Disease relevance of Fpgs

• Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally [1].
• Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS [1].
• The exclusive use of one of two alternative sets of initial coding exons in different tissues underlies this phenomenon, suggesting the design of antifolates specific for activation by individual FPGS isoforms and hence tissue-selective targeting of antifolate therapy for cancer, arthritis, or psoriasis [2].
• Folylpolyglutamyl synthetase (FPGS), partially purified from murine L1210 leukemia and Sarcoma 180 cells and the proliferative fraction of luminal epithelium from mouse small intestine (the site of limiting toxicity to folate analogues), was examined for its ability to utilize various 4-aminofolates as substrates [3].
• These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs [4].

High impact information on Fpgs

• Folates and folate antimetabolites are metabolically trapped in mammalian cells as polyglutamates, a process catalyzed by folylpoly-gamma-glutamate synthetase (FPGS) [2].
• Using 5'-rapid amplification of cDNA ends, RNase protection assays, transfection of cDNAs into FPGS-deficient cells, and kinetic analysis of recombinant enzymes expressed in insect cells, it was determined that the species of active FPGS in mouse liver and kidney was different from that in mouse tumor cells, bone marrow, and intestine [2].
• For FPGS derived from proliferative intestinal epithelium, aminopterin was also the preferred substrate, but the value for Vmax (derived with crude cell-free extract) was 6-fold lower than for tumor cell FPGS [3].
• Values for Vmax (derived with partially purified FPGS) for the other 4-aminofolate analogues and folic acid were similar (methotrexate) or 2-fold (10-ethyl-10-deazaaminopterin) and 5-fold (folic acid) lower than for aminopterin [3].
• In contrast, the relative rates of polyglutamylation in Manca cells were in the order 10-ethyl-10-deazaaminopterin approximately equal to aminopterin greater than 10-deazaaminopterin greater than methotrexate, suggesting that folylpolyglutamyl synthetase may have varying substrate preferences in different cell types [5].

Chemical compound and disease context of Fpgs

• Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be beta sheet 5, based on the crystal structure of the Lactobacillus casei enzyme [6].

Biological context of Fpgs

• These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2 (cen leads to Cl) [7].
• These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2 [8].
• Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end [8].
• Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98% [6].
• When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored [6].

Anatomical context of Fpgs

• The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners [9].
• Differing specificities for 4-aminofolate analogues of folylpolyglutamyl synthetase from tumors and proliferative intestinal epithelium of the mouse with significance for selective antitumor action [3].
• Moreover, FPGS mRNA from the variant cells was significantly less effective in mediating formation of the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types [10].

Associations of Fpgs with chemical compounds

• Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat-), were employed as recipients in microcell-mediated chromosome transfer experiments [7].
• We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+) [1].
• Our search for water-soluble quinazoline TS inhibitors that are transported into cells via the RFC, but are not substrates for FPGS, led us to the synthesis of dipeptide analogues of ICI 198583 diglutamate [11].
• The value for Km derived with aminopterin was similar to that derived for either tumor cell FPGS [3].
• Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Vmax with no change in apparent Km [10].

Analytical, diagnostic and therapeutic context of Fpgs

• Molecular cloning of murine folylpoly-gamma-glutamate synthetase [12].
• Findings with FPGS derived from L1210 cells were confirmed by high-pressure liquid chromatography analysis of product formation during the reaction with the parent compounds [3].
• In addition, Northern blotting of poly(A)+ RNA did not reveal any difference in the size or level of FPGS mRNA among these various cell types [10].
• Decreases in a 60-61-kDa protein as shown by immunoblotting with anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compared with parental cells [10].

References