Biji Chatterjee, K. Ghosh, N. Yadav, Santosh R. Kanade
Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.
{"title":"A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells","authors":"Biji Chatterjee, K. Ghosh, N. Yadav, Santosh R. Kanade","doi":"10.1093/jb/mvw057","DOIUrl":"https://doi.org/10.1093/jb/mvw057","url":null,"abstract":"Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"24 1","pages":"87–97"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78160670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The crypt is a minimal functional unit in the intestinal epithelium. This unique structure is maintained by surrounding mesenchymal cells that focally interact with associated epithelial cells. Canonical and non-canonical Wnt ligands enable specific microenvironments localized to each end of the crypt major axis. While canonical Wnt-expressing cells are localized near the crypt bottom where intestinal stem cells reside, non-canonical Wnt-expressing cells are positioned beneath the luminal surface of epithelial cells. During wound healing, propagation and appropriate relocation of each cell population are thought to ensure subsequent crypt regeneration. In this review, I integrate information from recent studies on Wnt-expressing cells and intestinal fibroblast lineages and discuss their roles in homeostasis and wound healing. More information on the lineages of Wnt-expressing cells will help clarify the mechanisms of epithelial tissue formation.
{"title":"Wnt-expressing cells in the intestines: guides for tissue remodeling","authors":"H. Miyoshi","doi":"10.1093/jb/mvw070","DOIUrl":"https://doi.org/10.1093/jb/mvw070","url":null,"abstract":"The crypt is a minimal functional unit in the intestinal epithelium. This unique structure is maintained by surrounding mesenchymal cells that focally interact with associated epithelial cells. Canonical and non-canonical Wnt ligands enable specific microenvironments localized to each end of the crypt major axis. While canonical Wnt-expressing cells are localized near the crypt bottom where intestinal stem cells reside, non-canonical Wnt-expressing cells are positioned beneath the luminal surface of epithelial cells. During wound healing, propagation and appropriate relocation of each cell population are thought to ensure subsequent crypt regeneration. In this review, I integrate information from recent studies on Wnt-expressing cells and intestinal fibroblast lineages and discuss their roles in homeostasis and wound healing. More information on the lineages of Wnt-expressing cells will help clarify the mechanisms of epithelial tissue formation.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"199 1","pages":"19–25"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76957316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wnt/&bgr;-catenin signaling is activated repeatedly during an animal’s lifespan, and it controls gene expression through its essential nuclear effector, &bgr;-catenin, to regulate embryogenesis, organogenesis, and adult homeostasis. Although the &bgr;-catenin transcriptional complex has the ability to induce the expression of many genes to exert its diverse roles, it chooses and transactivates a specific gene set from among its numerous target genes depending on the context. For example, the &bgr;-catenin transcriptional complex stimulates the expression of cell cycle-related genes and consequent cell proliferation in neural progenitor cells, while it promotes the expression of neural differentiation-related genes in differentiating neurons. Recent studies using animal and cell culture models have gradually improved our understanding of the molecular basis underlying such context-dependent actions of the &bgr;-catenin transcriptional complex. Here, we describe eight mechanisms that support &bgr;-catenin-mediated context-dependent gene regulation, and their spatio-temporal regulation during vertebrate development. In addition, we discuss their contribution to the diverse functions of Wnt/&bgr;-catenin signaling.
