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1.  Molecular and Biochemical Characterization of a Novel β-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis 
Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.
doi:10.7150/ijbs.4406
PMCID: PMC3445047  PMID: 22991497
β-N-acetyl-D-hexosaminidase; insect; glycoside hydrolase; N-glycan; Ostrinia furnacalis.
2.  Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta▿  
Applied and Environmental Microbiology  2007;73(15):4805-4812.
Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.
doi:10.1128/AEM.00463-07
PMCID: PMC1951009  PMID: 17557860
3.  The N-glycan processing enzymes α-mannosidase and β-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum 
Journal of Experimental Botany  2010;62(2):571-582.
Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.
doi:10.1093/jxb/erq289
PMCID: PMC3003805  PMID: 21030387
Capsicum; climacteric; fruit softening; N-glycans; non-climacteric; RNAi; α-mannosidase; β-D-N-acetylhexosaminidase
4.  BIOSYNTHESIS OF TRUNCATED N-LINKED OLIGOSACCHARIDES RESULTS FROM NON-ORTHOLOGOUS HEXOSAMINIDASE-MEDIATED MECHANISMS IN NEMATODES, PLANTS AND INSECTS* 
The Journal of biological chemistry  2007;282(38):27825-27840.
In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e., glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II and, finally, β-N-acetylglucosaminidases. In Drosophila, the recently characterised enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the α1,3-antenna of N-glycans. In the present study, we examined the products of five β-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5 and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2 and AtHEX3, corresponding to reading frames At1g65590, At3g55260 and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme, but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes a members of a distinct sub-family; nevertheless, two of these enzymes displayed the same α1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesised that the genetic origin of paucimannosidic glycans in nematodes, plants and insects involves highly-divergent members of the same hexosaminidase gene family.
doi:10.1074/jbc.M704235200
PMCID: PMC2850174  PMID: 17636254
Arabidopsis; Caenorhabditis; hexosaminidase; paucimannosidic N-glycans
5.  Molecular characterization of a novel mycovirus in the cultivated mushroom, Lentinula edodes 
Virology Journal  2012;9:60.
Background
In the 1970s, mycoviruses were identified that infected the edible mushroom Lentinula edodes (shiitake), but they were not regarded as causal agents for mushroom diseases. None of their genes has been sequenced. In this study, the dsRNA genome of a mycovirus recently found in a shiitake commercial strain was sequenced and its molecular structure was characterized.
Methods
A cDNA library was constructed from a dsRNA purified from the fruiting body of L. edodes. The virus was tentatively named L. edodes mycovirus HKB (LeV). Based on the deduced RNA-dependent RNA polymerase (RdRp) sequence, phylogenetic analysis of LeV was conducted. Because no virion particles associated with the dsRNA were observed by electron microscopic observation, atomic force microscopy (AFM) observation was chosen for achieving molecular imaging of the virus.
Results
The 11,282-bp genome of LeV was obtained. The genome encoded two open reading frames (ORFs). ORF1 coded for a hypothetical protein and ORF2 for a putative RdRp, respectively. In addition, a region coding for a NUDIX domain was present in ORF1. There was a 62-bp intergenic region between ORF1 and RdRp. Similarity with coat protein of mycoviruses was not found within the whole sequence. Based on phylogenetic analysis of the putative RdRp sequence, LeV grouped into a clade with dsRNA found in the basidiomycetes Phlebiopsis gigantea and Helicobasidium mompa. The clade was placed apart from the Totiviridae and Chrysoviridae families. As suggested from the genome sequence, AFM revealed that the structure of LeV was linear unencapsidated dsRNA.
Conclusions
The results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.
doi:10.1186/1743-422X-9-60
PMCID: PMC3365873  PMID: 22390839
Mycovirus; dsRNA; AFM; Lentinula edodes; Mushroom; NUDIX domain
6.  Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family ▿ † 
Applied and Environmental Microbiology  2011;77(23):8350-8354.
The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
doi:10.1128/AEM.05581-11
PMCID: PMC3233070  PMID: 21965406
7.  Insights into transcriptional regulation of β-D-N-acetylhexosaminidase, an N-glycan-processing enzyme involved in ripening-associated fruit softening 
Journal of Experimental Botany  2014;65(20):5835-5848.
