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CELLULAR AND MOLECULAR

Protein Cross-Linkage Induced by Formaldehyde Derived from Semicarbazide-Sensitive Amine Oxidase-Mediated Deamination of Methylamine

Neuropsychiatry Research Unit, Departments of Psychiatry (D.G.-H., W.H., M.K., J.S.R., P.H.Y.) and Pharmacology (J.S.R.), University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Received for publication March 18, 2004
Accepted May 5, 2004.

	Abstract

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Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the conversion of methylamine to formaldehyde. This enzyme is located on the surface of the cytoplasmic membrane and in the cytosol of vascular endothelial cells, smooth muscle cells, and adipocytes. Increased SSAO activity has been found in patients with diabetes mellitus, chronic heart failure, and multiple types of cerebral infarcts and is associated with obesity. Increased SSAO-mediated deamination may contribute to protein deposition, the formation of plaques, and inflammation, and thus may be involved in the pathophysiology of chronic vascular and neurological disorders, such as diabetic complications, atherosclerosis, and Alzheimer's disease. In the present study, we demonstrate the induction of cross-linkage of formaldehyde with the lysine moiety of peptides and proteins. Formaldehyde-protein adducts were reduced with sodium cyanoborohydride, hydrolyzed in hydrochloric acid, and the amino acids in the hydrolysates were derivatized with fluorenylmethyl chloroformate and then identified with high-performance liquid chromatography. We further demonstrate that incubation of methylamine in the presence of SSAO-rich tissues, e.g., human brain meninges, results in formaldehyde-protein cross-linkage of particulate bound proteins as well as of soluble proteins. This cross-linkage can be completely blocked by a selective inhibitor of SSAO. Our data support the hypothesis that the SSAO-induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders.

SSAO, a copper-containing enzyme with topaquinone as the cofactor, is distinctly different from mitochondrial monoamine oxidase (MAO) (Holt et al., 1998). SSAO catalyzes the deamination of aliphatic amines, including methylamine and aminoacetone, whereas MAO deaminates catecholamines, serotonin, and long-chain aliphatic amines. Methylamine, a major metabolite of creatine (Mitchell and Zhang, 2001), can also be derived from adrenaline, lecithin, dietary sources, and cigarette smoke (Yu, 1998). SSAO converts methylamine into formaldehyde, hydrogen peroxide, and ammonium, all of which are cytotoxic. Aminoacetone is derived from threonine and glycine, and its deamination leads to the production of cytotoxic methylglyoxal. Formaldehyde and methylglyoxal are extremely reactive chemicals, which can interact with free amino or amide groups to form Schiff's base, subsequently methylene bridges, and produce irreversible covalently cross-linked complexes between proteins, as well as between proteins and single-stranded DNA (Bolt, 1987).

Increased protein aggregation and deposition is associated with the aging process and with numerous pathological conditions, such as the formation of plaques in the pancreas, brain, kidney, and other organs. It would therefore be interesting to know whether formaldehyde and methylglyoxal produced from SSAO-catalyzed deamination of methylamine and aminoacetone, respectively, are involved in the process of protein cross-linkage. In animal studies, chronic methylamine treatment causes an increase in oxidative stress and vascular damage (Deng et al., 1998), while inhibiting SSAO activity reduces atherogenesis (Yu et al., 2002). SSAO-mediated deamination has been shown to be toxic toward cultured endothelial cells, and selective SSAO inhibitors can prevent such toxicity (Yu and Zuo, 1993). Massive exposure to methylamine after an industrial accident was lethal to humans (Yang et al., 1995). It has been hypothesized that the continuous presence of an elevated level of SSAO-mediated aldehydes may be responsible for the chronic inflammation and plaque formation associated with the cerebral vasculature, where SSAO is located (Yu, 2001). The present study demonstrates that formaldehyde derived from the action of SSAO on methylamine cross-links proteins.

	Materials and Methods

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Materials. Benzylamine, methylamine, lysine, N-methyl-lysine, N,N-dimethyl-lysine, N,N,N-trimethyl-lysine, fluorenylmethyl chloroformate (FMOC-Cl), bovine serum albumin (BSA), clorgyline, and (-)-deprenyl were purchased from Sigma-Aldrich (St. Louis, MO); [7-¹⁴C]benzylamine and [¹⁴C]methylamine were obtained from American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer Life and Analytical Sciences (Boston, MA), respectively. H-Lys-Leu-OH.HBr was from Bachem California (Torrance, CA). MDL-72974A [(E)-2-4-fluorophenethyl-3-flouroallylamine HCl] was previously provided by Marion-Merrel-Dow Inc. (Cincinnati, OH). The other chemicals were of analytical grade.

