Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
THASANEEYA KUBOKI Last modified date:2020.07.02

Assistant Professor / Laboratory of Biomedical and Biophysical Chemistry / Department of Applied Molecular Chemistry / Institute for Materials Chemistry and Engineering


Papers
1. Thasaneeya Kuboki, Hiroyuki Ebata, Tomoki Matsuda, Yoshiyuki Arai, Takeharu Nagai, and Satoru Kidoaki , Hierarchical development of motile polarity in durotactic cells just crossing an elasticity boundary, Cell Structure and Function, 2019.12.
2. Hiroyuki Ebata, Kousuke Moriyama, Thasaneeya Kuboki, Satoru Kidoaki, General cellular durotaxis induced with cell-scale heterogeneity of matrix-elasticity, Biomaterials, 2019.11, Stiffness-gradient-induced cellular taxis, so-called durotaxis, has been extensively studied on a substrate with a single broad or steep stiffness gradient. However, in actual living tissues, cells should sense cell-scaled heterogeneous elasticity distribution in the extracellular matrix. In this study, to clarify the effect of the cell-scale heterogeneity of matrix-elasticity on durotaxis, we examined the motility of different types of cells on microelastically-striped patterned gels with different cell-sized widths. We found that cells accumulated in stiff regions with specific width on cell-type-dependency, even when a stiffness gradient is too small to induce usual durotaxis with a monotonic stiffness gradient. Fibroblast cells accumulated in a wide stiff region of multicellular size, while mesenchymal stem cells localized in a narrow stiff region of single-cell size. It was revealed that durotactic activity is critically affected not only with the cell type but also with the cell-scale heterogeneity of matrix-elasticity. Based on the shape-fluctuation-based analysis of cell migration, the dynamics of the pseudopodia were found to play a key role in determining the behaviors of general durotaxis. Our results suggest that design of cell-scale heterogeneity of matrix-elasticity is pivotal in controlling directional cell migration, the spontaneous cell-patterning, and development of the tissue on the biomaterials surfaces..
3. Hiroyuki Ebata, Aki Yamamoto, Yukie Tsuji, Saori Sasaki, Kousuke Moriyama, Thasaneeya Kuboki & Satoru Kidoaki, Persistent random deformation model of cells crawling on a gel surface. , Sci Rep, 8, 2018.03, In general, cells move on a substrate through extension and contraction of the cell body. Though cell
movement should be explained by taking into account the efect of such shape fuctuations, past
approaches to formulate cell-crawling have not sufciently quantifed the relationship between cell
movement (velocity and trajectory) and shape fuctuations based on experimental data regarding actual
shaping dynamics. To clarify this relationship, we experimentally characterized cell-crawling in terms of
shape fuctuations, especially extension and contraction, by using an elasticity-tunable gel substrate to
modulate cell shape. As a result, an amoeboid swimmer-like relation was found to arise between the cell
velocity and cell-shape dynamics. To formulate this experimentally-obtained relationship between cell
movement and shaping dynamics, we established a persistent random deformation (PRD) model based
on equations of a deformable self-propelled particle adopting an amoeboid swimmer-like velocityshape
relationship. The PRD model successfully explains the statistical properties of velocity, trajectory
and shaping dynamics of the cells including back-and-forth motion, because the velocity equation
exhibits time-reverse symmetry, which is essentially diferent from previous models. We discuss the
possible application of this model to classify the phenotype of cell migration based on the characteristic
relation between movement and shaping dynamics..
4. Kantawong F, Saksiriwisitkul C, Riyapa C, Limpakdee S, Wanachantararak P, Kuboki T, Reprogramming of mouse fibroblasts into neural lineage cells using biomaterials., Bioimpact, 8, c, 129-138, 2018.01.
5. Eiji Usukura, Yuhki Yanase, Ayumi Ishijima, Thasaneeya Kuboki, Satoru Kidoaki, Koichi Okamoto, Kaoru Tamada, LSPR-mediated high axial-resolution fluorescence imaging on a silver nanoparticle sheet, PLOSONE, 12, 2017.12.
6. Masuda S, Yanase Y, Usukura E, Sou Ryuzaki, Wang P, K. Okamoto, Kuboki T, Satoru Kidoaki, Kaoru Tamada, High-resolution imaging of a cell-attached nanointerface using a gold-nanoparticle two-dimensional sheet, Sci Reports, 2017.09.
7. Kantawong F, Singhatong S, Srilamay A, Boonyuen K, Mooti N, Wanachantararak P, Kuboki T,, Properties of macerated herbal oil, Bioimpact, 6, 7(1), 13-23, 2017.02.
8. Shimada N, Saito N, Shukuri S, Kuroyanagi S, Kuboki T, Satoru Kidoaki, Nagai T, Maruyama A, Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers, ACS Appl. Mater. Interfaces, 8, 46, 31524-31529, 2016.11.
