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,1
* Department of Microbiology and Molecular Genetics, New Jersey Medical School, International Center for Public Health, Newark, New Jersey, USA;
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Pozna
, Poland;
Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands; and
Clinic for Special Children, Strasburg, Pennsylvania, USA
1Correspondence: Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, 225 Warren St., Newark, NJ 07101-1709, USA. E-mail: jakubows{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: genetic hyperhomocysteinemia • homocysteine thiolactone • fibrinogen • atherothrombosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Severe hyperhomocysteinemia observed in CBS or MTHFR deficiency is associated with pathologies affecting multiple organs, including brain and cardiovascular system, and leads to premature death due to vascular complications (1
, 2)
. Treatments with vitamin B6 in combination with folate or betaine lower plasma Hcy and improve vascular outcomes in CBS-deficient patients (3)
, which suggests that Hcy plays a causal role in atherothrombosis. For example, untreated CBS-deficient patients suffer 1 vascular event per 25 patient-years (1)
, whereas treated CBS-deficient patients suffer only 1 vascular event per 263 patient-years (relative risk 0.091, P<0.001) (3)
. Hcy-lowering betaine therapy started early in life prevents brain disease from severe MTHFR deficiency (2
, 4)
.
How CBS or MTHFR deficiency leads to specific clinical manifestations is not entirely clear. One hypothesis suggests that metabolic conversion to Hcy-thiolactone contributes to the pathophysiology of Hcy excess (5
, 6)
and is involved in atherothrombotic disease in humans (7
, 8)
. Consistent with this hypothesis, Hcy-thiolactone is elevated in CBS- or MTHFR-deficient patients and in mice fed a high-methionine diet (9)
and is more toxic to cultured human cells than Hcy itself (reviewed in refs. 10
, 11
).
Hcy-thiolactone could be detrimental because of its ability to modify proteins by forming adducts in which Hcy is N-linked to the 
-amino group of protein lysine residues (5
, 12)
. This modification affects the protein structure, impairs or alters its physiological function, and is directly cytotoxic (reviewed in refs. 7
, 8
, 10
, 11
). Major pathophysiological consequences of protein N-homocysteinylation include increased susceptibility to thrombogenesis (caused by N-Hcy-fibrinogen) (13
, 14)
and an autoimmune response elicited by N-Hcy-proteins, associated with stroke and coronary artery disease (8
, 15
, 16)
.
Whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans was not known. In the present work, using a new highly sensitive assay (17)
, we found that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also found that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen, possibly accounting for increased thrombogenesis observed in these patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Of these patients, 12 were 833T>C (Ile278Thr) homozygotes; 1 was a 456C>G (I152M) homozygote; 1 was a 494G>A (C165Y) homozygote; 1 was a 1330G>A (D444N) homozygote; 10 were compound heterozygotes for the 833T>C and 1111G>A (Val371Met) (n=2), 456C>G (I152M) (n=2), 494G>A (C165Y) (n=3), or unidentified (n=3) mutations; 2 were compound heterozygotes for the 373C>T (Arg125Trp) and 1301C>A (Thr434Asn) mutations; 1 was a compound heterozygote for the 539T>C (V180A) and an unidentified mutation; and 1 was a homozygote for two mutations, 1105C>T (R369C) + 1471C>T (R491C). CBS activity in fibroblasts derived from these patients was <2.5% of the control CBS activity from unaffected individuals (18)
. Patients were initially diagnosed at ages 2 to 54 on the basis of clinical manifestations of CBS deficiency (ectopia lentis, marfanoid appearance, osteoporosis), in combination with a quantitative determination of severe hyperhomocysteinemia and hypermethioninemia. Five CBS-deficient patients survived a vascular event before diagnosis. All patients were on Hcy-lowering treatment (vitamin B6) following diagnosis. Blood samples were taken into Vacutainer EDTA tubes for the preparation of plasma as described previously (19
, 20)
.
MTHFR-deficient human subjects
The MTHFR-deficient patients were from the Amish population centered on Somerset County, PA, USA (4
, 9)
. Four patients had hyperhomocysteinemia due to homozygous 1129C>T (Arg377Cys) missense mutation in the MTHFR gene diagnosed at ages 18 yr, 4 yr, 15 months, or 1 wk. The 1129C>T mutation was detected by DNA sequencing of the entire coding region of the MTHFR gene or by the polymerase chain reaction (21)
. Following diagnosis, the patients were on Hcy-lowering therapy (betaine). Six unaffected siblings or parents, heterozygous for the 1129C>T MTHFR mutation, were also studied. Eleven unrelated healthy adult subjects with normal plasma tHcy levels, confirmed by genotyping to normal at the 1129 locus, were used as controls.
