The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase.
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The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 A. Arginine and asparagine could provide only one hydrogen bond of 2. 52 and 3.02 A, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 +/- 7. 0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 +/- 8.0 and 129 +/- 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 +/- 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 +/- 71. 2 and 2960 +/- 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal. (+info)
Genetic basis of transferase-deficient galactosaemia in Ireland and the population history of the Irish Travellers.
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Transferase-deficient galactosaemia, resulting from deficient activity of galactose-1-phosphate uridyltransferase (GALT), is relatively common among the Travellers, an endogamous group of commercial/industrial nomads within the Irish population. This study has estimated the incidence of classical transferase-deficient galactosaemia in Ireland and determined the underlying GALT mutation spectrum in the Irish population and in the Traveller group. Based upon a survey of newborn screening records, the incidence of classical transferase-deficient galactosaemia was estimated to be 1 in 480 and 1 in 30,000 among the Traveller and non-Traveller communities respectively. Fifty-six classical galactosaemic patients were screened for mutation in the GALT locus by standard molecular methods. Q188R was the sole mutant allele among the Travellers and the majority mutant allele among the non-Travellers (89.1%). Of the five non-Q188R mutant alleles in the non-Traveller group, one was R333G and one F194L with three remaining uncharacterized. Anonymous population screening has shown the Q188R carrier frequency to be 0.092 or 1 in 11 among the Travellers as compared with 0.009 or 1 in 107 among the non-Travellers. The Q188R mutation was shown to be in linkage disequilibrium with a Sac I RFLP flanking exon 6 of the GALT gene. This represents the first molecular genetic description of classical transferase-deficient galactosaemia in Ireland and raises intriguing questions concerning the genetic history of the Irish Travellers. (+info)
Expression of human inositol monophosphatase suppresses galactose toxicity in Saccharomyces cerevisiae: possible implications in galactosemia.
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A suppressor of galactose toxicity in a gal7 yeast strain (lacking galactose 1-phosphate uridyl transferase) has been isolated from a HeLa cell cDNA library. Analysis of the plasmid clone indicated that the insert has an ORF identical to that of hIMPase (human myo-inositol monophosphatase). The ability of hIMPase to suppress galactose toxicity is sensitive to the presence of Li(+) in the medium. A gal7 yeast strain harboring a plasmid containing cloned hIMPase grows on galactose as a sole carbon source. hIMPase mediated galactose metabolism is dependent on the functionality of GAL1 as well as GAL10 encoded galactokinase and epimerase respectively. These results predicted that the UDP-glucose/galactose pyrophosphorylase mediated pathway may be responsible for the relief of galactose toxicity. Experiments conducted to test this prediction revealed that expression of UGP1 encoded UDP-glucose pyrophosphorylase can indeed overcome the relief of galactose toxicity. Moreover, expression of UGP1 allows a gal7 strain to grow on galactose as a sole carbon source. Unlike the hIMPase mediated relief of galactose toxicity, UGP1 mediated relief of galactose toxicity is lithium insensitive. Based on our results and on the basis of available information on galactose toxicity, we suggest an alternative explanation for the molecular mechanism of galactose toxicity. (+info)
Molecular basis for phenotypic heterogeneity in galactosaemia: prediction of clinical phenotype from genotype in Japanese patients.
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We identified 14 mutations in 15 Japanese subjects from 13 families with galactose-1-phosphate uridyltransferase (GALT) deficiency using denaturing gradient gel electrophoresis (DGGE) and direct sequence analysis. These mutations accounted for 22 (96%) of 23 mutant alleles in 15 Japanese subjects. The mutational spectrum included nine missense mutations (M142V, G179D, A199T, R231H, W249R, N314D, P325L, R333Q, and R333W), two deletions (L275fsdelT and Q317fsdelC), a nonsense mutation (W249X), and two splicing mutations (V85-N97fsdel38bp and IVS4nt+1). Ten of the 14 mutations have not been reported in Caucasians. Differences in frequency and spectrum of GALT mutations suggest that the mutations may have occurred after racial divergence of Caucasians and Asians. The Duarte variant in Japanese was associated with the N314D mutation, g.1105G > C, g.1323G > A, and g.1391G > A (SacI -) polymorphisms, as in Caucasians. The Duarte variant may have occurred before racial divergence, and was an ancient mutation. In vitro GALT activities of nine missense mutations were determined by a COS cell expression system, and indicated between 1.3% and 35% of wild-type control. Patients with R333Q (29% in vitro GALT activity) or A199T (35%) showed mild clinical phenotypes, i.e. no ovarian failure or neurological deterioration. Genotype determination is useful for predicting biochemical and clinical phenotypes in classic galactosaemia, and can be of further help in managing patients with this disorder. (+info)
A case-control study of galactose consumption and metabolism in relation to ovarian cancer.
