrin Ⅲ atherosclerosis Ⅲ thrombosis H omocysteine is a sulfhydryl-containing amino acid formed during the metabolism of methionine. In healthy well-nourished individuals, homocysteine metabolism is efficient and regulated, and the concentration of plasma total homocysteine is usually Յ12 mol/L. 1 Inborn errors of homocysteine metabolism, drugs that interfere with homocysteine metabolism, deficiencies of folic acid, vitamin B 6 or vitamin B 12 , and certain disease states, such as chronic renal failure, increase plasma total homocysteine levels. 2 The homocystinurias are a heterogeneous group of autosomal recessive diseases caused by inborn errors of homocysteine metabolism. [3][4][5] Patients with untreated homocystinuria have severe hyperhomocysteinemia (50 to 500 mol/L) and lifethreatening premature cardiovascular disease. Thrombosis, with or without embolism, is the major cause of death. Postmortem studies show multiple thrombotic occlusions and widespread premature atherosclerosis with intimal thickening. However, even mild hyperhomocysteinemia (15 to 25 mol/L), regardless of the underlying cause, is a strong independent risk factor for occlusive vascular disease, as shown by numerous case-control and prospective studies involving thousands of subjects. 1,2,6 The mechanisms of homocysteine-induced atherosclerosis and thrombosis have not been fully elucidated. Conclusions-TheseIn normal individuals, Ͼ70% of circulating homocysteine is disulfide-bonded to plasma proteins. 7,8 The primary carrier of disulfide-bound homocysteine is albumin, 9 -12 but other proteins with accessible cysteine residues are also potential carriers. The binding of homocysteine to cysteine residues may disrupt normal protein structure and impair...

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“…26) The remaining sulfhydryl groups are freely accessible (mercaptalbumin) and represent the largest fraction of free thiols in blood. Mercaptalbumin and probably also part of albumin forming mixed disulfides are able to bind nitric oxide and metal ions such as Ag ϩ , Hg 2ϩ , and Au ϩ as well as homocysteine 27) and several drugs. Among the latter are bucillamine derivatives, 28) aurothiomalate, 29) auranofin, 30) D-penicillamine, 31,32) captopril, 32,33) ethacrynate, 34) and cisplatin (which also binds to methionine residues).…”

Section: Ligand Bindingmentioning

confidence: 99%

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

Kragh‐Hansen

Chuang

Otagiri

2002

Biological & Pharmaceutical Bulletin

933

776

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Recent work with approaches like recombinant mutants and X-ray crystallography has given much new information about the ligand-binding properties of human serum albumin (HSA). The information increases the understanding of this unique transport and depot protein and could give a structural basis for the possible construction of therapeutic agents with altered HSA-binding properties. A tabulation of high-affinity binding sites for both endogenous and exogenous compounds has been made; it could be useful for the above-mentioned purpose, but it could also be of value when trying to predict potential drug interactions at the protein-binding level. Drug displacement is not always a complication to therapy; it can be used to increase the biological effect of a drug. However, due to rebinding at other sites, the increase in the free concentration of a displaced ligand can be less than expected. Drugs and drug metabolites can also interact covalently with HSA; thiol-containing drugs often bind to the single free cysteine residue of HSA, and glucuronidated drugs react irreversibly with other residues of the protein. Reversible binding of ligands is often stereospecific, and therefore immobilized HSA can be used to separate drug isomers. Albumin-containing dialysates are useful for extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. HSA has different types of hydrolytic activities, which also can be stereospecific. The esterase-like property seems especially useful in converting prodrugs to active drugs in plasma.

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Albumin Thiolate Anion Is an Intermediate in the Formation of Albumin-S–S-Homocysteine

Abstract: An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds.

Cited by 158 publications

References 39 publications

Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation

Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation

Homocysteine Binds to Human Plasma Fibronectin and Inhibits Its Interaction With Fibrin

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

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