Abstract
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.
Highlights
Anserine (b-alanyl-N-p-methyl-L-histidine) and balenine are naturally occurring derivatives of carnosine (b-alanyl-L-histidine) that have been reported to be present in skeletal muscle and the central nervous system of vertebrates [1]
Results of the SDS-PAGE analysis indicated that carnosine Nmethyltransferase activity was coeluted with three major polypeptide bands of about 23–26 and 37 kDa in the last purification step
The analysis indicated that the bands contained numerous proteins, but surprisingly, none of them appeared to be a methyltransferase. Since these negative results might have resulted from either a poor extraction of peptides from the gel or the absence of carnosine Nmethyltransferase sequence in the Uniprot database, both the gel bands and the whole fraction 17th of Superdex 75-purification step were reanalyzed by tandem mass spectrometry against a data bank containing all chicken protein sequences available at NCBI Protein database
Summary
Anserine (b-alanyl-N-p-methyl-L-histidine) and balenine (balanyl-N-t-methyl-L-histidine) are naturally occurring derivatives of carnosine (b-alanyl-L-histidine) that have been reported to be present in skeletal muscle and the central nervous system of vertebrates [1]. Balenine has been found exclusively in snake muscles and marine mammals such as whales and dolphins (up to 45 mM), while anserine was reported to be a major L-histidine-containing dipeptide in avian tissues (up to 43 mM in chicken pectoral muscle) [3] It has been detected in muscle of fish (2.5 up to 41 mM), cats (8 mM) and rabbits (17 mM), but not in frogs and humans [1,2]. Because of a limited presence of balenine in vertebrates, much effort has been put to understand the physiological role of both carnosine and anserine These two dipeptides have been postulated to serve as buffers neutralizing lactic acid produced in working muscle due to their abundance and pKa which is close to the physiological pH [4]. No definitive explanation of their physiological importance has been provided
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