Fibrinogen purification
Fibrinogen was purified from plasma by the glycine precipitation method (22)
. Glycine (33 mg) (Sigma-Aldrich, St. Louis, MO, USA) was added to ice-cold plasma (200 µl). The sample, kept on ice, was occasionally mixed until glycine dissolved. The fibrinogen pellet, recovered by microcentrifugation (20 min, 18,000 g, 4°C), was washed 2 times with 50 µl 2.1 M glycine, 20 mM sodium phosphate (pH 7.4), 0.1 M NaCl, 2 mM EDTA; dissolved in 400 µl 20 mM sodium phosphate (pH 7.4), 0.1 M NaCl, 2 mM EDTA, 4 mM dithiothreitol (DTT); quantified by A280 and A320 measurements; and concentrated to 25 µl by ultrafiltration. The fibrinogen preparations were >95% pure as determined by SDS-PAGE.
Determination of protein N-linked Hcy
The assay is based on a previous procedure (23)
with the following modifications (17)
. Human plasma (10–20 µl) or purified fibrinogen (25 µl) was diluted with phosphate-buffered saline supplemented with 5 mM DTT (200 µl). After 5 min at room temperature, free Hcy was removed by microcentrifugation at 4°C (30 min, 10,000 g) using a Millipore 10-kDa cutoff ultrafiltration device (Millipore, Billerica, MA, USA). Plasma proteins were diluted and washed 2 more times with 200 µl 5 mM DTT in phosphate-buffered saline. The DTT-washed plasma protein sample was transferred to a 1 ml Wheaton Gold Band glass ampoule (Wheaton Science Products, Millville, NJ, USA), diluted to 40 µl with 0.2 M DTT (20 µl) and water as needed. Next, 40 µl 12 N HCl was added, the sample was frozen at –80°C, the ampoule was sealed under vacuum, and the sample was hydrolyzed at 120°C for 1 h. This procedure quantitatively converts protein N-linked Hcy to Hcy-thiolactone.
The hydrolysates were lyophilized, dissolved in 10 µl water containing 20,000 cpm D,L-[35S]Hcy-thiolactone (40,000 Ci/mol) (24)
as a tracer, and 3 µl aliquots were subjected to two-dimensional thin-layer chromatography on 5 x 4 cm cellulose plates (Analtech, Newark, DE, USA). Hcy-thiolactone, localized by autoradiography using Kodak BioMax MR film (Eastman Kodak, Rochester, NY, USA; 3 h exposure at 4°C), was eluted with 60 µl 2 mM HCl, and quantified by HPLC. Similarly processed N-Hcy-albumin (0.5–16 µM) was used as a standard.
HPLC analyses were performed using PolySulfoethyl Aspartamide cation exchange column (2.1x35 mm, 5 µm, 300 Å; PolyLC, Inc., Columbia, MD, USA) and system Gold Noveau HPLC instrumentation (Beckman-Coulter, Fullerton, CA, USA) (9
, 17
, 19
, 20)
. Elution was isocratic, and 10 mM sodium phosphate (pH 6.6) and 30 mM NaCl was used as solvent (5 min run time). Detection was by fluorescence (excitation at 370 nm, emission at 480 nm) after a postcolumn derivatization with o-phthaldialdehyde.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Significantly more plasma N-Hcy-protein was present in heterozygous than in wild-type individuals (1.06±0.22 vs. 0.49±0.08 µM, P=0.003). Because the MTHFR-deficient patients were on Hcy-lowering therapy (see Materials and Methods), these N-Hcy-protein concentrations represent minimal values. In one patient from whom plasma sample was obtained prior to Hcy-lowering therapy, the therapy resulted in a lowering of plasma N-Hcy-protein from 15.40 to 9.52 µM [Hcy-thiolactone and total Hcy (tHcy) were lowered from 47.3 to 16.6 nM and from 208.0 to 66.2 µM, respectively, after the therapy; ref 9
].
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Plasma Hcy-thiolactone concentrations in the homozygous MTHFR-deficient patients were elevated (11.8±8.8 nM) relative to heterozygous subjects (0.5±0.29 nM) and wild-type individuals (0.2±0.14 µM) (Table 1)
. A significant linear relation was found between plasma N-Hcy-protein and Hcy-thiolactone (R2=0.94, P<0.0001) (not shown) in the MTHFR-deficient patients.
Plasma tHcy concentrations in the homozygous MTHFR-deficient patients were elevated (50.1±15.1 µM) in comparison with heterozygous (7.8±2.8 µM) and wild-type individuals (6.7±1.9 µM) (Table 1)
. A significant linear relation was found between plasma N-Hcy-protein and tHcy (R2=0.86, P<0.0001) (Fig. 1
).
