Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.     

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease

β-hexosaminidase A ($\text{β-N-}\text{acetyl-}\text{d}\text{-hexosaminidase}$, EC 3.2.1.52) is a lysosomal hydrolase composed of an α- and a β-subunit. It is responsible for the degradation of G_M2 ganglioside. Mutations in the HEXB gene encoded β-subunit cause a form of G_M2 gangliosidosis known as Sandhoff disease. Although this is a rare disease population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16–29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5′ donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compound of the intron 2 G → A susbtitution and a second allele due to a 4-bp deletion in exon 7. The β-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid procedure for 5′ donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc₁ complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8^m). Subsequently replacing ARG8^m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit⁻). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Design and efficient synthesis of novel GM2 analogues with respect to the elucidation of the function of GM2 activator

To elucidate the mechanism underlying the hydrolysis of the GalNAcβ1→4Gal linkage in ganglioside GM2 [GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1′ Cer] by β-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues—6′-NeuAc-GM2 and α-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.     

Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+-ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis

Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24-fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3-fold increased cholesterol content, and a 1.4-fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+,K+-ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+,K+-ATPase.     

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.     

Rapid identification of lymnaeid snails and their infection with Fasciola gigantica in Thailand

Freshwater snails of the family Lymnaeidae are the intermediate hosts of the liver fluke Fasciola worldwide. While distinct species have been identified at the molecular level in other parts of the world such data have not been published for Thailand. In this study we collected Lymnaeidae from different localities across Thailand and analyzed their 16S rDNA sequences as a molecular signature for classification. In addition to the ubiquitous Radix rubiginosa, we have confirmed the presence of Austropeplea viridis and Radix swinhoei, for the latter of which the ribosomal rDNA sequences are reported for the first time, in North-Thailand. Based on the obtained 16S rDNA data three primer pairs were designed that allowed rapid identification of these snail species by PCR. To determine their infection status, PCR primers for F.gigantica cathepsin L were used in parallel with the snail 16S rDNA species-specific primers in multiplex PCR analyses. Western blot analysis of total snail protein with a monoclonal anti-F.gigantica cathepsin L antibody confirmed positive cathepsin L PCR results. The developed diagnostic PCR will be of use in risk assessment for transmission of fascioliasis in Thailand.     

Biochemical Basis of Type AB GM2 Gangliosidosis in a Japanese Spaniel

The biochemical basis of a case of GM2 gangliosidosis in a Japanese Spaniel was studied. This dog had a massive accumulation of GM2 ganglioside in the brain. The beta-hexosaminidase activity in this affected dog brain was approximately 12 times higher than that of normal brain. However, the activity toward p-nitrophenyl-6-sulfo-2-acetamido-2-deoxyglucopyranoside was only four times higher in the affected brain than in normal brain. The GM2 activator preparation obtained from the normal dog brain could stimulate the hydrolysis of GM2 ganglioside by beta-hexosaminidase isolated from the affected dog. However, the corresponding activator fraction from the affected dog could not stimulate such a reaction. It was concluded that the biochemical basis of the GM2 gangliosidosis in this Japanese Spaniel was due to the attenuation in the stimulatory activity of GM2 activator. This case represents the first animal form similar to the activator deficiency (or defect) of Type AB GM2 gangliosidosis in humans.     

Characterization of seven novel mutations on the HEXB gene in French Sandhoff patients

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5′-end 16 kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.     

Tay-Sachs disease with atypical chronic course and limited brain storage: Alpha-locus hexosaminidase genetic compound

A 19-year-old Irish-Jewish male had a slow neurologic regression starting at age 4 1/2 years with stuttering. The chronic course resembled that of Spielmeyer-Vogt (juvenile ceroid-lipofuscinosis) disease. The brain was atrophic with neuronal losses and huge compound inclusions in the remaining neurons. Lipid NANA was within normal limits in gray and white matter and G_M2 gangliosides were moderately elevated at 11.5% lipid NANA. Beta-hexosaminidase A activity was lipid composition showed nonspecific abnormalities. Exhaustive tissue extraction ruled out the possibility of tightly bound gangliosides to account for the relatively low G_M2 ganglioside concentration. The extract contained unidentified chromogenic substances interfering with the resorcinol reaction. The similarly affected patient's sister lived to age 26 years and her brain was even more atrophic. No biochemical abnormality to account for progressive neuronal losses and relative lack of G_M2 ganglioside storage was found.Deceased.Special issue dedicated to Dr. Leon S. Wolfe.     

An alpha-subunit loop structure is required for GM2 activator protein binding by beta-hexosaminidase A

The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are approximately 60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the alpha-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant alpha-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.     

The Protein Activator Specific for the Enzymic Hydrolysis of GM2 Ganglioside in Normal Human Brain and Brains of Three Types of GM2 Gangliosidosis

Abstract: In order to understand the etiology of Type AB GM2 gangliosidosis, we have purified and characterized the activator protein (GM2 activator) specific for the enzymic hydrolysis of GM₂ ganglioside from normal human brain. The purified activator from human brain moved as one major protein band in various electrophoretic systems. We have also prepared the antiserum against this activator. The levels and the nature of GM₂ activator and β-hexosaminidase A were examined in the brains of five cases of GM₂. gangliosidosis—one Type B, two Type O, and two Type AB. We found that the levels of GM₂ activator in both Type B and Type O cases were markedly elevated, and that the two Type AB cases were the results of different causes. One case had a defective β hexosaminidase A and an elevated level of GM₂ activator. Although this defective β-hexosaminidase A could hydrolyze synthetic substrates, it was inactive in the cleavage of natural glycosphingolipids in the presence of the GM₂ activator. It could, however, hydrolyze asialo-GM₂ and GbOse₄Cer in the presence of sodium taurodeoxycholate. The other case had normal β-hexosaminidase A, but had a very low level of GM₂ activator when analyzed by in vitro assay, suggesting the deficiency of this activator. By immunoelectrophoresis, this case was found to be completely devoid of the protein that cross-reacts with the antiserum against the GM₂ activator.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Molecular identification of aiiA homologous gene from endophytic Enterobacter species and in silico analysis of putative tertiary structure of AHL-lactonase

The aiiA homologous gene known to encode AHL- lactonase enzyme which hydrolyze the N-acylhomoserine lactone (AHL) quorum sensing signaling molecules produced by Gram negative bacteria. In this study, the degradation of AHL molecules was determined by cell-free lysate of endophytic Enterobacter species. The percentage of quorum quenching was confirmed and quantified by HPLC method (p < 0.0001). Amplification and sequence BLAST analysis showed the presence of aiiA homologous gene in endophytic Enterobacter asburiae VT65, Enterobacter aerogenes VT66 and Enterobacter ludwigii VT70 strains. Sequence alignment analysis revealed the presence of two zinc binding sites, "HXHXDH" motif as well as tyrosine residue at the position 194. Based on known template available at Swiss-Model, putative tertiary structure of AHL-lactonase was constructed. The result showed that novel endophytic strains of Enterobacter genera encode the novel aiiA homologous gene and its structural importance for future study.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides

Several novel N₂-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N₂-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.     

More constraint on ParaHox than Hox gene families in early metazoan evolution

Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early evolution, we performed multiple systematic comparative phylogenetic analyses, which identified derived sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx-Pdx/Xlox-PG2-PG3] families in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario whereby H/P genes originated from a [Gsx-Pdx/Xlox-PG2-PG3]-related ProtoHox gene, the «posterior» and «anterior» H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX clusters in the Cnidaria-Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as neurogenesis rather than axis specification.     

A cis-encoded sRNA controls the expression of fabH2 in Yersinia

YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.