Preparation of SSAO-Rich Tissues. Samples of aorta from Wistar rats (200 g), small intestine from male CD 1 Swiss mice (28-30 g), and human brain meninges were used. All procedures involving animals were approved by the Ethics Committee of the University of Saskatchewan. After dissection, the tissues were rinsed thoroughly with saline (0.9% NaCl), sliced into small pieces, and homogenized with a Polytron homogenizer (PT-10^-35) for 1 min on ice in chilled 0.02 M phosphate buffer (pH 7.4) [1:10% (w/v)]. The crude homogenates were centrifuged at 900g for 10 min, and the low-speed supernatants were further centrifuged at 100,000g for 30 min. The resulting supernatants and membrane fractions were stored at -70°C and used as the enzyme samples in the study. To obtain a higher concentration of SSAO, we concentrated (50x) the high-speed supernatant with the Minicon B15 sample concentrator (Millipore, Mississauga, ON, Canada). Tissue slices (250 x 250 µm) were prepared from saline-washed meninges, aorta, or small intestine with a McIwain tissue chopper (Michle Laboratory Engineer, Surry, UK).

Determination of SSAO Activity. SSAO activity was assessed by a radioenzymatic procedure using ¹⁴C-labeled benzylamine as the substrate (Yu and Zuo, 1993). The SSAO enzyme preparations were incubated with clorgyline (10^-6 M) and (-)-deprenyl (10^-6 M) at 37°C for 20 min to ensure that any MAO activity was completely blocked. Aliquots of the enzyme preparation were then incubated with 50 µl of [¹⁴C]benzylamine (4 x 10^-4 M, 1 µCi/ml) in a final volume of 200 µl in phosphate buffer (0.1 M, pH 7.4) at 37°C for 30 min. The enzyme reaction was terminated by adding 250 µl of 2 M citric acid. The oxidized products were extracted into 1 ml of toluene/ethyl acetate [1:1 (v/v)], of which 600 µl was transferred to a counting vial containing 10 ml of ACS scintillation cocktail (American Radiolabeled Chemicals). Radioactivity was assessed in an LS-7500 liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA).

Cross-Linkage of Formaldehyde with Proteins. To test formaldehyde-induced protein cross-linkage, we used a bipeptide, Lys-Leu-OH, and BSA. The peptide or BSA (2 mg/ml) was incubated in the presence of 10 mM formaldehyde in 0.02 M phosphate buffer (pH 7.4) at 37°C for 4 h, followed by further incubation with 10 mM NaCNBH₃ for 24 h. The reaction mixture regarding BSA-formaldehyde adducts was then dialyzed extensively (Spectr/Por cellulose membrane, molecular weight cuttoff 3.5 kDa; Spectrum Laboratories, Rancho Dominguez, CA) with three changes of 0.2 M phosphate buffer (pH 8.0). The formaldehyde-peptide or formaldehyde-protein adducts were hydrolyzed in 6 N HCl at 110°C for 24 h. The amino acids and altered amino acids were derivatized with FMOC-Cl and assessed by an HPLC-UV procedure (Bank et al., 1996).

Precolumn Derivatization of Amino Acids with FMOC-Cl. The hydrolyzed samples were neutralized with 10 M NaOH. To each 100-µl sample, 50 µl of potassium borate buffer (0.8 M, pH 10) was added, and the solution was vigorously vortexed for 1 min. One hundred microliters of FMOC-Cl solution (10 mM in dehydrated acetonitrile) was added, and the solution was immediately vortexed for 1 min. One milliliter of hexane was then added, and the reaction mixture was vigorously shaken for 45 s, centrifuged, and the hexane phase containing excess reagent was discarded. The extraction was repeated twice. A 10-µl aliquot of acetic acid [20% (v/v)] was added, the tube was mixed, and the sample was applied to HPLC analysis.

