Fig 2 - uploaded by Md. Anowar Hossain
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An organization of Man-Le gene. The gene located at the chromosome 6 under BAC clone C06Hba0086B01.1 found in the GenBank is illustrated from 5 0 (left) to 3 0 (right). Exons 130 are indicated by gray boxes. Total length of the gene was 10,007 bp.
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It has been reported that acidic α-mannosidase activity increases during tomato fruit ripening, suggesting the turnover of N-glycoproteins is deeply associated with fruit ripening. As part of a study to reveal the relationship between the plant α-mannosidase activity and fruit maturation at the molecular level, we have already purified and characte...
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... tion using another pair of primer (K and L) for obtain- ing short PCR products. The genomic DNAPCR product size (1,850 bp) was larger than RTPCR prod- uct (370 bp) suggested that genomic DNA contained much more introns in genomic DNA (data not shown). The presence of introns in Man-Le gene was also supported by the exonintron organization (Fig. 2). To obtain the 5 0 -and 3 0 -end of cDNA, the 5 0 -and 3 0 -RACE-PCR and nested-PCR were performed using gene-specific primers (CF). The amplified fragments were cloned into pGEM-T easy vector and determined the sequence of three clones. We used another pair of primer, G and H for the PCR amplification to obtain the full-length cDNA, ...
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... that ESTs clones, SGN-U343973 and SGN-U327376, were the part of the Man-Le mRNA. The Man-Le mRNA had maximum identities found in NCBI BLAST nucleotide search with the mRNA sequences of Ricinus communis (77%, XM_002512794.1), Populus trichocarpa (76%, XM_ 002321039.1) and Vitis vinifera (76 %, XM_ 002276056.1). 30 exons as well as 29 introns (Fig. 2). The first exon started at 26,091 bp and 30th exon ended at 36,097 bp. (Fig. 1). Therefore, the mature Man-Le is a single polypeptide containing 1,004 amino acid residues with a predicted molecular mass of 114 kDa and pI 6.19. A typical poly (A) þ tail was found at the 3 0 untranslated region. Further analysis showed that the deduced ...
Citations
... The open reading frame (ORF) of all FaMAN sequences was between 3024 and 3069 bp, and they had a protein length of 1008 to 1023 amino acids (Table 1). In Solanum esculentum, an αman gene of 30 exons has been described with an ORF of 3084 bp and a 1028 amino acid sequence length [25]. The difference in exon numbers did not result in significant protein length variations ( Table 1). ...
... Hossain et al. [25] conducted the first phylogenetic analysis of α-man from plants, insects, and animals, and grouped 14 sequences from Oryza sativa, Ricinus communis, and Arabidopsis thaliana into two main groups, with one group further subdivided into three subgroups. In our study of 76 sequences, we identified three primary groups: I, II, and III ( Figure 2 and Supplementary Table S1). ...
Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.
... GO analysis of the 2964 genes revealed that these predicted target genes of TCP TFs were involved in flavonoid transport, mannosidase activity, and purine nucleobase transport (Figure 6b; Table S4). In particular, mannosidase is widely distributed in animals and plants (Hossain et al., 2010) and is considered critical for plant development or fruit maturation (Meli et al., 2010;Strasser et al., 2006). ...
The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.
... GO analysis of the 2964 genes revealed that these predicted target genes of TCP TFs were involved in flavonoid transport, mannosidase activity, and purine nucleobase transport (Figure 6b; Table S4). In particular, mannosidase is widely distributed in animals and plants (Hossain et al., 2010) and is considered critical for plant development or fruit maturation (Meli et al., 2010;Strasser et al., 2006). ...
... However, excessive expression of α-mannosidases commonly leads to abnormal function in plants and humans [5]. α-Mannosidase gives rise to diseases caused by an abnormal function of α-mannosidase or effect on fruit ripening and storage [6,7]. For example, the yield of α-mannosidase in ripe peppers or tomato shows that lower expression will extend the storage of pepper [8,9]. ...
... In this study, swainsonine strongly inhibited the activity, and deoxymannojirimycin had almost no effect on enzyme activity. This property was consistent with most GH38 α-mannosidases [7,32]. ...
This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0–6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co2+ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu2+, Zn2+, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins.
... GHs are important cell wall polysaccharide-modified enzymes that participate in the division and expansion of plant cells and their substrates are pectin and hemicellulose [40−46] . GH16, GH31 and GH51 may act on modification of xylans in cell wall, and GH28 can hydrolyze pectin [47,48] . GH38 may be involved in the modification of the mannose and GH32 as the invertase functions in carbohydrate allocation [49−51] . ...