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Consumption or metabolism of dairy sugar and ovarian cancer have been linked based on evidence that galactose may be toxic to ovarian germ cells and that ovarian cancer is induced in animals by depletion of oocytes. We assessed consumption of dairy products and obtained blood for biochemical and molecular genetic assessment of galactose metabolism in 563 women with newly diagnosed epithelial ovarian cancer and 523 control women selected either by random digit dialing or through lists of residents in eastern Massachusetts and New Hampshire. We observed no significant differences between cases and controls in usual consumption of various types of dairy products or total daily lactose (the principal source of galactose in the diet); nor did we find that RBC activity of either galactose-1-phosphate uridyl transferase (GALT) or galactokinase differed. The mean (and SE) activity of uridine diphospho-galactose 4'-epimerase (in micromoles per hour per gram of hemoglobin) was, however, significantly lower (P < 0.005) in cases compared with controls, 20.32 (0.31) versus 21.64 (0.36). Ovarian cancer cases were also more likely to carry the N314D polymorphism of the GALT gene, generally predisposing to lower GALT activity. The difference was most evident for endometrioid and clear cell types of ovarian cancer, in which 3.9% of cases were found to be homozygous for N314D compared with 0.4% of controls, yielding an odds ratio and 95% confidence interval of 14.17 (2.62-76.60). We conclude that, whereas adult consumption of lactose carries no clear risk for the disease, certain genetic or biochemical features of galactose metabolism may influence disease risk for particular types of ovarian cancer. (+info)
Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase.
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Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (hGALT) results in the potentially lethal disorder classic galactosemia. Although a variety of naturally occurring mutations have been identified in patient alleles, few have been well characterized. We have explored the functional significance of a common patient mutation, F171S, using a strategy of conservative substitution at the defined residue followed by expression of the wild-type and, alternatively, substituted proteins in a null-background strain of yeast. As expected from patient studies, the F171S-hGALT protein demonstrated <0.1% wild-type levels of activity, although two of three conservatively substituted moieties, F171L- and F171Y-hGALT, demonstrated approximately 10% and approximately 4% activity, respectively. The third protein, F171W, demonstrated severely reduced abundance, precluding further study. Detailed kinetic analyses of purified wild-type, F171L- and F171Y-hGALT enzymes, coupled with homology modeling of these proteins, enabled us to suggest that the effects of these substitutions resulted largely from altering the position of a catalytically important residue, Gln-188, and secondarily, by altering the subunit interface and perturbing hexose binding to the uridylylated enzyme. These results not only provide insight into the functional impact of a single common patient allele and offer a paradigm for similar studies of other clinically or biochemically important residues, but they further help to elucidate activity of the wild-type human GALT enzyme. (+info)
'Durate variant with clinical signs' has alpha1 -antitrypsin genotype ZZ.
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A patient with neonatal jaundice and cirrhosis who was previously reported homozygous for the Durate variant of galactose-1-phosphate uridyl transferase has the ZZ genotype for alpha1-antitrypsin. A sister of the patient, also with ZZ genotype, is less severly affected with liver disease and is a heterozygote for the Durate variant. Since a number of patients with ZZ genotype of alpha1-antitrypsin have been previously reported to have liver disease, the latter genotype is the more probable explanation for the patients' clinical state. A question is raised, however, whether the Duarte variant may be specifically associated with the development of liver disease in ZZ individuals. (+info)
Quantitative Beutler test for newborn mass screening of galactosemia using a fluorometric microplate reader.
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BACKGROUND: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of the assay. METHODS: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min. RESULTS: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 degrees C for 3 days and to 40% after storage at 37 degrees C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate. CONCLUSIONS: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia. (+info)