2-Acetamido-2-deoxy-d-glucono-1,5-lactone Sulfonylhydrazones: Synthesis and Evaluation as Inhibitors of Human OGA and HexB Enzymes.

Inhibition of the human O-linked ß-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure-activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO2 oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH3/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, Ki = 27 nM). This compound had a Ki of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein-ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds.

Clinical and Molecular Characteristics of Two Chinese Children with Infantile Sandhoff Disease and Review of the Literature.

Infantile Sandhoff disease is an autosomal recessive inherited disease primarily characterized by cherry red spots in the retina, muscle weakness, seizure, truncal hypotonia, hyperacusis, developmental delay and regression. The pathogenic genetic defects of the HEXB gene, which encodes the ß subunit of the hexosaminidase A (ɑß) and hexosaminidase B (ßß) enzymes, cause deficiency of both the Hex A and Hex B enzymes, resulting in the deposition of GM2 ganglion glycerides in the lysosomes of the central nervous system and somatic cells. The aim of this study was to discover disease-causing variants of the HEXB gene in two Chinese families through the use of exome sequencing. By characterizing three novel variants by molecular genetics, bioinformatics analysis, and three-dimensional structure modeling, we showed that all these novel variants influenced the protein structure. The results broaden the variant spectrum of HEXB in different ethnic groups. Furthermore, not all patients diagnosed with infantile Sandhoff disease had characteristic cranial imaging findings, which can only be used as supplementary information for diagnosis. The results of this study may contribute to clinical management, genetic counseling, and gene-targeted treatments for Sandhoff disease.

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

The adult form of Sandhoff disease with the motor neuron disease phenotype is a rare neurodegenerative disorder caused by mutations in HEXB encoding the ß-subunit of ß-hexosaminidase, yet the properties of mutant ß-subunits of the disease have not been fully determined. We identified a novel mutation (H235Y) in the ß-sheet of the (ß/α)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 ß-subunits demonstrated that the H235Y mutation abolished both α-ß and ß-ß dimer formation without increasing ß-hexosaminidase activity, whereas other reported mutant ß-subunits (Y456S, P504S or R533H) associated with the motor neuron disease phenotype formed dimers. Structural analysis suggested that the H235Y mutation in the ß-sheet of the (ß/α)8-barrel domain changed the conformation of the ß-subunit by causing a clash with the E288 side chain. In summary, H235Y is the first mutation in the ß-sheet of the (ß/α)8-barrel domain of the ß-subunit that abolishes α-ß and ß-ß 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.

Therapeutic potential of intracerebroventricular replacement of modified human ß-hexosaminidase B for GM2 gangliosidosis.

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of ß-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human ß-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

GalNAcß1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with ß-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcß-nLc4Cer² sequence.

Molecular and functional analysis of the HEXB gene in Italian patients affected with Sandhoff disease: identification of six novel alleles.

We report the molecular characterization of 12 unrelated Italian patients affected with Sandhoff disease (SD), a recessively inherited disorder caused by mutations in HEXB gene. We identified 11 different mutations of which six are novel: one large deletion of 2,406 nt, (c.299+1471_408del2406), one frameshift mutation c.965delT (p.I322fsX32), one nonsense c.1372C>T (p.Q458X), and three splicing mutations (c.299G>T, c.300-2A>G and c.512-1G>T). One allele was only characterized at the messenger RNA (mRNA) level (r = 1170_1242del). Real-time polymerase chain reaction analysis of the HEXB mRNA from fibroblasts derived from patients carrying the novel point mutations showed that the presence of the premature termination codon in the transcript bearing the mutation c.965delT triggers the nonsense-mediated decay (NMD) pathway, which results in the degradation of the aberrant mRNA. The presence of the c.299G>T mutation leads to the degradation of the mutated mRNA by a mechanism other than NMD, while mutations c.300-2A>G and c.512-1G>T cause the expression of aberrant transcripts. In our group, the most frequent mutation was c.850C>T (p.R284X) representing 29% of the alleles. Haplotype analysis suggested that this mutation did not originate from a single genetic event. Interestingly, the common 16-kb deletion mutation was absent. This work provides valuable information regarding the molecular genetics of SD in Italy and provides new insights into the molecular basis of the disease.

A novel HEXB mutation and its structural effects in juvenile Sandhoff disease.

Mutations in HEXB, encoding the beta-subunit common to hexosaminidases A and B, cause the neurodegenerative condition, Sandhoff disease. A homozygous missense HEXB mutation (p. D459A) was discovered in six patients with a rare juvenile variant: we show that this disrupts a salt bridge between aspartate D459 and arginine 505 at the subunit interface; R505 mutations are reported in late-onset Sandhoff disease. Identification of D459A contributes to diagnosis and molecular understanding of attenuated Sandhoff disease variants.