{"title":"Context-dependent regulation of the &bgr;-catenin transcriptional complex supports diverse functions of Wnt/&bgr;-catenin signaling","authors":"Takamasa Masuda, T. Ishitani","doi":"10.1093/jb/mvw072","DOIUrl":"https://doi.org/10.1093/jb/mvw072","url":null,"abstract":"Wnt/&bgr;-catenin signaling is activated repeatedly during an animal’s lifespan, and it controls gene expression through its essential nuclear effector, &bgr;-catenin, to regulate embryogenesis, organogenesis, and adult homeostasis. Although the &bgr;-catenin transcriptional complex has the ability to induce the expression of many genes to exert its diverse roles, it chooses and transactivates a specific gene set from among its numerous target genes depending on the context. For example, the &bgr;-catenin transcriptional complex stimulates the expression of cell cycle-related genes and consequent cell proliferation in neural progenitor cells, while it promotes the expression of neural differentiation-related genes in differentiating neurons. Recent studies using animal and cell culture models have gradually improved our understanding of the molecular basis underlying such context-dependent actions of the &bgr;-catenin transcriptional complex. Here, we describe eight mechanisms that support &bgr;-catenin-mediated context-dependent gene regulation, and their spatio-temporal regulation during vertebrate development. In addition, we discuss their contribution to the diverse functions of Wnt/&bgr;-catenin signaling.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"73 1","pages":"9–17"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86348661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Y. Kawakami, Mai Kinoshita, Y. Mori, Shuji Okochi, Shiori Hirano, Ichika Shimoda, Keita Kanzaki, T. Suzuki-Yamamoto, M. Kimoto, M. Sugahara, T. Hori, H. Saino, M. Miyano, Shozo Yamamoto, Yoshitaka Takahashi
The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4. Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4. The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.
{"title":"The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4","authors":"Y. Kawakami, Mai Kinoshita, Y. Mori, Shuji Okochi, Shiori Hirano, Ichika Shimoda, Keita Kanzaki, T. Suzuki-Yamamoto, M. Kimoto, M. Sugahara, T. Hori, H. Saino, M. Miyano, Shozo Yamamoto, Yoshitaka Takahashi","doi":"10.1093/jb/mvw055","DOIUrl":"https://doi.org/10.1093/jb/mvw055","url":null,"abstract":"The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4. Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4. The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"52 1","pages":"79–86"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90284185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yan Zhang, Chunyu Yang, A. Dancis, Eiko Nakamaru-Ogiso
Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe–2S]) cluster and one tetranuclear ([4Fe–4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe–2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe–4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe–4S] cluster (gx,y,z = 1.830, 1.947 and 2.018) and for the [2Fe–2S] cluster (gx,y,z =1.919, 1.962 and 2.001). We also observed spin–spin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe–4S] cluster converting to a [2Fe–2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys Fe–S cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.
{"title":"EPR studies of wild type and mutant Dre2 identify essential [2Fe–-2S] and [4Fe–-4S] clusters and their cysteine ligands","authors":"Yan Zhang, Chunyu Yang, A. Dancis, Eiko Nakamaru-Ogiso","doi":"10.1093/jb/mvw054","DOIUrl":"https://doi.org/10.1093/jb/mvw054","url":null,"abstract":"Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe–2S]) cluster and one tetranuclear ([4Fe–4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe–2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe–4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe–4S] cluster (gx,y,z = 1.830, 1.947 and 2.018) and for the [2Fe–2S] cluster (gx,y,z =1.919, 1.962 and 2.001). We also observed spin–spin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe–4S] cluster converting to a [2Fe–2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys Fe–S cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"28 1","pages":"67–78"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82168416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ami Sotokawauchi, M. Kato‐Murayama, K. Murayama, T. Hosaka, I. Maeda, M. Onjo, N. Ohsawa, D. Kato, K. Arima, M. Shirouzu
Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the &agr;–&bgr; sandwich motif with four-stranded antiparallel &bgr;-sheets, two helices and a strand of the C-terminal region. The &bgr;-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.
{"title":"Structural basis of cucumisin protease activity regulation by its propeptide","authors":"Ami Sotokawauchi, M. Kato‐Murayama, K. Murayama, T. Hosaka, I. Maeda, M. Onjo, N. Ohsawa, D. Kato, K. Arima, M. Shirouzu","doi":"10.1093/jb/mvw053","DOIUrl":"https://doi.org/10.1093/jb/mvw053","url":null,"abstract":"Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the &agr;–&bgr; sandwich motif with four-stranded antiparallel &bgr;-sheets, two helices and a strand of the C-terminal region. The &bgr;-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"99 1","pages":"45–53"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79260639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Takada, S. Fujimori, Takuma Shinozuka, R. Takada, Yusuke Mii
During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.
