Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area.

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families, five of them Druze, residing in a very small area of northern Israel. Since such clustering suggests a classic founder effect, the presence of three mutant alleles of the IDUA gene was unexpected.     

Canavan disease: mutations among Jewish and non-jewish patients.

Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point mutations--693C-->A, 854A-->C, and 914C-->A--were identified in the coding sequence. The 693C-->A and 914C-->A base changes, resulting in nonsense tyr231-->ter and missense ala305-->glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A-->C transversion converted glu to ala in codon 285. The glu285-->ala mutant ASPA has 2.5% of the activity expressed by the wild-type enzyme. A fourth mutation, 433 --2(A-->G) transition, was identified at the splice-acceptor site in intron 2. The splice-site mutation would lead to skipping of exon 3, accompanied by a frameshift, and thus would produce aberrant ASPA. Of the 128 unrelated Canavan chromosomes analyzed, 88 were from probands of Ashkenazi Jewish descent. The glu285-->ala mutation was predominant (82.9%) in this population, followed by the tyr231-->ter (14.8%) and 433 --2(A-->G) (1.1%) mutations. The three mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305-->glu mutation was found exclusively in non-Jewish probands of European descent and constituted 60% of the 40 mutant chromosomes. Predominant occurrence of certain mutations among Ashkenazi Jewish and non-Jewish patients with Canavan disease would suggest a founding-father effect in propagation of these mutant chromosomes.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

The hemochromatosis 845 G-->A and 187 C-->G mutations: prevalence in non-Caucasian populations.

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation.     

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1).

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.     

Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients.

We describe four new mutations in the beta-galactosidase gene. These are the first mutations causing infantile and juvenile GM1-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM1-gangliosidosis were analyzed. Northern blot analysis showed the acid beta-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys577-->Arg, Arg590-->His, and Glu632-->Gly. The fourth mutation, Arg208-->Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system.     

Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: toward mutation mapping of the iduronate-2-sulfatase gene.

Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C-->T, severe phenotype); P358R (1073C-->G, severe); R468W (1402C-->T, mild); P469H (1406C-->A, mild); and Y523C (1568A-->G, mild). Non-sense mutations were identified in two patients: R172X (514C-->T, severe) and Q389X (1165C-->T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (> 95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395 C-->G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented.     

Linkage of a new mutation in the proteolipid protein (PLP) gene to Pelizaeus-Merzbacher disease (PMD) in a large Finnish kindred.

The purpose of this study was to confirm linkage of the proteolipid protein gene (PLP) and Pelizaeus-Merzbacher disease (PMD). A T-->A transversion in nucleotide pair 35 of exon 4 of PLP was found in a large Finnish kindred with PMD. This mutation results in the substitution Val165-->Glu165. We used a combination of single-strand conformational polymorphism and PCR primer extension to determine the presence or absence of the point mutation in family members. A lod score of 2.6 (theta = 0) was found for linkage of the gene and the disease. We examined 101 unrelated X chromosomes and found none with the transversion. This is the second report of linkage of PMD to a missense mutation in PLP. These findings support the hypothesis that PMD in this family is a result of the missense mutation present in exon 4 of PLP.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263.

The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.     

Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase. In order to characterize mutations, RNA was isolated from cultured fibroblasts from 13 unrelated patients with neonatal citrullinemia. Ten mutations were identified by sequencing of amplified cDNA. Seven single base missense mutations were identified: Gly14----Ser, Ser180----Asn, Arg157----His, Arg304----Trp, Gly324----Ser, Arg363----Trp, and Gly390----Arg. Six of these missense mutations involved conversion of a CpG dinucleotide in the sense strand to TpG or CpA, and six of the seven mutations alter a restriction enzyme site in the cDNA. Two mutations were observed in which the sequences encoded by a single exon (exon 7 or 13) were absent from the cDNA. One mutation is a G----C substitution in the last position of intron 15 resulting in splicing to a cryptic splice site within exon 16. There is extreme heterogeneity of mutations causing citrulinemia. This heterogeneity may prove typical for less common autosomal recessive human genetic diseases.     

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.     