Highlight text
Ripening-specific expression of β-Hex, a gene involved in the fruit-softening process, is transcriptionally regulated by the MADS-box transcription factor RIN in SlASR1-dependent and -independent manners.
Tomato (Solanum lycopersicum) fruit ripening-specific N-glycan processing enzyme, β-D-N-acetylhexosaminidase (β-Hex), plays an important role in the ripening-associated fruit-softening process. However, the regulation of fruit ripening-specific expression of β-Hex is not well understood. We have identified and functionally characterized the fruit ripening-specific promoter of β-Hex and provided insights into its transcriptional regulation during fruit ripening. Our results demonstrate that RIPENING INHIBITOR (RIN), a global fruit ripening regulator, and ABSCISIC ACID STRESS RIPENING 1 (SlASR1), a poorly characterized ripening-related protein, are the transcriptional regulators of β-Hex. Both RIN and SlASR1 directly bound to the β-Hex promoter fragments containing CArG and C2-3(C/G)A cis-acting elements, the binding sites for RIN and SlASR1, respectively. Moreover, β-Hex expression/promoter activity in tomato fruits was downregulated once expression of either RIN or SlASR1 was suppressed; indicating that RIN and SlASR1 positively regulate the transcription of β-Hex during fruit ripening. Interestingly, RIN could also bind to the SlASR1 promoter, which contains several CArG cis-acting elements, and SlASR1 expression was suppressed in rin mutant fruits, indicating that RIN also acts as a positive regulator of SlASR1 expression during fruit ripening. Taken together, these results suggest that RIN, both directly and indirectly, through SlASR1, regulates the transcription of β-Hex during fruit ripening. The fruit ripening-specific promoter of β-Hex could be a useful tool in regulating gene expression during fruit ripening.
doi:10.1093/jxb/eru324
PMCID: PMC4203122  PMID: 25129131
Fruit ripening; fruit ripening-specific promoter; β-Hex; RIN; SlASR1; transcriptional regulation.
8.  Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies 
Background
Fungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.
Results
The complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.
Conclusion
Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.
doi:10.1186/1472-6807-7-32
PMCID: PMC1885261  PMID: 17509134
9.  A Sperm–Plasma β-N-Acetyl-D-Hexosaminidase Interacting with a Chitinolytic β-N-Acetyl-D-Hexosaminidase in Insect Molting Fluid 
PLoS ONE  2013;8(8):e71738.
Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm–plasma enzyme functioning in egg–sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval–larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.
doi:10.1371/journal.pone.0071738
PMCID: PMC3741120  PMID: 23951233
10.  Genomic Analysis and Initial Characterization of the Chitinolytic System of Microbulbifer degradans Strain 2-40 
Journal of Bacteriology  2003;185(11):3352-3360.
The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-d-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-β-d-N,N′-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-β-d-N,N′,N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-β-d-glucosaminidases and one cytoplasmic (HexC) N-acetyl-β-d-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.
doi:10.1128/JB.185.11.3352-3360.2003
PMCID: PMC155392  PMID: 12754233
11.  Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes 
BMC Research Notes  2013;6:307.
Background
Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.
Findings
Shiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.
Conclusions
We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.
doi:10.1186/1756-0500-6-307
PMCID: PMC3750829  PMID: 23915543
Assembly; Basidiomycota; Mapping; Mushroom; NGS; Pyrosequencing; QTL; Shiitake
12.  Assessing the severity of the small inframe deletion mutation in the α-subunit of β-hexosaminidase A found in the Turkish population by reproducing it in the more stable β-subunit 
Summary
GM2 gangliosidoses are a group of panethnic lysosomal storage diseases in which GM2 ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the α- or β-subunits of β-N-acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the α-subunit of Hex has been found in five Turkish patients with infantile Tay–Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive α-protein in its precursor form. Because the α- and β-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the β-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the α homodimer. Thus, mutations that completely destabilize the α-subunit can often be partially rescued if expressed in the aligned positions in the β-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267–1278) in the β-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the β-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable β-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.
doi:10.1023/B:BOLI.0000045759.12935.76
PMCID: PMC2910077  PMID: 15505380 CAMSID: cams1390
13.  Molecular Phylogeny and Predicted 3D Structure of Plant beta-D-N-Acetylhexosaminidase 
The Scientific World Journal  2014;2014:186029.
beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α)8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and Glu(331) could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom.
doi:10.1155/2014/186029
PMCID: PMC4129151  PMID: 25165734
14.  Crystal Structure of Human β-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay–Sachs Disease 
Journal of molecular biology  2003;327(5):1093-1109.
