Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

Simultaneous evaluation of molecular size and antigenic stability of PNEUMOVAX 23, a multivalent pneumococcal polysaccharide vaccine.

A technique using high performance size exclusion chromatography (HPSEC) with rate nephelometry (RN) detection has been developed to simultaneously measure the relative molecular size and antigenicity of the bacterial polysaccharide components in a multivalent pneumococcal vaccine, PNEUMOVAX 23. This assay was used to establish stability profiles for each of the 23 pneumococcal polysaccharide serotypes in this vaccine formulation, based on concurrent analyses of vaccine lots up to nine years of age. The exceptional inter-assay precision (<1% RSD) permitted detailed analysis of the data and a more accurate measure of the stability of this product that heretofore has not been available. While 21 of the 23 serotypes in the vaccine show essentially no change in molecular size over several years, serotypes 19A and 19F show changes in relative molecular size of approximately 2% per year. Similar decreases in relative molecular size for serotypes 19A and 19F stored in aqueous formulation have also been observed in other commercially available pneumococcal vaccine products. Additionally, stability profiles of relative antigenicity for nine of the 23 serotypes are reported based on information that is simultaneously obtained in the HPSEC/RN analysis. Of the nine serotypes examined, only serotypes 1, 9V and 18C demonstrate antigenic lability over time, in each case showing a decrease in antigenicity on the order of 5% to 10% per year. Overall, this method is a precise and efficient means of providing data on relative molecular size and relative antigenicity for each polysaccharide component of a multivalent vaccine product. Application of this method in stability studies of such vaccines provides critical information for evaluating time-dependent changes in these products.

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.

Development and validation of an NMR-based identity assay for bacterial polysaccharides.

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.