Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.     

Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family.

We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase

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.     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

Molecular characterization of two galactosemia mutations: correlation of mutations with highly conserved domains in galactose-1-phosphate uridyl transferase.

Galactosemia is an autosomal recessive disorder of human galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT). The molecular basis of this disorder is at present not well understood. We report here two missense mutations which result in low or undetectable enzymatic activity. First, we identified at nucleotide 591 a transition which substitutes glutamine 188 by arginine. The mutated glutamine is not only highly conserved in evolution (conserved also in Escherichia coli and Saccharomyces cerevisiae), but is also two amino acid residues downstream from the active site histidine-proline-histidine triad and results in about 10% of normal enzymatic activity. The arginine 188 mutation is the most common galactosemia mutation characterized to date. It accounts for one-fourth of the galactosemia alleles studied. Second, we report the substitution of arginine 333 by tryptophan, caused by a transition at nucleotide 1025. The area surrounding this missense mutation is the most highly conserved domain in the homologous enzymes from E. coli, yeast, and humans, and this mutation results in undetectable enzymatic activity, suggesting that this is a severe mutation. This second mutation appears to be rare, since it was found only in the patient we sequenced. Our data provide further evidence for the heterogeneity of galactosemia at the molecular level, heterogeneity which might be related to the variable clinical outcome observed in this disorder.     

Linkage disequilibrium between a SacI restriction fragment length polymorphism and two galactosemia mutations

We have identified a novel SacI restriction fragment length polymorphism (RFLP) in the human galactose-1-phosphate uridyl transferase (GALT) gene. This RFLP can be readily typed by the polymerase chain reaction (PCR). The polymorphic allele is found on about 11% of normal chromosomes and is in linkage disequilibrium with the two most common mutations identified in GALT thus far: Q188R and N314D. Q188R is found exclusively on chromosomes with the SacI restriction site, whereas N314D is found only on chromosomes lacking this site. This suggests that these two mutations arose independently in evolution on different chromosomal backgrounds. Galactosemia patients without the Q188R mutation have a frequency of the SacI polymorphism similar to normal controls suggesting that several different galactosemia mutations must be present in them. The SacI RFLP may also be useful in the prenatal diagnosis of galactosemia.     

Characterization of two missense mutations in human galactose-1-phosphate uridyltransferase: different molecular mechanisms for galactosemia.

We report the molecular characterization of two novel galactosemia mutations that exhibit different molecular phenotypes. Both are of the missense type with low or no residual enzyme activity. The R148W mutation results in an unstable protein, although messenger RNA is still produced. In contrast, the L195P mutation produces stable but inactive immunoreactive protein. The R148W mutation alters an amino acid that is not evolutionarily conserved, while the L195P mutation affects a well-conserved residue nine amino acids down-stream from the putative active site nucleophile. These mutations provide evidence that different mechanisms can result in galactosemia: destabilizing mutations in any given area of the protein and missense mutations in conserved domains of the enzyme resulting in low or no activity. These two mutant alleles represent the fifth and sixth galactosemia mutations and confirm the hypothesis that galactosemia results from a multiplicity of mutations at the molecular level.     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Characterization of two stop codon mutations in the galactose-1-phosphate uridyltransferase gene of three male galactosemic patients with severe clinical manifestation

Classical galactosemia, which is caused by deficiency of galactose-1-phosphate uridyltransferase, is characterized by acute problems of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. Galactose limitation reverses these symptoms immediately; however, the long-term complications, such as mental retardation and ovarian failures are major problems in most of these patients. In order to investigate the molecular basis for phenotype variation in galactosemia, we have screened the most common mutation in the GALT gene, Q188R. We have further examined those patients who are heterozygous for Q188R or negative for this mutation by SSCP analysis and direct sequencing. In three male patients, we have identified, for the first time, two stop-codon mutations in the GALT gene, G212X (exon 7) and E340X (exon 10). Two patients of 8 and 28 years of age, respectively, who are compound heterozygotes for Q188R and G212X, have severe mental retardation and their general clinical condition is more severe than that of patients with missense mutations. The third patient, who is 8 years of age and who is homozygous for E340X, the N314D polymorphism and a silent substitution L218L, presents with a relatively normal physical and mental condition to date.     