In humans, two major β-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits α and β (60% identity), whereas Hex B is a homodimer of β-subunits. Interest in human β-hexosaminidase stems from its association with Tay–Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of GM2-ganglioside (GM2). Hex A degrades GM2 by removing a terminal N-acetyl-D-galactosamine (β-GalNAc) residue, and this activity requires the GM2–activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4 Å) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2 Å) or NAG-thiazoline (2.5 Å). From these, and the known X-ray structure of the GM2–activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how α and β-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease (β-subunit mutations) and Tay–Sachs disease (α-subunit mutations).
PMCID: PMC2910754  PMID: 12662933 CAMSID: cams1386
hexosaminidase; Sandhoff; Tay–Sachs; anchimeric assistance; X-ray crystal structure
15.  Determination of Glucan Contents in the Fruiting Bodies and Mycelia of Lentinula edodes Cultivars 
Mycobiology  2014;42(3):301-304.
Shiitake mushrooms (Lentinula edodes) containing β-glucans may be beneficial for human health; they have been used in the treatment of cancer, hypertension, and high cholesterol levels. The objective of this study was to determine the β-glucan content in different sections of the fruiting bodies and mycelia of ten shiitake mushroom cultivars. The measured β-glucan content ranged from 20.06 ± 1.76% to 44.21 ± 0.13% in the pileus sections, and from 29.74 ± 1.40% to 56.47 ± 4.72% in the stipe sections. The results of this study indicate that the variance in β-glucan content dependent on the shiitake cultivar, and that the β-glucan content is higher in the stipe than in the pileus.
doi:10.5941/MYCO.2014.42.3.301
PMCID: PMC4206800  PMID: 25346611
β-Glucan; Cultivar; Lentinula edodes; Shiitake mushroom
16.  Evaluation of Receptor Binding Specificity of Escherichia coli K88 (F4) Fimbrial Adhesin Variants Using Porcine Serum Transferrin and Glycosphingolipids as Model Receptors  
Infection and Immunity  2002;70(5):2336-2343.
Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 μM), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a β-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a β-linked HexNAc. In addition, the presence of a terminal galactose β-linked to this HexNAc residue enhances K88 adhesin binding.
doi:10.1128/IAI.70.5.2336-2343.2002
PMCID: PMC127900  PMID: 11953368
17.  In vitro bone inducing effects of Lentinula edodes (shiitake) water extract on human osteoblastic cell cultures 
The effect of Lentinula edodes water extract (LE) on two osteoblastic cell cultures (HOS 58 and Saos-2) was investigated to determine if this edible medicinal mushroom has osteoinductive properties. Activity of alkaline phosphatase and mineralization were used as indicators for the vitality and maturation of the bone cells. Cultivation of human osteosarcoma cells HOS 58 for five days in presence of a serial dilution of the aqueous extract of L. edodes (0.8 μg/mL-125 μg/mL) resulted in a significant elevation of alkaline phosphatase activity (ALP) of the cells in comparison to untreated cells. Saos-2 cells, incubated with LE (20 μg/mL) and β-glycerol phosphate (2 mM) for 21 days, displayed a 2 fold level of mineralization than cells cultured soley with the positive control, β-glycerophosphate. The obtained results clearly indicate the activity of LE as a bone inducing agent in vitro. Therefore, the shiitake mushroom (L. edodes) deserves attention as a supportive dietary treatment or nutraceutical in the case of diseases accompanied with bone disorder, such as osteoporosis, osteopenia, and late complication of diabetes.
doi:10.1007/s13659-013-0070-3
PMCID: PMC4131604
medicinal mushrooms; Lentinula edodes; osteoporosis; alkaline phosphatase; bone mineralization; shiitake
18.  Breeding and Screening of Lentinula edodes Strains Resistant to Trichoderma spp. 
Mycobiology  2008;36(4):270-273.
