Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like).

Anserine (beta-alanyl-N(Pi)-methyl-L-histidine), a naturally occurring derivative of carnosine (beta-alanyl-L-histidine), is an abundant constituent of skeletal muscles and brain of many vertebrates. Although it has long been proposed to serve as a proton buffer, radicals scavenger and transglycating agent, its physiological function remains obscure. The formation of anserine is catalyzed by carnosine N-methyltransferase which exhibits unknown molecular identity. In the present investigation, we have purified carnosine N-methyltransferase from chicken pectoral muscle about 640-fold until three major polypeptides of about 23, 26 and 37 kDa coeluting with the enzyme were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in an identification of histamine N-methyltransferase-like (HNMT-like) protein as the only meaningful candidate. Analysis of GenBank database records indicated that the hnmt-like gene might be a paralogue of histamine N-methyltransferase gene, while comparison of their protein sequences suggested that HNMT-like protein might have acquired a new activity. Chicken HNMT-like protein was expressed in COS-7 cells, purified to homogeneity, and shown to catalyze the formation of anserine as confirmed by both chromatographic and mass spectrometry analysis. Both specificity and kinetic studies carried out on the native and recombinant enzyme were in agreement with published data. Particularly, several compounds structurally related to carnosine, including histamine and L-histidine, were tested as potential substrates for the enzyme, and carnosine was the only methyl group acceptor. The identification of the gene encoding carnosine N-methyltransferase might be beneficial for estimation of the biological functions of anserine

Time-course of anserine synthesis in lysates of control or HNMT-like protein-overexpressing COS-7 and HEK-293T cells.

<p>COS-7 and HEK-293T cells were transfected with either unmodified pEF6/Myc-His A vector (Control) or the same vector encoding chicken HNMT-like protein (HNMT-like) as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#s2" target="_blank">Materials and Methods</a>”. The cell-free lysates (2–3 µg of protein) were incubated for 0, 5, 10, 15 and 20 min in the reaction mixture containing 1 µM SAM (100 pmol), as well as 440×10³ cpm of (³H)SAM. The formation of radiolabeled anserine was determined after its chromatographic separation from (³H)SAM. Values are the means ± S.E. of two separate transfection experiments. The presence of recombinant protein in tested lysates was verified by Western-blot analysis. Lysates (15 µg of protein) were loaded reduced onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane which was then sequentially probed with a mouse primary antibody against His6 tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. COS, COS-7 cell lysate; HEK, HEK-293T cell lysate.</p

Mass spectrum of a product formed by chicken HNMT-like protein.

<p>Homogenous recombinant chicken HNMT-like protein was incubated for 12 h with 3 mM carnosine in the absence or presence of 2 mM SAM. The produced methylated dipeptide was separated from the substrates and analyzed by mass spectrometry. Both mass spectra, covering the mass range <i>m/z</i> 50–600, and tandem mass spectra (Q-TOF) for anserine precursor ion (<i>m/z</i> 241) were acquired.</p

Purification of chicken carnosine N-methyltransferase.

<p>The enzyme was purified by chromatography on DEAE-Sepharose (A), Q-Sepharose (not shown), Superdex 200 (B), and Superdex 75 (C) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#s2" target="_blank">Materials and Methods</a>” section. Fractions were tested for carnosine N-methyltransferase activity. Protein concentration was determined in the most active fractions with the Bradford assay. The indicated fractions eluted from the Superdex 75 column were analyzed by SDS-PAGE and the gel was stained with Coomassie Blue. The indicated bands were cut out of the gel, submitted to trypsin digestion and analyzed by tandem mass spectrometry.</p

HPLC-HILIC analysis of a product formed by chicken HNMT-like protein.

<p>Chromatograms of standard mixture of carnosine and anserine (10 nmol) (A), of deproteinized reaction mixtures obtained from incubation of homogenous recombinant chicken HNMT-like protein for 12 h with 3 mM carnosine in the absence (B) or presence of 2 mM SAM (C) and following the supplementation of the former deproteinized reaction mixture with 20 nmol of anserine standard (D). The identity of all indicated compounds was confirmed by mass spectrometry. The sample processing and chromatographic conditions are described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#s2" target="_blank">Materials and Methods</a>”. AdoHcy, S-Adenosyl-L-homocysteine.</p

Amino acid sequence alignment of human HNMT with its chicken orthologue and chicken HNMT-like protein.

<p>Sequences were obtained with following GenBank accession numbers: human HNMT (NP_008826.1), chicken HNMT (XP_422143.2) and HNMT-like protein (XP_001234740.1). The chicken HNMT-like protein sequence has been confirmed by PCR amplification of the cDNA and sequencing. Percentage of amino acid identities with chicken HNMT is given in the upper right. Fully conserved residues are highlighted with a black background. Residues of human HNMT interacting with either SAM or histamine are marked by hashes or asterisks, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#pone.0064805-Horton1" target="_blank">[19]</a>. The peptides identified by mass spectrometry in the protein purified from chicken pectoral muscle are underlined in the chicken sequence.</p

(³H)methylation of various imidazole group-containing compounds catalyzed by carnosine N-methyltransferase.

<p>The transfer of methyl group from SAM was determined with the use of homogenous recombinant chicken histamine N-methyltransferase-like protein (HNMT-like) and chicken muscle carnosine N-methyltransferase purified by chromatography on DEAE-Sepharose, Q-Sepharose, Superdex 200 and Superdex 75. Enzyme preparations were incubated for 10–15 min in the presence of 1 µM (¹H+³H)SAM and 10 mM of the indicated methyl-group acceptor with the exception of histamine that was added at 0.1 mM concentration. Values are the means ± S.E. of two or three separate experiments.</p

Purification of carnosine N-methyltransferase from chicken pectoral muscle.

#<p>The data represent values for the most purified fraction.</p

Amino acid sequence alignment of selected HNMT-like proteins with paralogue HNMT proteins.

<p>Sequences of turkey HNMT-like protein (GenBank Accession Number: XP_003207778.1), Zebra Finch HNMT-like protein (XP_002194298.1), the Green Anole HNMT-like protein (XP_003215153.1), the Nile Tilapia HNMT-like_1 and HNMT-like_2 proteins (XP_003445374.1 and XP_003445450.1, respectively), and the Sea Urchin HNMT-like_1 and HNMT-like_2 proteins (XP_786900.1 and XP_792696.1, respectively) were identified by Protein Blast searches with the use of chicken HNMT-like protein sequence (XP_001234740.1) and aligned with HNMT sequences of chicken (GenBank Accession Number: XP_422143.2), turkey (XP_003207782.1), Zebra Finch (XP_002194327.1) and the Green Anole (XP_003215142.1) using M-Coffee <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#pone.0064805-Wallace1" target="_blank">[27]</a>. Level of residues conservation is indicated by black (100%), dark grey (70% and more) and light gray (50% and more) background.</p