{"title":"Differences in the secretion and transport of Wnt proteins","authors":"S. Takada, S. Fujimori, Takuma Shinozuka, R. Takada, Yusuke Mii","doi":"10.1093/jb/mvw071","DOIUrl":"https://doi.org/10.1093/jb/mvw071","url":null,"abstract":"During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"54 1","pages":"1–7"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73682757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mohammad Ziaur Rahman, Megumi Maeda, Satsuki Itano, Md. Anowar Hossain, T. Ishimizu, Y. Kimura
In this study, we identified a gene in tomato that encodes an acidic α-fucosidase (LOC101254568 or Solyc03g006980, α-Fuc'ase S1-1), which may be involved in the turnover of plant complex-type N-glycans. Recombinant α-Fuc'ase S1-1 (rFuc'ase S1-1) was expressed using a baculovirus-insect cell expression system. rFuc'ase Sl-1 is 55 kDa in size and has an optimum pH around 4.5. It substantially hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Lea epitopes on plant complex-type N-glycans, but not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylaminated Fucα1-3GlcNAc. Furthermore, we found that this tomato α-Fuc'ase S1-1 was inactive toward the core penta-oligosaccharide unit [Manβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA] of plant complex-type N-glycans. Molecular 3D modelling of α-Fuc'ase Sl-1 and structure/sequence interpretation based on comparison with a homologous α-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) indicated that residues Asp193 and Glu237 might be important for substrate binding.
{"title":"Molecular characterization of tomato &agr;1,3/4-fucosidase, a member of glycosyl hydrolase family 29 involved in the degradation of plant complex type N-glycans","authors":"Mohammad Ziaur Rahman, Megumi Maeda, Satsuki Itano, Md. Anowar Hossain, T. Ishimizu, Y. Kimura","doi":"10.1093/jb/mvw089","DOIUrl":"https://doi.org/10.1093/jb/mvw089","url":null,"abstract":"In this study, we identified a gene in tomato that encodes an acidic α-fucosidase (LOC101254568 or Solyc03g006980, α-Fuc'ase S1-1), which may be involved in the turnover of plant complex-type N-glycans. Recombinant α-Fuc'ase S1-1 (rFuc'ase S1-1) was expressed using a baculovirus-insect cell expression system. rFuc'ase Sl-1 is 55 kDa in size and has an optimum pH around 4.5. It substantially hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Lea epitopes on plant complex-type N-glycans, but not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylaminated Fucα1-3GlcNAc. Furthermore, we found that this tomato α-Fuc'ase S1-1 was inactive toward the core penta-oligosaccharide unit [Manβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA] of plant complex-type N-glycans. Molecular 3D modelling of α-Fuc'ase Sl-1 and structure/sequence interpretation based on comparison with a homologous α-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) indicated that residues Asp193 and Glu237 might be important for substrate binding.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"20 1","pages":"421–432"},"PeriodicalIF":0.0,"publicationDate":"2016-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83336318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ananthanarayanan Kumar, M. Isumi, M. Sakuma, Shiwei Zhu, Yuuki Nishino, Yasuhiro Onoue, S. Kojima, Y. Miyanoiri, K. Imada, M. Homma
The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.
{"title":"Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus","authors":"Ananthanarayanan Kumar, M. Isumi, M. Sakuma, Shiwei Zhu, Yuuki Nishino, Yasuhiro Onoue, S. Kojima, Y. Miyanoiri, K. Imada, M. Homma","doi":"10.1093/jb/mvw076","DOIUrl":"https://doi.org/10.1093/jb/mvw076","url":null,"abstract":"The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"2 1","pages":"331–337"},"PeriodicalIF":0.0,"publicationDate":"2016-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82343376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains.
{"title":"The emerging complexity of ubiquitin architecture","authors":"F. Ohtake, Hikaru Tsuchiya","doi":"10.1093/jb/mvw088","DOIUrl":"https://doi.org/10.1093/jb/mvw088","url":null,"abstract":"Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"1 1","pages":"125–133"},"PeriodicalIF":0.0,"publicationDate":"2016-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81897454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}