Trichoderma spp. cause large crop losses of the cultivated shiitake mushroom, Lentinula edodes. We bred several shiitake strains that are resistant to Trichoderma spp. using di-mon mating to establish a useful method for controlling the greenmold disease. We examined the competitive ability of L. edodes against Trichoderma spp. using a dual culture system to select resistant strains. By screening Trichoderma-resistant strains, we found that among 11 parental strains, 4 strains, including KFRI 36, were confirmed resistant strains. They showed especially strong resistance to T. harzianum, which formed deadlock after mycelial contact and then invaded into the territory of T. harzianum. KFRI 171 also showed resistance to T. atroviride strains. Among 13 strains, which were made by hybridization of shiitake strains, 5 were confirmed to be resistant to Trichoderma, including KFRI 58-1. Their resistance was not correlated to the resistant activity of their parents' strains. Two strains lose resistance and two strains acquire resistance compared to their parents' strains. In SEM observation, the mycelium of L. edodes at the interaction zone of Lentinula-Trichoderma was rugged and swollen by T. harzianum.
doi:10.4489/MYCO.2008.36.4.270
PMCID: PMC3755208  PMID: 23997639
Hybrid strains; Lentinula edodes; Screening of resistant strains; Trichoderma species
19.  Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase 
PLoS ONE  2012;7(12):e52225.
The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp490, Glu328, Val327 in OfHex1, and Trp358, Tyr131 and Ile179 in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu328 together with Trp490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in Km for (GlcNAc)2 and a 42-fold increment in Ki for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu368 and Asp367) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu328 and Val327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.
doi:10.1371/journal.pone.0052225
PMCID: PMC3531433  PMID: 23300622
20.  Extracellular Enzyme Activities of the Monokaryotic Strains Generated from Basidiospores of Shiitake Mushroom 
Mycobiology  2008;36(1):74-76.
To obtain basic information on the biochemical property of basidiospores of shiitake mushroom (Lentinula edodes), the ability of producing extracellular enzyme was assessed using a chromogenic plate-based assay. For the aim, amylase, avicelase, β-glucosidase, CM-cellulase, pectinase, proteinase, and xylanase were tested against monokaryotic strains generated from forty basidiospores of two different parental dikaryotic strains of shiitake mushroom, Sanjo-101Ho and Sanjo-108Ho. These two parental strains showed different degree of extracellular enzyme activity. No identical patterns of the degree of enzyme activity were observed between monokaryotic strains and parental strains of the two shiitake cultivars. The degree of extracellular enzyme activity also varied among monokaryotic strains of the two shiitake cultivars. Our results showed that dikaryotic parental strains of shiitake mushroom produce monokaryotic basidiospores having very diverse biochemical properties.
doi:10.4489/MYCO.2008.36.1.074
PMCID: PMC3755257  PMID: 23997613
Basidiospores; Chromogenic media; Extracellular enzyme activity; Shiitake mushroom
21.  Characterization of an Exo-β-d-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1 
Journal of Bacteriology  2003;185(17):5175-5181.
We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc2) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc2. Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative β-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the β-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to β-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-β-d-glucosaminidase (GlcNase) activity but not β-galactosidase activity. This GlcNase (GlmATk), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmATk was induced by GlcNAc2 and chitin, strongly suggesting that GlmATk is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc2 from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.
doi:10.1128/JB.185.17.5175-5181.2003
PMCID: PMC181003  PMID: 12923090
22.  Pleurotus ostreatus inhibits proliferation of human breast and colon cancer cells through p53-dependent as well as p53-independent pathway 
International journal of oncology  2008;33(6):1307-1313.
