Molecular basis of an adult form of β-hexosaminidase B deficiency with motor neuron disease

Banerjee, Probal; Siciliano, L; Oliveri, Douglas R.; McCabe, Norah R.; Boyers, Michael J.; Horwitz, Allen L.; Li, Su-Chen; Dawson, Glyn

doi:10.1016/s0006-291x(05)81388-9

Cited by 29 publications

(21 citation statements)

References 20 publications

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“…Tyr 456 plays an important role in dimerization of two subunits in human HexB (42), where Tyr 456 of one subunit forms hydrophobic interactions with Ile 454 and Tyr 492 of the other subunit. The importance of Tyr 456 was indicated by the absence of active HexB in cultured fibroblasts of a Sandhoff disease patient with a Y456S mutation and a defect in homodimerization of the mutant ␤-chains (46). As shown in Fig.…”

Section: Expression Of Sf Hex From Its Cdna In Sf9mentioning

confidence: 98%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

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Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a ␤-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a ␤-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the ␤(1,2)-linked N-acetylglucosamine from the Man␣(1,3) branch compared with the Man␣(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal ␤-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing ␤-Nacetylhexosaminidase acting on N-glycans from Sf9 cells.␤-N-Acetylhexosaminidase (hexosaminidase, EC 3.2.1.52) catalyzes the hydrolysis of nonreducing terminal N-acetyl-Dhexosamine residues in N-acetyl-␤-D-hexosaminides. Hexosaminidases belong to the glycosyl hydrolase (GH) 2 3, GH20, or GH84 family (1-4). Of these, family 20 hexosaminidases include mammalian lysosomal hexosaminidases, fungal exochitinases, bacterial chitobiases, and insect chitinolytic hexosaminidases.The hexosaminidase activity of insects and insect cells is of particular interest because of the role that the enzyme may play in altering the structures of N-glycans generated by these cells. The N-glycan synthesis pathway in insects differs from that in mammals in that insects and insect cells produce appreciable amounts of paucimannosidic glycans (reviewed in Ref. 5). The intracellular N-glycan processing pathway in the endoplasmic reticulum of insects has been observed to include the addition of a Glc 3 Man 9 GlcNAc 2 group to the acceptor Asn residue, followed by the subsequent trimming of the initial oligosaccharide to generate Man 5 GlcNAc 2 . Insect cells also contain significant levels of N-acetylglucosaminyltransferase I, which adds a GlcNAc residue to the Man␣(1,3) branch, followed by the removal of two Man residues to produce the GlcNAc␤(1,2)Man␣(1,3)(Man(1,6))-Man␤(1,4)GlcNAc␤(1,4)GlcNAc structure. Unlike ma...

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Section: Expression Of Sf Hex From Its Cdna In Sf9mentioning

confidence: 98%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

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“…Amino acid substitutions causing a folding defect around this lesion may result in a transport defect and the expressed mutant protein is likely to be degraded quickly. I207V was previously reported as a disease-causing mutation (Banerjee et al 1991;Banerjee et al 1994), but it is now thought to be a silent polymorphism from the results of recent analyses (Hara et al 1998;Redonnet-Vernhet et al 1996). In this study, we constructed a mutant model with I207V and the results revealed that an amino acid substitution does not have any significant influence on the protein structure.…”

Section: Discussionmentioning

confidence: 99%

“…Gene analysis revealed that the patient was homozygous for R505Q, which was observed in cis with I207V. I207V had been reported to be a disease-causing mutation (Banerjee et al 1991(Banerjee et al , 1994, but recent reports (Hara et al 1998;Redonnet-Vernhet et al 1996) have ruled this out, and it is now thought to be a neutral polymorphism.…”

Section: Patientsmentioning

confidence: 97%

Molecular and structural studies of the GM2 gangliosidosis 0 variant

Sakuraba

Matsuzawa²,

Aikawa³

et al. 2002

J Hum Genet

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“…In contrast, a variety of less severe and chronic phenotypes of Sandhoff disease with a small percentage of residual Hex A activity have been reported, including motor neuron disease, spinocerebellar ataxia, intellectual disability and dysfunction of the autonomic nervous system (210). Only four missense mutations (Y456S, P504S, R533H and P417L) associated with the adult form of Sandhoff disease presenting the motor neuron disease phenotype have been reported (3,7,9,1113). In this study, we report the molecular and biochemical study of a Japanese male patient with the adult form of Sandhoff disease with the motor neuron disease phenotype.…”

mentioning

confidence: 99%

“…The red spheres represent the residues mentioned in this article, including mutated residues such as H235. This figure was generated using UCSF Chimera (19) Y456S-, P504S-and R533H-mutant b-subunits identified from the adult form of Sandhoff disease with the motor neuron disease phenotype form dimers with the wild-type a-and b-subunits of Hex Three missense mutations (Y456S, P504S and R533H) in HEXB have been identified in patients with the adult form of Sandhoff disease with the motor neuron disease phenotype (3,9,1113). To analyse dimer formation between these wild-type or mutant b-subunits and the wild-type a-or b-subunit, we performed immunoprecipitation using anti-FLAG M2 antibodyconjugated gels as depicted in Fig.…”

mentioning

confidence: 99%

Characterization of the mutant β-subunit of β-hexosaminidase for dimer formation responsible for the adult form of Sandhoff disease with the motor neuron disease phenotype

Yamada

Takado

Kato

et al. 2012

The Journal of Biochemistry

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The adult form of Sandhoff disease with the motor neuron disease phenotype is a rare neurodegenerative disorder caused by mutations in HEXB encoding the b-subunit of b-hexosaminidase, yet the properties of mutant b-subunits of the disease have not been fully determined. We identified a novel mutation (H235Y) in the b-sheet of the (b/a) 8 -barrel domain, in addition to the previously reported P417L mutation that causes aberrant splicing, in a Japanese patient with the motor neuron disease phenotype. Enzyme assays, gel filtration studies and immunoprecipitation studies with HEK293 cells transiently expressing mutant b-subunits demonstrated that the H235Y mutation abolished both ab and bb dimer formation without increasing b-hexosaminidase activity, whereas other reported mutant b-subunits (Y456S, P504S or R533H) associated with the motor neuron disease phenotype formed dimers. Structural analysis suggested that the H235Y mutation in the b-sheet of the (b/ a) 8 -barrel domain changed the conformation of the b-subunit by causing a clash with the E288 side chain. In summary, H235Y is the first mutation in the b-sheet of the (b/a) 8 -barrel domain of the b-subunit that abolishes ab and bb dimer formation; the presented patient is the second patient to exhibit the motor neuron disease phenotype with P417L and a non-functional allele of HEXB.

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Molecular basis of an adult form of β-hexosaminidase B deficiency with motor neuron disease

Cited by 29 publications

References 20 publications

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Molecular and structural studies of the GM2 gangliosidosis 0 variant

Characterization of the mutant β-subunit of β-hexosaminidase for dimer formation responsible for the adult form of Sandhoff disease with the motor neuron disease phenotype

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