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N-Hcy-protein levels in CBS-deficient patients
Plasma N-Hcy-protein in the CBS-deficient patients was elevated (3.02±2.27 µM) (Table 1)
. Because the CBS-deficient patients, like the MTHFR-deficient patients, were on Hcy-lowering therapy (see Materials and Methods), these N-Hcy-protein concentrations represent minimal values. Indeed, in one noncompliant patient N-Hcy-protein concentration was 12.1 µM (Table 1)
. Plasma Hcy-thiolactone and tHcy in the CBS-deficient patients were also elevated, 14.4 ± 30.4 nM and 48.5 ± 57.5 µM, respectively (Table 1)
. A significant relation was found between plasma N-Hcy-protein and tHcy in a group of CBS-deficient patients. On average, N-Hcy-protein represented 3% and 7% of plasma tHcy, in CBS and MTHFR-deficient patients, respectively (Fig. 1)
.
N-Hcy-fibrinogen levels in CBS-deficient patients
Plasma N-Hcy-fibrinogen in the CBS-deficient patients was significantly elevated, compared to unaffected individuals (72.5±58.1 vs. 35.8±14.3 nM, P=0.01) (Table 2
). Total fibrinogen concentrations in CBS-deficient patients and unaffected controls were similar (6.95±1.65 vs. 6.56±1.98 µM, P=0.32). Relative N-Hcy-fibrinogen levels (N-Hcy-fibrinogen/total fibrinogen) were also significantly elevated in CBS-deficient patients, compared with controls (1.00±0.64 vs. 0.61±0.21%, P=0.01). Because these CBS-deficient patients were on Hcy-lowering therapy (see Materials and Methods), their N-Hcy-fibrinogen concentrations represent minimal values. Indeed, in one noncompliant patient N-Hcy-fibrinogen concentration was 314.8 nM, or 3.78% relative to total fibrinogen (Table 2)
.
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Significant positive relations were found between plasma N-Hcy-fibrinogen and total Hcy (R2=0.72, P<0.001) (Fig. 2
, middle panel), or N-Hcy-protein (R2=0.67, P<0.001) (Fig. 2
, right panel), and total fibrinogen (R2=0.23, P=0.005). The relation between N-Hcy-fibrinogen and tHcy remained significant after normalization for the variations in plasma total fibrinogen levels (R2=0.73, P<0.001). Using the values for plasma Hcy-thiolactone measured for CBS-deficient patients in our previous work (9)
, we have also found a positive correlation between plasma N-Hcy-fibrinogen and Hcy-thiolactone (R2=0.37, P<0.01) (Fig. 2
, left panel). However, the correlation was relatively week, most likely reflecting a lower number of samples (n=14) available for Hcy-thiolactone assays (9)
, compared with the number of samples (n=29) used for N-Hcy-fibrinogen assays.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Accumulating evidence strongly suggests that Hcy-thiolactone is involved in the pathology of hyperhomocysteinemia. For example, Hcy-thiolactone is greatly elevated in hyperhomocysteinemic CBS- or MTHFR-deficient patients (9)
. Hcy-thiolactone is also elevated in mice fed a proatherogenic high-methionine diet (9)
. Furthermore, proteins modified by Hcy-thiolactone (N-Hcy-protein) accumulate in atheromas, as shown by immunohistochemical staining for N-Hcy-protein within atherosclerotic lesions in sections prepared from aortas of ApoE–/– mice fed a normal chow diet. The N-Hcy-protein immunostaining increases in aortic lesions from ApoE–/– mice fed a high methionine diet (25)
. In other animal models, treatments with Hcy-thiolactone cause pathophysiological changes reminiscent of those observed in human genetic hyperhomocysteinemia. For example, infusions with Hcy-thiolactone, or an Hcy-thiolactone-supplemented diet produce atherosclerotic changes in baboons (26)
or rats (27)
. Furthermore, treatments with Hcy-thiolactone cause developmental abnormalities in eyes of chick embryos, including optic lens dislocation (28)
, a diagnostic feature prevalent in CBS-deficient human patients (1)
.
Our present findings that N-Hcy protein levels are significantly elevated in CBS- or MTHFR-deficient patients provide support to a hypothesis that protein N-homocysteinylation by Hcy-thiolactone contributes to the pathophysiology of hyperhomocysteinemia (5
, 7
, 10
, 11
, 29)
. N-Hcy-proteins are detrimental to the human body and contribute to two important aspects of pathophysiology: immune activation and thrombogenesis. For example, the modification by Hcy-thiolactone renders proteins highly immunogenic in rabbits, whereas in humans endogenous N-Hcy-proteins induce anti-N-Hcy-protein autoantibodies. Furthermore, the levels of anti-N-Hcy-protein autoantibodies positively correlate with plasma total Hcy levels and are predictors of stroke and coronary artery disease (8)
.