The structural and molecular biology of type I galactosemia: Enzymology of galactose 1-phosphate uridylyltransferase

Reduced galactose 1-phosphate uridylyltransferase (GALT) activity is associated with the genetic disease type I galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GALT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (II) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GALT is required to assist greater understanding of the effects of disease-associated mutations.     

Galactosemia: a strategy to identify new biochemical phenotypes and molecular genotypes.

We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recessive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E203K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E203K mutation created a unique isoform-banding pattern. An asymptomatic sister''s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intraallelic complementation when in cis.     

Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.     

Reversion from deficiency of galactose-1-phosphate uridylytransferase (GALT) in an SV40-transformed human fibroblast line

Control SV40-transformed human fibroblasts can be readily adapted to growth on medium containing galactose as sole hexose source (galactose-MEH). However, most cells from a line of SV40-transformed skin fibroblasts from a patient with galactosemia (galactose-1-phosphate uridylyltransferase (GALT) deficiency) died in galactose-MEM. Surviving cells of this line either grew in completely sugar-free media or had acquired significant amounts of GALT activity. Two presumptive revertant cell lines with GALT activity were characterized in detail. The expression of GALT in these two lines was stable in nonselective conditions. Each had different reaction maximum velocities with respect to uridine diphosphoglucose (UDPg) concentration as compared to residual activity in the parental cell strain or control cells. Both appeared to demonstrate heat-inactivation profiles for GALT than differed from the parental cells or controls. UDPG concentration was found to significantly alter the thermostability of GALT. A competitive radioimmunoassay for GALT showed that these two lines had amounts of the GALT protein comparable to that of the parental cell strain or control cells. The electrophoretic mobility of GALT from the two presumptive revertants was found to differ from control cells. It was concluded that structural gene changes were probably responsible for the apparent reversion in these lines.     

Relationship between genotype, activity, and galactose sensitivity in yeast expressing patient alleles of human galactose-1-phosphate uridylyltransferase

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.     

Large-scale molecular screening for galactosemia alleles in a pan-ethnic population

DNA samples from 4,796 subjects from diverse ethnic groups were screened for five frequently encountered galactose-1-phosphate uridyl transferase (GALT) mutations: S135L (cDNA nt 404C-->T, as numbered from the initiator ATG codon, with A=1); Q188R (cDNA nt 563A-->G); K285 N (cDNA nt 855G-->T); the Duarte variant, N314D (cDNA nt 940A-->G); and the Los Angeles variant, which contains L218L (cDNA nt 652C-->T) and N314D. Among Whites, the gene frequency of the Q188R mutation was 0.29%, and that of the K285 N mutation was 0.062%. Only one S135L mutation was encountered among 505 African-Americans (gene frequency 0.10%). The pan-ethnic gene frequencies of the Duarte and the Los Angeles variants were 5.1% and 2.7%, respectively. Both of these frequencies were significantly less among African-Americans and Asians than among Whites and Hispanics. Native Americans revealed a higher incidence of the both variants. Based upon the gene frequency of the Q188R mutation in the White population, the birth incidence of classic galactosemia is estimated at one patient per 47,000 in the White population. This prevalence would be increased by inbreeding. It agrees well with the results from newborn screening programs and is only minimally higher than that reported in most studies, suggesting that most, if not all, infants with the galactosemia genotype are born and survive sufficiently long to be screened.     

