Transport of Rat Liver Glycine N-Methyltransferase into Rat Liver Nuclei

Abstract

Rat liver cytosolic glycine N-methyltransferase (GNMT) catalyzes the S-adenosylmethionine-dependent methylation of glycine to sarcosine. It is comprised of four identical 292-amino acid residue subunits. Recently, evidence has been provided to show that GNMT is identical to the cytosolic receptor for benzo[a]pyrene, which induces cytochrome P450 1A1 gene expression. In the present study we show that chemical modification of purified rat liver GNMT with fluorescein isothiocyanate (FITC) resulted in dissociation of the tetrameric enzyme and was accompanied by loss of enzyme activity. Amino acid sequence analysis of the FITC-labeled peptides obtained by hydrolysis of the modified protein with Staphylococcus aureus V8 protease revealed that lysines 45, 89, 92, 96, 122, and 147 were modified. Lys-122 and Lys-147 were derivatized in tetrameric, dimeric, and monomeric forms of the enzyme. Lysines 45, 89, 92, and 96 were derivatized only in monomeric GNMT, suggesting that modification of these residues resulted in GNMT dissociation. The modified monomeric GNMT was quickly transported into isolated rat liver nuclei. This transport was specific for the GNMT monomer, since neither tetramer nor dimer was able to enter the nuclei. Bovine carbonic anhydrase, similar in size to the GNMT monomer, was labeled with FITC to a similar extent but was not transported into the nuclei. Disruption of the nuclei containing fluorescein-labeled GNMT and subsequent extraction of the nuclear lysate with both high and low salt buffers recovered FITC-GNMT only in the chromatin pellet. Our study supports the suggestion of an additional function for GNMT, probably connected with regulation of cytochrome P450 1A1 gene expression.

A Novel Tumor Suppressor Function of Glycine N-Methyltransferase Is Independent of Its Catalytic Activity but Requires Nuclear Localization

“…coli and purified by several subsequent chromatographic steps as we previously described [14]. Site-directed mutagenesis was carried out using a QuickChange site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing of the mutant constructs at the Nucleic Acid Analysis Core Facility, MUSC.…”

“…Although each of the four subunits has a catalytic center, the structural organization of the enzyme as a tetramer is important for its catalytic activity. In fact, GNMT dimers or monomers are catalytically inactive [12,14]. The oligomeric structure of the enzyme is also important for its regulation by folate since the GNMT tetramer binds only two molecules of 5-methyltetrahydrofolate (5-MTHF).…”

“…Interestingly, the translocation of GNMT to the nucleus has been reported after binding of B[a]P or aflatoxin, as well as after tetramer dissociation induced by disrupting lysine salt bridges on the monomer interface [14,29]. Though the role of GNMT in the nucleus is not clear at present, the protein is tightly bound to chromatin after nuclear translocation [14]. …”

Haplotypes, Loss of Heterozygosity, and Expression Levels of Glycine N-Methyltransferase in Prostate Cancer

“…GNMT, which is primarily expressed in cell cytoplasm (6,9,20), is a polycyclic aromatic hydrocarbon-binding protein (27). Previously, we and other groups showed that if the cells were treated with benzo(a)pyrene, GNMT will be translocated into the nuclei (8,28,29). Because cigarette smoking has been identified as a possible prostate cancer risk factor and GNMT inhibits benzo(a)pyrene-7,8-diol-9,10-epoxide-DNA adduct formation (8), the GNMT expression in the prostate may exert a protective effect for individuals exposed to benzo(a)pyrene and its down-regulation may be an early sign for prostate cancer carcinogenesis.…”

Glycine N-Methyltransferase and Regulation of S-Adenosylmethionine Levels

“…Other studies have questioned the role of GNMT as the 4 S aryl hydrocarbon receptor (8). Studies in which GNMT was incubated with isolated rat liver nuclei showed that native tetrameric GNMT was unable to enter the purified liver nuclei (10). The possible function of GNMT in the nucleus is still a matter of speculation, and if GNMT in the nucleus is not simply a result of cytoplasmic contamination, its function there is not clear because it does not appear to be involved in the induction of the cytochrome P-450 family of enzymes.…”