Chromatography. The HPLC system consisted of a Shimadzu LC-10AD VP delivery system, a DGU-14A degasser, a SIL-10AD VP auto-injector (Man-Tech, Guelph, ON, Canada), and an integrator (Spectra Physics, San Jose, CA). The separation was performed using a reverse-phase Ultrasphere LP analytical column (4.6 x 250 mm, 5 µm; Beckman Coulter, Toronto, ON, Canada). Elution was either isocratic with 0.05 M sodium-acetate buffer [pH 5.0; 43% (v/v)] in acetonitrile (flow rate 1.4 ml/min), or a ternary gradient system, where solvent A was 20 mM citric acid containing 5 mM tetramethylammonium chloride adjusted to pH 2.85 with 20 mM sodiumacetate, solvent B was composed of 80% (v/v) 20 mM sodium-acetate solution containing 5 mM tetramethylammonium (adjusted to pH 4.5 with concentrated phosphoric acid) and 20% (v/v) methanol, and solvent C was acetonitrile. The gradient program is according to Bank et al. (1996). The separation was performed at room temperature. Spectrophotometric detection was conducted using a -Max model 481 LC spectrophotometer (Millipore) at a wavelength of 265 nm. Data represent the average of at least three analyses.

Formaldehyde-Protein Adducts Derived from SSAO-Mediated Reactions. For the detection of cross-linkage of proteins by formaldehyde derived from the deamination of methylamine, SSAO-rich tissue homogenates [200 µl; 1:10 (w/v) in 0.02 M phosphate buffer, pH 7.4] were incubated with methylamine (10^-3 M) at 37°C for 4 h. Then, 1 ml of 10 mM NaCNBH₃ was added, and the samples were mixed and further incubated at 37°C for 24 h. For controls, tissue homogenates were preincubated (37°C for 30 min) with the specific SSAO inhibitor MDL-72974A (1 x 10^-6 M). In other experiments, [¹⁴C]methylamine was used to trace the deamination and the subsequent cross-linkage of formaldehyde with proteins in vitro and in vivo. The tissue homogenates were ultracentrifuged (100,000g for 30 min). Because SSAO is present in both the soluble and the particulate fractions, both fractions were used in cross-linkage experiments. The soluble fractions (high-speed supernatant) were further purified by gel filtration via a Sephadex G-25 PD-10 column (Amersham Biosciences AB, Uppsala, Sweden). For the in vivo studies, a 100-µl injection of [¹⁴C]methylamine (2 µCi) was administered via the tail vein, and tissues were collected after different time periods.

HPLC-Mass Spectrometry. N-methylated lysine-FMOC derivatives were analyzed by reversed phase HPLC (Alliance model 2695; Waters, Milford, MA) coupled to electrospray mass spectrometry (Quattro UltimaTM; Micromass, Manchester, UK). The HPLC fractions (1.4 ml), which were photometrically detected, were collected. Amino acid derivatives in these fractions were then extracted with 1:1 ethyl acetate/toluene, and the organic fraction was transferred to a new vial and dried under nitrogen. These extracted fractions were then reconstituted with 1 ml of 1:1 acetonitrile/water. Ten-microliter aliquots of these extracted fractions were injected onto an analytical column (100 x 2.1 mm, 4-µm Genesis C18; model FK10960EJ; Jones Chromatography, Hengoed, UK) by an integrated HPLC pump and auto-sampler (Alliance model 2695; Waters) at a flow rate of 0.20 ml/min with an isocratic mobile phase of 75:25 acetonitrile/water. Mass spectrometric analysis was conducted in both positive and negative ion MS1-mode (m/z 50-850). Source temperature was 120°C, and capillary voltage was 2.53 kV with a cone voltage of 45 V. Spectra were matched to those of standards where possible.

	Results

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Methylamine Induced Production of Formaldehyde-Protein Adducts. As can be seen in Fig. 1, the administration of [¹⁴C]methylamine to mice causes a long-lasting radioactive deposition in different tissues that is substantially reduced by the highly selective SSAO inhibitor MDL-72974A. This is consistent with the notion that formaldehyde derived from the deamination of methylamine induces protein cross-linkages.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Residual radioactivity detected in different mouse tissues after administration of [14C]methylamine. The animals were pretreated with saline or SSAO inhibitor MDL-72974A (5 mg/kg i.p.) 2 h before administration of [14C]methylamine (5 µCi, 64 pmol/mouse, via tail vein injection). The residual radioactivity in different tissues was assessed 48 h after administration of the 14C-labeled methylamine. The radioactivity (dpm per gram of fresh tissue) was measured in a Beckman Coulter scintillation counter. Values are means ± standard error of the mean of five animals. Statistical comparison in different groups of experiments was performed using a one-way analysis of variance, followed by Newman-Keuls multiple comparisons; *, p < 0.01.

