Mari Maeda

A missense mutation in the Na(+)/glucose cotransporter gene SGLT1 in a patient with congenital glucose-galactose malabsorption: normal trafficking but inactivation of the mutant protein.

The Na(+)/glucose cotransporter gene SGLT1 was analyzed in a Japanese patient with congenital glucose-galactose malabsorption. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 15 exons of SGLT1. The amplification products were cloned and sequenced. About half of the exon 5 clones of the patient contained a C-->T transition, resulting in an Arg(135)-->Trp mutation, whereas the remaining clones contained the normal exon 5 sequence. In addition, whereas some exon 12 clones exhibited the normal sequence, others showed a CAgtaggtatcatc-->CAgacc mutation at the splice donor site of intron 12 that may result either in the skipping of exon 12 or in read-through of intron 12. Neither the Arg(135)-->Trp mutant nor either of the possible intron 12 mutant proteins exhibited Na(+)-dependent glucose transport activity when expressed in Xenopus oocytes. Immunocytochemical analysis indicated, however, that the Arg(135)-->Trp mutant was localized to the oocyte plasma membrane. DNA sequence analysis revealed that the missense mutation in exon 5 and the splice site mutation in intron 12 were inherited from the probands father and mother, respectively. These results indicate that the patient is a compound heterozygote for this disease, and that the Arg(135)-->Trp mutant of SGLT1 undergoes normal trafficking to the plasma membrane but is non-functional.

Amino Acid Residues Responsible for Galactose Recognition in Yeast Gal2 Transporter

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter ofSaccharomyces cerevisiae. A mixture of approximately 25,000 distinct plasmids that encode all the combinations of 12 amino acids in transmembrane 10 that are different in Gal2 and the homologous glucose transporter Hxt2 was synthesized. Selection of galactose transport-positive clones on galactose limited agar plates yielded 19 clones, all of which contained the Tyr446 residue found in Gal2. 14 of the 19 clones contained Trp455 found in Gal2, whereas the other 5 contained Cys455, a residue not found in either Gal2 or Hxt2. When Tyr446 of Gal2 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr446 plays an essential role in the transport of this sugar. Replacement of 2 amino acids of Hxt2 with the corresponding Tyr446 and Trp455 of Gal2 allowed the modified Hxt2 to transport galactose. The K m of galactose transport for the modified transporter was 8-fold higher than that of Gal2. These results and other evidence unequivocally show that Tyr446 is essential and Trp455 is important for the discrimination of galactose versus glucose.

Transmembrane segment 10 is important for substrate recognition in Ga12 and Hxt2 sugar transporters in the yeast Saccharomyces cerevisiae

A systematic series of chimeras between Gal2 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH‐terminus that has been previously identified as the critics substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport‐active chimeras were found to possess Ga12‐derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM10 region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.

Contribution to Substrate Recognition of Two Aromatic Amino Acid Residues in Putative Transmembrane Segment 10 of the Yeast Sugar Transporters Gal2 and Hxt2

The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter ofSaccharomyces cerevisiae has shown that Tyr446is essential and Trp455 is important for galactose recognition by Gal2. Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721–16724). Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2. The role of Phe431 in Hxt2 was similarly studied. Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20%. In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity. The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp. Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr. These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong.

Identification by Comprehensive Chimeric Analysis of a Key Residue Responsible for High Affinity Glucose Transport by Yeast HXT2

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu59 and Leu61 (TM1), Leu201 (TM5), Asn331 (TM7), and Phe366 (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn331 yielded transporters with various affinities, with those of the Ile331, Val331, and Cys331 mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn331 or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile331, Val331, Cys331) face the central cavity and are within van der Waals distances of Phe208 (TM5), Leu357 (TM8), and Tyr427 (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.

Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Asn(331) in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile(287) in human GLUT1, which corresponds to Asn(331) in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile(287) generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile(287) is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

ABSTRACT Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Mechanism of Action of ME1111, a Novel Antifungal Agent for Topical Treatment of Onychomycosis

ABSTRACT Despite the existing treatment options for onychomycosis, there remains a strong demand for potent topical medications. ME1111 is a novel antifungal agent that is active against dermatophytes, has an excellent ability to penetrate human nails, and is being developed as a topical agent for onychomycosis. In the present study, we investigated its mechanism of action. Trichophyton mentagrophytes mutants with reduced susceptibility to ME1111 were selected in our laboratory, and genome sequences were determined for 3 resistant mutants. The inhibitory effect on a candidate target was evaluated by a spectrophotometric enzyme assay using mitochondrial fractions. Point mutations were introduced into candidate genes by a reverse genetics approach. Whole-genome analysis of the 3 selected mutants revealed point mutations in the structural regions of genes encoding subunits of succinate dehydrogenase (complex II). All of the laboratory-generated resistant mutants tested harbored a mutation in one of the subunits of succinate dehydrogenase (SdhB, SdhC, or SdhD). Most of the mutants showed cross-resistance to carboxin and boscalid, which are succinate dehydrogenase inhibitors. ME1111 strongly inhibited the succinate-2,6-dichlorophenolindophenol reductase reaction in Trichophyton rubrum and T. mentagrophytes (50% inhibitory concentrations [IC50s] of 0.029 and 0.025 μg/ml, respectively) but demonstrated only moderate inhibition of the same reaction in human cell lines. Furthermore, the target protein of ME1111 was confirmed by the introduction of point mutations causing the amino acid substitutions in SdhB, SdhC, and SdhD found in the laboratory-generated resistant mutants, which resulted in reduced susceptibility to ME1111. Thus, ME1111 is a novel inhibitor of the succinate dehydrogenase of Trichophyton species, and its mechanism of action indicates its selective profile.

Detection and identification of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real‐time PCR

A real‐time PCR method for detection and identification of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single‐colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real‐time PCR assay had 100% specificity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real‐time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.

Nephrocalcinosis in glucose-galactose malabsorption: nephrocalcinosis and proximal tubular dysfunction in a young infant with a novel mutation of SGLT1.

We report an association of proximal renal tubular dysfunction in a 50-day-old girl with glucose-galactose malabsorption who was found to have nephrocalcinosis, but no sign of nephrolithiasis. A novel homozygous nonsense mutation at 267Arg →stop (CGA→TGA) in the Na+-dependent glucose transporter (SGLT1) was found in loop 5 connecting transmembrane segments 6 and 7, indicating the complete loss of glucose transport activity. This case indicates that hypercalcaemia, nephrocalcinosis and proximal tubular dysfunction may be seen in association with glucose-galactose malabsorption and that most of these abnormalities improve with a glucose-galactose-free diet.