Glycosylation is a significant post-translational modification of proteins, and some glycoproteins serve as players in plant cell wall synthesis and modification. Wood is a highly developed cell wall organization, and protein glycosylation as a regulatory mechanism may be involved in wood formation. Here, a lectin affinity-based glycoproteome was performed in stem developing xylem of poplar. After enrichment, trypsin digestion, LC-MS/MS analysis and peptide identification, we identified 154 glycoproteins from poplar developing xylem, which were classified into nine functional groups mainly including protein acting on carbohydrates, oxido-reductase, proteases, and protein kinases. Further, N- and/or O-glycosylation sites of the identified proteins were analyzed using bioinformatic tools, and deglycosylation experiments in the selected PtSOD and PtHAD proteins verified the reliability of the identified glycoproteins. Analysis of protein subcellular localization showed that a total of 63% of the identified glycoproteins were extracellular proteins or located in the plasma membrane. Poplar eFP and RT-qPCR data showed that a number of the genes encoding these glycoproteins such as laccase, peroxidase and cysteine protease, have highly preferential expression profiles in the developing xylem. Together with previously published research, most identified glycoproteins could be involved in wood cell wall synthesis and modification in poplar. Thus, our study provides some potential wood formation-related glycoproteins to be determined during tree stem development.
... Man 5 GLcNAc 2 is modi ed by N-acetyl glucosaminyl transferase, which links with an N-acetylglucosaminyl transferase to form GLcMan 5 GlcNAc 2 . Then, it is transported to a Golgi apparatus cavosurface by II-type a-mannose glycosidase shear, losing two mannose forms of GLcMan3GlcNAc2 [22]. Combined, these results support an essential role for the activity of glycosyl transferase, and glycosidase was signi cantly decreased as an extension of time for SW in the N-glycan process. ...
Background: Glycosylation and glycan composition play an important role in the biological properties of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Swainsonine (SW) causes oligosaccharide accumulation and incomplete glycoprotein processing. Previous research has shown that SW reduces serum progesterone concentrations in pregnant livestock. However, we do not know how SW affects the secretion of reproductive hormones. Materials: We used primary culture of mouse endometrial epithelial cells, Real-time polymerase chain reaction, Western blot, Periodic acid-Schiff (PAS) staining, Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), ELLSA, etc. method. Results: In this study, we observed a significant decrease in the number of surviving fetal mice in the SW treatment group mice and a high level of carbohydrate deposition in PAS stained uterine tissue in the SW treatment group mice. Extensive vacuole degeneration can also be observed in mouse primary endometrial epithelial cell cultures in vitro after SW poisoning. MALDI-TOF-MS shows that the time of exposure to SW is prolonged, the number of di-, tri-, and tetra-complex glycosylated gradually decreases, and the number of tri- hybrid type glycosylated increases, while significantly reduced activity of glycosyltransferases and glycosidases are observed in endometrial epithelial cells. FSH and LH were significantly decreased at 7 and 15 days after pregnancy and 7 days after childbirth, the content of E2 and P4 decreased significantly at 7 days after childbirth, and the expression levels of mRNA and protein of 3β-hydroxysteroid dehydrogenase (HSD-3β) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) also showed a significant decrease at 15 days after pregnancy and 7 days after childbirth. Conclusions: In summary, The glycan chain structure of FSH and LH glycoprotein hormones were changed by swainsonine, which they are decreased on the expression of the limiting enzyme of steroid hormones indirectly, and ultimately lead to a decrease in the content of E2 and P4. The rate of female mice were decreased by dysfunction of the reproductive hormones.
... The reaction mixtures were incubated at 37°C overnight. After stopping the enzyme reactions in boiling water, the enzyme products were analyzed by SF-HPLC using an Asahipak NH2P-50 column (0.46 × 25 cm) as described previously [21][22][23]. As shown in Figure 2, activity of all four exoglycosidases was detected in xylem sap. ...
... These results suggested that the GN2 PTC-FNGs produced by aPNGase are degraded in the xylem sap by a combination of several exoglycosidases, although the degradation rate of M3FX by α-Man'ase seems to be slow, which explains the predominance of this structure in the xylem sap. The accumulation of free M3FX in the tomato xylem sap seems to suggest that the occurrence of the β1-2 xylosyl and α1-3-fucosyl resides on the core pentasaccharide structure (Man 3 GlcNAc 2 ) should decrease the reaction rate of the tomato α-mannosidase as described in the previous report [21]. Figure 2. Analyses of exoglycosidase activities (α-Fuc'ase, β-Gal'ase, β-GlcNAC'ase, and α-Man'ase) by SF-HPLC. ...
Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant.