Lysine as a Target for Cross-Linkage of Formaldehyde and Proteins. The interaction of formaldehyde with proteins is rather complicated. To demonstrate that formaldehyde, derived from the deamination of methylamine, cross-links with proteins, a simplified model is required. In the present study, lysine was selected as the reaction target, although formaldehyde reacts with several other amino acid residues in proteins as well. The interaction of formaldehyde with a bipeptide, H-Lys-Leu-OH, was initially used. Figure 2 shows chromatograms demonstrating that formaldehyde induces H-Lys-Leu-OH cross-linkage (Fig. 2C). This cross-linkage is unstable upon acid hydrolysis (i.e., in 6 N HCl at 110°C for 24 h), because only lysine and leucine residues are detected in the hydrolysates either pretreated or untreated with formaldehyde (Fig. 2, D and E). This is expected, because formaldehyde first forms relatively unstable Schiff's bases between two free amino groups of lysine. However, when the Schiff's bases are reduced by sodium cyanoborohydride, N-methyl-lysine and formyl-lysine peaks are detected (Fig. 2E). N-Methyl-lysine was therefore used as a marker for formaldehyde-induced protein cross-linkage in the subsequent experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Cross-linkage of formaldehyde with bipeptide H-Lys-Leu-OH. Amino acids derived from Lys-Leu-OH with or without cross-linkage with formaldehyde and/acid hydrolysis were derivatized with FMOC-Cl and separated by HPLC and assessed by UV detection ( = 265 nm). A, amino acid standards. B, H-Lys-Leu-OH alone. C, H-Lys-Leu-OH (6 mM) + formaldehyde (10 mM). D, HCl hydrolysates of H-Lys-Leu-OH, E, HCl hydrolysates of H-Lys-Leu-OH-formaldehyde adduct. F, HCl hydrolysates of Lys-Leu-OH-formaldehyde adduct pretreated NaCNBH3 (10 mM).

We also demonstrated the cross-linkage of formaldehyde with BSA, a protein containing 14% lysine residues. Based on the detection of N-methyl-lysine in the acid hydrolysates of formaldehyde-BSA adducts lysine was found to be a major target for formaldehyde (Fig. 3). However, under the present chromatography condition, dimethyl-lysine and trimethyllysine peaks were not detected due to interference of amino acids in the BSA hydrolysate. The extent of cross-linkage is dose-dependent, namely, cross-linkage can be detected at low micromolar formaldehyde and becomes saturated around 5 mM formaldehyde. The sensitivity of the spectrometric FMOC method regarding N-methyl-lysine is around 5 ng/injection. To achieve higher sensitivity, we traced the reactions with ¹⁴C-labeled formaldehyde and used mass spectrometry. This substantially enhanced the sensitivity of detection and therefore has been used in subsequent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Cross-linkage of formaldehyde with BSA. A, BSA (2 mg/ml) alone. B, BSA in the presence of formaldehyde (1 mM). The reaction mixtures were incubated at pH 7.4 for 4 h. Adducts were pretreated NaCNBH3 (10 mM) before HCl hydrolysis. Amino acids derived from the formaldehyde-BSA adducts in the hydrolysates were derivatized with FMOC-Cl, separated by HPLC, and assessed by UV detection ( = 265 nm). Under these chromatographic conditions, the FMOC derivatives of N-methy-lysine, lysine, and tyrosine are separated from other amino acids.

Identification of Methylated Lysine in the Formaldehyde-Protein Adducts. The methylated lysine residues collected from the HPLC fractions were identified by HPLCMS. As can be seen in Fig. 4, the mass spectra of the FMOC derivatives of lysine, N-methyl-lysine, N,N-dimethyl-lysine, and N,N,N-trimethyl-lysine are characterized by intense ion peaks at 591, 605, 619, and 411, respectively. The masses are consistent with the attachment of two FMOC moieties to each residue except for trimethylamine, which has only one FMOC. Other major constituents shown in the mass scans consist of fragments that have lost one or both FMOC moieties. Further analyses of the mass spectra of the molecular ions reveal fragments corresponding to lysine, methyl-lysines as well as lysines with one FMOC remaining. The results unambiguously identify methylated lysine as a result of formaldehyde-protein interaction.