In spite of the global consumption of mushrooms, only two epidemiological studies demonstrated an inverse correlation between mushroom intake and the risk of cancer. Therefore, in the present study we evaluated whether extracts from edible mushrooms Agaricus bisporus (portabella), Flammulina velutipes (enoki), Lentinula edodes (shiitake) and Pleurotus ostreatus (oyster) affect the growth of breast and colon cancer cells. Here, we identified as the most potent, P. ostreatus (oyster mushroom) which suppressed proliferation of breast cancer (MCF-7, MDA-MB-231) and colon cancer (HT-29, HCT-116) cells, without affecting proliferation of epithelial mammary MCF-10A and normal colon FHC cells. Flow cytometry revealed that the inhibition of cell proliferation by P. ostreatus was associated with the cell cycle arrest at G0/G1 phase in MCF-7 and HT-29 cells. Moreover, P. ostreatus induced the expression of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21(CIP1/WAF1), whereas inhibited the phosphorylation of retinoblastoma Rb protein in MCF-7 cells. In addition, P. ostreatus also up-regulated expression of p21 and inhibited Rb phosphorylation in HT-29 cells, suggesting that that P. ostreatus suppresses the proliferation of breast and colon cancer cells via p53-dependent as well as p53-independent pathway. In conclusion, our results indicated that the edible oyster mushroom has potential therapeutic/preventive effects on breast and colon cancer.
PMCID: PMC2796484  PMID: 19020765
mushroom; breast cancer; prostate cancer; cell proliferation
23.  Nucleotide sequence of the Streptococcus pneumoniae hexB mismatch repair gene: homology of HexB to MutL of Salmonella typhimurium and to PMS1 of Saccharomyces cerevisiae. 
Journal of Bacteriology  1989;171(10):5332-5338.
The Hex mismatch repair system of Streptococcus pneumoniae acts both during transformation (a recombination process that directly produces heteroduplex DNA) to correct donor strands and after DNA replication to remove misincorporated nucleotides. The hexB gene product is one of at least two proteins required for mismatch repair in this organism. The nucleotide sequence of a 2.7-kilobase segment from the S. pneumoniae chromosome that includes the 1.95-kilobase hexB gene was determined. The gene encodes a 73.5-kilodalton protein (649 residues). The spontaneous hex Rx chromosomal mutant allele with which a mutator phenotype has been associated is shown to result from a single base substitution (TAC to TAA) leading to a truncated HexB polypeptide (484 residues). The HexB protein is homologous to the MutL protein, which is required for methyl-directed mismatch repair in Salmonella typhimurium and Escherichia coli, and to the PMS1 gene product, which is likely to be involved in a mismatch correction system in Saccharomyces cerevisiae. The conservation of HexB-like proteins among procaryotic and eucaryotic organisms indicates that these proteins play an important common role in the repair process. This finding also suggests that the Hex, Mut, and PMS systems evolved from a common ancestor and that functionally similar mismatch repair systems could be widespread among procaryotic as well as eucaryotic organisms.
PMCID: PMC210370  PMID: 2676973
24.  Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. 
Journal of Bacteriology  1988;170(1):190-196.
The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. An open reading frame that could encode a 17-kilodalton polypeptide (OrfC) was located just upstream of the gene encoding a polypeptide of 95 kilodaltons corresponding to HexA. Shine-Dalgarno sequences and putative promoters were identified upstream of each protein start site. Insertion mutations showed that only HexA functioned in mismatch repair and that the promoter for hexA transcription was located within the OrfC-coding region. The HexA polypeptide contains a consensus sequence for ATP- or GTP-binding sites in proteins. Comparison of the entire HexA protein sequence to that of MutS of S. typhimurium, which was determined by Haber et al. in the accompanying paper (L. T. Haber, P. P. Pang, D. I. Sobell, J. A. Mankovitch, and G. C. Walker, J. Bacteriol. 170:197-202, 1988), showed the proteins to be homologous, inasmuch as 36% of their amino acid residues were identical. This homology indicates that the Hex and Mut systems of mismatch repair evolved from an ancestor common to the gram-positive streptococci and the gram-negative enterobacteria. It is the first direct evidence linking the two systems.
PMCID: PMC210625  PMID: 3275608
25.  Presence of galactosylated core fucose on N-glycans in the planaria Dugesia japonica 
Journal of Mass Spectrometry  2011;46(6):561-567.
Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan–lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man5GlcNAc2 structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex4HexNAc2Fuc1Me2 was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex5HexNAc2Fuc1Me3 structure. This is the first report of these types of structures in a platyhelminth and indicates that the ‘GalFuc’ modification of N-glycans is not just restricted to molluscs and nematodes. Copyright © 2011 John Wiley & Sons, Ltd.
doi:10.1002/jms.1925
PMCID: PMC3155867  PMID: 21630384
Galβ1,4Fuc epitope; planaria; N-glycan; methylhexose; mass spectrometry

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