Our findings that N-Hcy-fibrinogen is elevated in CBS-deficient patients can explain increased susceptibility to thrombogenesis observed in these patients. Fibrinogen is known to undergo facile N-homocysteinylation by Hcy-thiolactone in vitro (13
, 23)
, and the present work shows that this process occurs in vivo in humans. Other data show that N-Hcy-fibrinogen has prothrombotic properties. For example, clots formed from N-Hcy-fibrinogen in vitro have a more compact structure, are less permeable, and lyse slower than clots from normal fibrinogen (13)
. Some of the lysine residues susceptible to N-homocysteinylation are close to tPA and plasminogen binding or plasmin cleavage sites, which can explain abnormal characteristics of clots formed from N-Hcy-fibrinogen (13)
. The in vitro prothrombotic effects of N-Hcy-fibrinogen are similar to the prothrombotic effects of fibrinogen mutations in humans, which introduce a cysteine thiol group, e.g., A
Arg16
Cys; Bβ Arg14Cys; C
Arg275Cys, Tyr354Cys (30)
. The prothrombotic effects of elevated plasma tHcy on fibrin clot permeability and lysis in humans are also consistent with a mechanism involving fibrinogen modification by Hcy-thiolactone (14)
.
The present work also demonstrates that insights into N-Hcy-protein metabolism obtained in previous ex vivo studies with cultured human and rodent cells are valid for the human body. For example, cultured human CBS-deficient fibroblasts have been previously shown to synthesize more N-Hcy protein, in addition to Hcy-thiolactone and Hcy, than normal fibroblasts (5)
; the present work shows a similar effect of CBS deficiency on N-Hcy protein levels in the human body (Table 1)
. Similarly, previous work has shown that limiting availability of folic acid greatly enhances N-Hcy protein synthesis in cultured human fibroblasts (5)
and vascular endothelial cells (29)
, whereas the present work shows a similar effect of 5-methyltetrahydofolate deficiency (caused by the MTHFR mutation) on N-Hcy protein synthesis in human subjects (Table 1)
. Furthermore, previous in vitro and ex vivo tissue culture studies have also demonstrated the substrate product relations between Hcy, Hcy-thiolactone, and N-Hcy-protein (reviewed in refs. 6
, 7
, 31
). Linear relations between plasma N-Hcy-protein and tHcy (Fig. 1)
and N-Hcy-fibrinogen and tHcy (Fig. 2
, middle panel) or Hcy-thiolactone (Fig. 2
, left panel) in MTHFR- and CBS-deficient patients described in the present work suggest that the pathway involving methionyl-tRNA synthetase-catalyzed conversion of Hcy to Hcy-thiolactone followed by nonenzymatic protein N-homocysteinylation by Hcy-thiolactone, deduced from the in vitro and ex vivo tissue culture studies (5
, 12
, 29
, 32
, 33)
, is present in the human body.
Our findings suggest that increased formation of N-homocysteinylated proteins is responsible for increased thrombogenesis observed in patients with genetic hyperhomocysteinemia according to the following scheme: Hcy Hcy thiolactone protein modification (N-homocysteinylation) N-Hcy-fibrinogen atherothrombosis. Because plasma tHcy levels (mean 48.5 µM, Table 1
) in our treated CBS-deficient patients are within a range corresponding to mild hyperhomocysteinemia (tHcy levels below 100 µM), and mildly elevated plasma tHcy exerts concentration-dependent prothrombotic effects (14)
, our conclusions are also relevant for the pathogenesis of mild hyperhomocysteinemia in the general population.
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ACKNOWLEDGMENTS |
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Received for publication May 8, 2008. Accepted for publication July 17, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  J. Perla-Kajan and H. Jakubowski Paraoxonase 1 protects against protein N-homocysteinylation in humans FASEB J, March 1, 2010; 24(3): 931 - 936. [Abstract] [Full Text] [PDF]
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 J. Beltowski, G. Wojcicka, and H. Jakubowski Modulation of paraoxonase 1 and protein N-homocysteinylation by leptin and the synthetic liver X receptor agonist T0901317 in the rat J. Endocrinol., February 1, 2010; 204(2): 191 - 198. [Abstract] [Full Text] [PDF]
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H. Jakubowski, J. Perla-Kajan, R. H. Finnell, R. M. Cabrera, H. Wang, S. Gupta, W. D. Kruger, J. P. Kraus, and D. M. Shih Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice FASEB J, June 1, 2009; 23(6): 1721 - 1727. [Abstract] [Full Text] [PDF]
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