View larger version (20K): [in this window] [in a new window]   Fig. 4. FMOC derivatization and identification of lysine and N-methylated lysines by mass spectrometry. Spectra indicate that the molecular ions of the FMOC derivatives of lysine, methyl-lysines linked with two moieties of FMOC are 590, 604, and 618, respectively. Only one FMOC moiety was attached to trimethyl-lysine.

Cross-Linkage of Formaldehyde, Derived from SSAO-Catalyzed Deamination of Methylamine, with BSA. In this preliminary experiment, mouse intestine, which possess very high SSAO activity, was used. The tissues were homogenized and extracts were centrifuged and concentrated using a protein concentrator (Amicon Corp., Danvers, MA). Then, the enzyme extracts were incubated in the presence of methylamine (1 mM) and BSA (2 mg/ml) at 37°C for 4 h, and N-methyl-lysine in the hydrolyzed proteins was detected. Approximately, 2% of the total lysine residues were methylated. In the controls, i.e., tissue extracts + BSA in the absence of methylamine, no methyl-lysine was detected. When the tissue extracts were pretreated with the highly selective SSAO inhibitor MDL-72974A, formaldehyde-induced cross-linkage was not detected. The spectrophotometric HPLC/FMOC method does not allow the assessment of N,N-dimethyl-lysine and N,N,N-trimethyl-lysine, because the corresponding peaks cochromatograph with other amino acids. However, radioactive N-methyl-lysine, N,N-dimethyl-lysine, and N,N,N-trimethyl-lysine can be traced if ¹⁴C-labeled methylamine was used as substrate. As shown in Fig. 5, the radioactivity in HPLC fraction 18 is due to N,N-dimethyllysine and N,N,N-trimethyl-lysine, whereas fraction 28 corresponds to N-methyl-lysine. The amount of methyl-lysine produced is approximately 3-fold that of N,N-dimethyl-lysine and N,N,N-trimethyl-lysine combined. The SSAO inhibitor MDL-72974A again effectively blocks the generation of methylated lysine. Radioactivity was also detected in fraction 3. Although MDL-72974A also inhibits the formation of these radioactive products, these compounds have not been identified.

View larger version (22K): [in this window] [in a new window]   Fig. 5. SSAO-mediated formation of formaldehyde-protein adducts of the mouse intestine. Concentrated extracts of mouse small intestine tissue were incubated with [14C]methylamine (1 x 10-3 M; 1 µCi) in the presence or absence of MDL-72974A (1 x 10-6 M) at 37°C for 4 h, and then with 10 mM NaCNBH3 at 37°C for 24 h. After dialysis (0.2M Na2HPO4 for 24 h) and hydrolysis (6 N HCl, at 110°C for 24 h), the samples were separated by HPLC (as described under Materials and Methods), fractions of 1.4 ml/min were collected, and the radioactivity was determined. A, chromatographic separation patterns of lysine FMOC-derivatives (100 ng/50-µl injection volume). B, radioactivity detected in the HPLC fractions. Fraction 18 contains N,N-dimethyl-lysine and N,N,N-trimethyl-lysine, and fraction 28 represents the N-methyl-lysine.

Evidence of SSAO-Mediated Formaldehyde-Protein Cross-Linkage in Human Tissue. Tissue slices (250-µm² pieces) of human brain meninges, which are also rich in SSAO, were incubated with ¹⁴C-labeled methylamine (1 mM) in the presence or absence of the SSAO inhibitor MDL-72974A. BSA was not included in this experiment. After incubation for 60 min at 37°C, the tissue preparations were homogenized and then separated into soluble and particulate fractions by centrifugation at 100,000g for 30 min. The soluble fractions were subjected to gel filtration via a PD-10 Sephadex G-25 M column. Column fractions 7 to 12 contain the major radioactivity of small molecules, which are excess methylamine and unbound formaldehyde (Fig. 6). Labeled macromolecules were detected in the void volume. When SSAO activity was blocked with MDL-72974A, no labeled macromolecules were found and the radioactivity in small molecule fractions increased.