Abbreviations: RP-HPLC: reversed-phase HPLC; SF-HPLC: size-fractionation HPLC; PA-: pyridylamino; PCT: plant complex type; Hex: hexose; HexNAc: N-acetylhexosamine; Pen: pentose; Deoxyhex: deoxyhexose; Man: D-mannose; GlcNAc: N-acetyl-D-glucosamine; Xyl: D-xylose; Fuc: L-fucose; Lea: Lewis a (Galβ1-3(Fucα1-4)GlcNAc); PCT: plant complex type; M3FX: Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA; GN2M3FX: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA; (Lea)1GN1M3FX: Galβ1-3(Fucα1-4)GlcNAc1-2 Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA or GlcNAc1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAc1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA.
... Introduction a-Mannosidases are ubiquitous in which have been purified and well characterized from various plants, microbial and animal sources. These enzymes are an abundant constituent of plant hydrolytic system [1,2]. Mannosidases are accumulated in vacuoles and presumed to be involved in catabolism and turnover of N-linked glycoproteins [3,4]. ...
... Another peak marker, IWB67907, is within a predicted TRIAE_CS42_6AL_TGACv1_472781_AA1525920 gene that codes Alphamannosidase. In other plants, alphamannosidase has enzymatic function on Nglycans [36,37]. IWB7004, also within confidence interval for 6A.2, is linked to TRIAE_CS42_1BS_TGACv1_051115_AA0177850 gene for uncharacterized protein in wheat (http://plants.ensembl.org). ...
Stable quantitative trait loci (QTL) are important for deployment in marker assisted selection in wheat (Triticum aestivum L.) and other crops. We reported QTL discovery in wheat using a population of 217 recombinant inbred lines and multiple statistical approach including multi-environment, multi-trait and epistatic interactions analysis. We detected nine consistent QTL linked to different traits on chromosomes 1A, 2A, 2B, 5A, 5B, 6A, 6B and 7A. Grain yield QTL were detected on chromosomes 2B.1 and 5B across three or four models of GenStat, MapQTL, and QTLNetwork while the QTL on chromosomes 5A.1, 6A.2, and 7A.1 were only significant with yield from one or two models. The phenotypic variation explained (PVE) by the QTL on 2B.1 ranged from 3.3–25.1% based on single and multi-environment models in GenStat and was pleiotropic or co-located with maturity (days to heading) and yield related traits (test weight, thousand kernel weight, harvest index). The QTL on 5B at 211 cM had PVE range of 1.8–9.3% and had no significant pleiotropic effects. Other consistent QTL detected in this study were linked to yield related traits and agronomic traits. The QTL on 1A was consistent for the number of spikes m⁻² across environments and all the four analysis models with a PVE range of 5.8–8.6%. QTL for kernels spike⁻¹ were found in chromosomes 1A, 2A.1, 2B.1, 6A.2, and 7A.1 with PVE ranged from 5.6–12.8% while QTL for thousand kernel weight were located on chromosomes 1A, 2B.1, 5A.1, 6A.2, 6B.1 and 7A.1 with PVEranged from 2.7–19.5%. Among the consistent QTL, five QTL had significant epistatic interactions (additive × additive) at least for one trait and none revealed significant additive × additive × environment interactions. Comparative analysis revealed that the region within the confidence interval of the QTL on 5B from 211.4–244.2 cM is also linked to genes for aspartate-semialdehyde dehydrogenase, splicing regulatory glutamine/lysine-rich protein 1 isoform X1, and UDP-glucose 6-dehydrogenase 1-like isoform X1. The stable QTL could be important for further validation, high throughput SNP development, and marker-assisted selection (MAS) in wheat.
... The presence of identical subunits (homo dimer) has been reported for α-mannosidase from two closely related species Phaseolus vulgaris (110 kDa) (Paus 1977) and D. lablab (116 kDa) (Gnanesh Kumar et al. 2013). However, the tomato enzyme is a hetero tetramer with pair of 70 and 47 kDa subunits (Hossain et al. 2010). The N-terminal sequence of smaller subunit identified in tomato α-mannosidase is different from that of JBM and in both active site amino acid tyrosine is located in the smaller subunit (Y 625 for JBM and Y 661 for tomato α-mannosidase) (Supplementary data,Figure S6). ...
Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y1 ions (peptide+HexNAc)(+n) revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man11-15GlcNAc2 at Asn13, Man8-12GlcNAc2 at Asn87, Man9-14GlcNAc2 at Asn168 and phosphorylated Man12-17GlcNAc2 as well as Man11-16GlcNAc2 at Asn368. The presence of N-glycans with Man <18GlcNAc2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn13 or Asn168 followed by at Asn368. However, glycans at Asn87 which comprises Man8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn87 with the polypeptide chain implicating it in the folding of the protein.