Novel splice, missense, and nonsense mutations in the fumarylacetoacetase gene causing tyrosinemia type 1.

In six unrelated patients with hereditary tyrosinemia type 1 (HT1), three different disease-causing mutations were found by DNA sequencing. Two Pakistani patients, with acute and intermediate forms of HT1, were homozygous for a G192-->T mutation in the last nucleotide of exon 2. This caused aberrant splicing with partial intron 2 retention and premature termination. Three Turkish patients with chronic and intermediate forms of HT1 were homozygous for an A698-->T mutation substituting aspartic acid 233 with valine. A Norwegian patient with an intermediate clinical phenotype was heterozygous for G786-->A, introducing a TGA stop codon for Trp262 (W262X). Site-directed mutagenesis and expression in a rabbit reticulocyte lysate system demonstrated that the nonsense and missense mutations abolished fumarylacetoacetase activity and gave reduced amounts of a truncated and a full-length protein, respectively. Simple tests were established to identify the three mutations by restriction digestion of PCR-amplified genomic DNA. Among 30 additional HT1 patients investigated, 2 were found to be homozygous and 1 heterozygous for G192-->T. Two other patients were homozygous and one was heterozygous for W262X.     

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2)

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.     

Mutants at Ser277 of Xenopus cdc2 protein kinase induce oocyte maturation in the absence of the positive regulatory phosphorylation site Thr161.

The cdc2 protein kinase is an important regulatory protein for both meiosis and mitosis. Previously, we demonstrated that simultaneous mutation of Thr14-->Ala14 and Tyr15-->Phe15 in the Xenopus cdc2 protein results in an activated cdc2 mutant that induces maturation in resting oocytes. In addition, we confirmed the importance of the positive regulatory phosphorylation site, Thr161, by demonstrating that cdc2 mutants containing additional mutations of Thr161-->Ala161 or Glu161 are inactive in the induction of oocyte maturation. Here, we have analyzed the importance of an additional putative cdc2 phosphorylation site,Ser277. Single mutation of Ser277-->Asp277 or Ala277 had no effect on activity, and these mutants were unable to induce Xenopus oocyte maturation. However, the double mutant Ala161/Asp277 was capable of inducing oocyte maturation, suggesting that mutation of Ser277-->Asp277 could compensate for the mutation of Thr161-->Ala161. The Asp277 mutation could also compensate for the Ala161 mutation in the background of the activating mutations Ala14/Phe15. Although mutants containing the compensatory Ala161 and Asp277 mutations were capable of inducing oocyte maturation, these mutant cdc2 proteins lacked detectable in vitro kinase activity. Tryptic phosphopeptide mapping of mutant cdc2 protein and comparison with in vitro synthesized peptides indicated that Ser277 is not a major site of phosphorylation in Xenopus oocytes; however, we cannot rule out the possibility of phosphorylation at this site in a biologically active subpopulation of cdc2 molecules. The data presented here, together with prior reports of Ser277 phosphorylation in somatic cells, suggest an important role for Ser277 in the regulation of cdc2 activity. The regulatory role of Ser277 most likely involves its indirect effects on the nearby residue Arg275, which participates in a structurally important ion pair with Glu173, which lies in the same loop as Thr161 in the cdc2 protein.     

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

beta-Hexosaminidase isozymes from cells cotransfected with alpha and beta cDNA constructs: analysis of the alpha-subunit missense mutation associated with the adult form of Tay-Sachs disease.

In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the alpha-subunit of beta-hexosaminidase A (alpha beta) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of alpha-chain mutations is not straightforward. We examine three approaches utilizing previously identified mutations affecting alpha-chain folding. These involve transfection of (1) the alpha cDNA alone; (2) a beta cDNA construct encoding a beta-subunit substituted at a position homologous to that of the alpha-subunit, and (3) both alpha and beta cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an "active" alpha Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. We conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some alpha-chain mutations. Using this technique, we demonstrate that the adult-onset Tay-Sachs mutation, alpha Gly --> Ser269, does not directly affect alpha beta dimerization but exerts an indirect effect on the dimer through destabilizing the folded alpha-subunit at physiological temperatures. Two other alpha mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.