View larger version (15K): [in this window] [in a new window]   Fig. 6. SSAO-mediated formation of formaldehyde-protein adducts in the soluble fraction from human meninges. Human brain meninges tissue slices were incubated with [14C]methylamine (10-3 M; 1 µCi) in the presence or absence of a selective SSAO inhibitor MDL-72974A (1 x 10-6 M) at 37°C for 4 h. The tissue slices were then homogenized using a Polytron and centrifuged at 100,000g for 30 min. Aliquot of the supernatant was applied to a small Sephadex G-25 column (PD-10), and 1-ml fractions were collected. Radioactivity in different fractions was assessed.

A substantial amount of radioactivity was found to deposit in the particulate fractions. MDL-72974A reduces the deposit of radioactivity in these fractions. The radioactively labeled protein adducts in the particulate preparations were treated with sodium cyanoborohydride, followed by hydrolysis in HCl and derivatization with FMOC-Cl. The HPLC profile is shown in Fig. 7. Radioactively labeled methylated lysines (i.e., in fractions 18 and 28) and methylarginine (fraction 16) were detected. Unknown radioactively labeled compounds in fractions 3, 12, and 20 were also detected. The formation of these radioactively labeled products was reduced by MDL-72974A.

View larger version (19K): [in this window] [in a new window]   Fig. 7. SSAO-mediated formation of formaldehyde-protein adducts in the particulate bound fraction and identification of the formaldehyde cross-linkagesites.Human meninges tissue slices were incubated with[14C]methylamine (1 x 10-3 M; 1 µCi) in the presence (closed circle) or absence (open square) of a selective SSAO inhibitor MDL-72974A (1 x 10-6 M) at 37°C for 4 h. The reaction mixtures were subsequently incubated in the presence of 10 mM NaCNBH3 at 37°C for 24 h. After dialysis against 0.2 M phosphate buffer (pH 7.2) for 24 h, the samples were hydrolyzed in 6 N HCl at 110°C for 24 h. The samples were then derivatized with FMOC-Cl and separated by HPLC as described in text. Fractions of 1.4 ml/min were collected, and the radioactivity was determined. N-methylated lysines and arginine are indicated in the figure.

	Discussion

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Formaldehyde is produced via SSAO-catalyzed oxidative deamination of methylamine (Yu, 1990). Methylamine is a readily available endogenous substrate derived from e.g., creatine and adrenaline (Yu et al., 1997; Yu and Deng, 2000). When radioactively labeled methylamine was administered to rodents, irreversibly bound methylamine metabolites were detected by an autoradiographic examination (Gronvall et al., 1998). The formation of such protein adducts was blocked by selective SSAO inhibition. To test the involvement of formaldehyde derived from methylamine in protein cross-linkage, it is necessary to assess formaldehyde-protein cross-linkage sites. Although it is known that formaldehyde forms methylene bridges between protein residues and produces intramolecular and intermolecular cross-linkage and possibly advanced protein aggregation (Francel-Conrat and Olcott, 1948), the identification of such an interaction is not well established, especially under the condition of low formaldehyde concentrations. We have therefore developed a simple method for identification of formaldehyde-protein cross-linkage.

Under physiological conditions (i.e., 37°C, pH 7.4), formaldehyde readily interacts with the bipeptide H-Lys-Leu-OH and with BSA. Although cross-linkage of formaldehyde with H-Lys-Leu-OH occurs, after acid hydrolysis only lysine and leucine residues are produced. N-Methyl-lysine was detected only when the molecule was reduced by NaCNBH₃. The -amino amino group of lysine reacts with formaldehyde to form hydroxymethylamine, which is unstable to either extensive dialysis or acidic hydrolysis. Hydroxymethylamine combines slowly with free amino, amid, guanidyl, phenolic, or imidazole groups of amino acids in the same or another protein molecule to form Schiff's base, which would not be detected after hydrolysis of the proteins unless the Schiff's base is reduced to stable intra- or intermolecular methylene cross-links (Francel-Conrat and Olcott, 1948). N-Methyl-lysine is a very useful marker to substantiate SSAO-mediated formaldehyde-protein cross-linkage. Inhibition of SSAO activity also causes an increase in labeled hydrophilic compounds, which are radioactively labeled and eluted in fractions of the solvent front. They are probably free formaldehyde, or chemically altered formaldehyde, released from sites loosely bound to proteins.