Galactose-1-phosphate uridyl transferase in density-fractionated erythrocytes

Summary Galactose-1-phosphate uridyl transferase (GALT), the deficient enzyme in classical galactosemia, was studied by Percoll-gradient age-fractionation of erythrocytes. For normal GALT, a rapid and substantial decrease in GALT activity and loss of most of two isozymes was found to occur in the reticulocyte fractions. The loss of activity was then followed by relative stabilization of both GALT-specific activity and microheterogeneity in mature and aging erythrocytes. When applied to the study of mutant GALT from galactosemic patients, the Percoll-gradient fractionation method permitted detection in the reticulocyte-enriched fractions of up to 5% of normal GALT-specific activity and an isoelectric focusing pattern essentially the same as that of normal GALT. Percoll-gradient fractionation of erythrocytes offers a simple and direct method to study characteristics of GALT activity and microheterogeneity in normal and galactosemic human erythrocytes.     

GALT Protein Database, a Bioinformatics Resource for the Management and Analysis of Structural Features of a Galactosemia-related Protein and Its Mutants

We describe the GALT-Prot database and its related web-based application that have been developed to collect information about the structural and functional effects of mutations on the human enzyme galactose-1-phosphate uridyltransferase (GALT) involved in the genetic disease named galactosemia type Ⅰ. Besides a list of missense mutations at gene and protein sequence levels, GALT-Prot reports the analysis results of mutant GALT structures. In addition to the structural information about the wild-type enzyme, the database also includes structures of over 100 single point mutants simulated by means of a computational procedure, and the analysis to each mutant was made with several bioinformatics programs in order to investigate the effect of the mutations. The web-based interface allows querying of the database, and several links are also provided in order to guarantee a high integration with other resources already present on the web. Moreover, the architecture of the database and the web application is flexible and can be easily adapted to store data related to other proteins with point mutations. GALT-Prot is freely available at http://bioinformatica.isa.cnr.it/GALT/.     

A common mutation associated with the Duarte galactosemia allele.

The human cDNA and gene for galactose-1-phosphate uridyl transferase (GALT) have been cloned and sequenced. A prevalent mutation (Q188R) is known to cause classic galactosemia (G/G). G/G galactosemia has an incidence of 1/38,886 in 1,396,766 Georgia live-born infants, but a more common variant of galactosemia, Duarte, has an unknown incidence. The proposed Duarte biochemical phenotypes of GALT are as follows: D/N, D/D, and D/G, which have approximately 75%, 50%, and 25% of normal GALT activity respectively. In addition, the D allele has isoforms of its enzyme that have more acidic pI than normal. Here we systematically determine (a) the prevalence of an A-to-G transition at base pair 2744 of exon 10 in the GALT gene, transition that produces a codon change converting asparagine to aspartic acid at position 314 (N314D), and (b) the association of this mutation with the Duarte biochemical phenotype. The 2744G nucleotide change adds an AvaII (SinI) cut site, which was identified in PCR-amplified DNA. In 111 biochemically unphenotyped controls with no history of galactosemia, 13 N314D alleles were identified (prevalence 5.9%). In a prospective study, 40 D alleles were biochemically phenotyped, and 40 N314D alleles were found. By contrast, in 36 individuals known not to have the Duarte biochemical phenotype, no N314D alleles were found. We conclude that the N314D mutation is a common allele that probably causes the Duarte GALT biochemical phenotype and occurs in a predominantly Caucasian, nongalactosemic population, with a prevalence of 5.9%.     

Molecular analysis of 11 galactosemia patients.

Galactosemia is a human inborn error of galactose metabolism due to deficiency of galactose-1-phosphate uridyl transferase. In this paper, I describe the molecular analysis of genomic DNA, mRNA and protein from 11 different galactosemic patients by Southern, Northern and Western blotting. The results of these experiments lead me to conclude that galactosemia is caused mostly by missense mutations. The unusual preponderance of missense mutations in galactosemia led me to investigate its cause. I demonstrate that all 9 patients I investigated have detectable residual enzyme activity (ranging from 0.7-6.9% of normal). This finding is of potential importance in addressing the long-term complications of galactosemia.