Blood vessels and small intestine tissues possess very high SSAO activity. They were used for SSAO-induced formaldehyde-protein cross-linkage studies. Methylamine, after incubation with SSAO-containing tissue extracts or slices, is converted into formaldehyde and thus reacts with lysine residues and induces protein cross-linkage. SSAO blocks such reactions. After incubation of ¹⁴C-labeled methylamine with tissue slices of SSAO-rich human brain meninges, the majority of the radioactivity was associated with the particulate bound proteins. The lysine residue seems to be the primary target for formaldehyde interaction. It is interesting to note that the generation of formaldehyde-protein adducts in the meninges tissue slices occurs in the absence BSA. This suggests that endogenous proteins in or on meninges are readily available for interaction with formaldehyde derived from SSAO-mediated reactions. It is unclear whether it is the membrane-bound, or the cytoplasmic SSAO that is primarily responsible for the production of the formaldehyde involved in the production of formaldehyde-protein adducts. It seems to be reasonable to suggest that formaldehyde generated in the immediate compartment adjacent to the outer membrane SSAO sites would favor the interaction of formaldehyde with membrane proteins. If deamination of methylamine takes place within the cells, formaldehyde would be quickly metabolized by cytosolic aldehyde dehydrogenase. Furthermore this newly generated formaldehyde would be quickly diffused and diluted by the cytoplasm. We have shown that formaldehyde at concentrations lower than 10 µM become more difficult to cross-link with proteins. Both of these factors would reduce the interaction of formaldehyde with proteins and this suggests that the cytoplasmic SSAO may play a minor role in the production of formaldehyde induced protein deposition.

The membrane-bound SSAO was independently identified as an endothelial surface vascular adhesion protein-1 involved in lymphocyte trafficking (Salmi et al., 2002). Formaldehyde generated extracellularly via SSAO-catalyzed deamination of methylamine would lead to the formylation and methylation of adjacent membrane-bound proteins and could even play a role in lymphocyte adhesion (Salmi and Jalkanen, 2002). Although this formaldehyde-induced alteration of proteins may not be sufficient to cause acute toxicity, the effect of chronic elevated levels of formaldehyde-protein cross-linkage may cause an accumulation of protein deposits, the formation of extracellular plaques, and result in the slow, silent damage to vascular tissues associated with various pathological conditions, such as diabetic complications and Alzheimer's disease (Yu et al., 2003).

It is known that some proteins undergo post-translational methylated by protein methyltransferase using S-adenosylmethionine as the methyl group donor (Paik and Kim, 1980). The modification of proteins by methyl groups can alter protein function due to changes in charges, steric relations, or hydrophobicity at the site of the attached methyl group (Alleta et al., 1998). The present study demonstrates an additional mechanism, namely, formylation and subsequent methylation, of proteins, which may result in altered function of proteins. Recently, it has been demonstrated that formaldehyde derived from SSAO-mediated deamination of methylamine dilates human blood vessels (Conklin et al., 2004) and may play a role in the regulation of blood pressure.

In conclusion, the present study demonstrates that formaldehyde produced by SSAO-catalyzed deamination of methylamine, forms adducts with cytoplasmic as well as membrane-bound proteins. Such cross-linkage may affect vascular function or cause pathological consequences. Future investigations are required in to understand the physiological and pathological implications of protein alterations induced by the endogenous production of formaldehyde.

	Footnotes

 
This work was supported by grants from the Canadian Institute of Health Research, Saskatchewan Health, and Saskatchewan Alzheimer's Society.

doi:10.1124/jpet.104.068601.

ABBREVIATIONS: SSAO, semicarbazide-sensitive amine oxidase; MAO, monoamine oxidase; FMOC, fluorenylmethyl chloroformate; BSA, bovine serum albumin; MDL-72974A, (E)-2-4-fluorophenethyl-3-fluoroallylamine HCl; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. Peter H. Yu, Neuropsychiatry Research Unit, University of Saskatchewan, Saskatoon, SK S7N 5E4, Canada. E-mail: yup{at}usask.ca

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