9. Kuboki T, Satoru Kidoaki, Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture., Methods in Molecular Biology, 425-441, 2016.05, Surface elasticity or stiffness of an underlying substrate may regulate cellular functions such as adhesion, proliferation, signaling, differentiation, and migration. Recent studies have reported on the development of biomaterials to control stem cell fate determination via the stiffness of the culture substrates. In this chapter, we provide a detailed protocol for fabricating elasticity-tunable gelatinous hydrogels for stem cell culture with photo-induced or thermo-induced crosslinking of well-developed styrenated gelatin (StG). We also include the detailed application of gelatinous gel for mesenchymal stem cell (MSC) culture and sample collection for transcriptional and proteomic analysis..
10. Kantawong F, Kuboki T, Kidoaki S, Redox gene expression of adipose-Derived stem cells in response to soft hydrogel, Turkish Journal of Biology, 39, 682-691, 2015.08, Adipose-derived stem cells (ADSCs) showed morphological change to a neuron-like shape, and presented neuronal lineage bias when cultured on a very soft surface. To gain basic insight into gene expression relating to neuronal lineage bias in ADSCs, we examined the correlation between the gene expression levels of neuronal markers and redox proteins that were considered to have a close relation with lineage specification in stem cells. ADSCs were cultured on gelatinous soft hydrogel for 1-2 weeks. The time course changes in the expressions of neuronal genes (TUBB3 and NSE) and redox genes (TRX1, SOD1, SOD2, PRX2, GSTT1, and GSTP1) were monitored using real-time PCR. It was found that the TUBB3 gene had significantly upregulated compared to the control condition for tissue culture polystyrene, indicating that the neuronal gene expression of ADSCs could be achieved on soft hydrogel without the addition of any supplement. The expressions of the TRX1 and SOD1 genes were also observed to have significantly upregulated on the soft hydrogels. The results demonstrate that the upregulation of the neural marker of TUBB3 in the ADSCs correlates well with the upregulation of the redox genes of TRX1 and SOD1, when cultured on appropriate soft hydrogel substrates..
11. Kuboki T, Chen W, Kidoaki S, Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band., Langmuir, 10.1021/la501058j, 30, 6187-6196, 2014.05, Durotaxis, biased cell movement up a stiffness gradient on culture substrates, is one of the useful taxis behaviors for manipulating cell migration on engineered biomaterial surfaces. In this study, long-term durotaxis was investigated on gelatinous substrates containing a soft band of 20, 50, and 150 μm in width fabricated using photolithographic elasticity patterning; sharp elasticity boundaries with a gradient strength of 300 kPa/50 μm were achieved. Time-dependent migratory behaviors of 3T3 fibroblast cells were observed during a time period of 3 days. During the first day, most of the cells were strongly repelled by the soft band independent of bandwidth, exhibiting the typical durotaxis behavior. However, the repellency by the soft band diminished, and more cells crossed the soft band or exhibited other mixed migratory behaviors during the course of the observation. It was found that durotaxis strength is weakened on the substrate with the narrowest soft band and that adherent affinity-induced entrapment becomes apparent on the widest soft band with time. Factors, such as changes in surface topography, elasticity, and/or chemistry, likely contributing to the apparent diminishing durotaxis during the extended culture were examined. Immunofluorescence analysis indicated preferential collagen deposition onto the soft band, which is derived from secretion by fibroblast cells, resulting in the increasing contribution of haptotaxis toward the soft band over time. The deposited collagen did not affect surface topography or surface elasticity but did change surface chemistry, especially on the soft band. The observed time-dependent durotaxis behaviors are the result of the mixed mechanical and chemical cues. In the studies and applications of cell migratory behavior under a controlled stimulus, it is important to thoroughly examine other (hidden) compounding stimuli in order to be able to accurately interpret data and to design suitable biomaterials to manipulate cell migration..
12. Umemiya-Shirafuji R, Boldbaatar D, Liao M, Battur B, Rahman MM, KUBOKI T, Galay RL, Tanaka T, Fujisaki K, Target of rapamycin (TOR) controls vitellogenesis via activation of the S6 kinase in the fat body of the tick, Haemaphysalis longicornis. Int J Parasitol. , Int J Parasitol, 42, 11, 991-998, 2012.10, Vitellogenin (Vg) synthesis, vitellogenesis, is an essential process for the development and reproduction of ticks. Our previous finding led to the hypothesis that target of rapamycin (TOR) pathway is important for vitellogenesis in the hard tick, Haemaphysalis longicornis. The TOR pathway controls cellular activity according to nutrient availability in eukaryotes. TOR, a member of the phosphatidylinositol 3-kinase family, is a central player in this pathway. Here, we present preliminary evidence that H. longicornis TOR (HlTOR) controls vitellogenesis via activation of S6 kinase (S6K) in the fat body. RNA interference (RNAi)-mediated gene silencing of HlTOR was undertaken to elucidate the involvement of HlTOR in the vitellogenesis of the tick. HlTOR-RNAi caused inhibition of S6K phosphorylation in the fat body. HlTOR-RNAi also altered not only the expression levels of GATA mRNA and protein but also the intracellular localisation of GATA in the fat body. The expression levels of Vg mRNA and protein in the fat body of HlTOR-RNAi ticks were significantly lower than those in control ticks. In the pre-ovipositional stage, the ovaries of control ticks had brown oocytes developing, but those of HlTOR-RNAi ticks were white and immature. The haemolymph colour indicated that the amount of Vg was lower in HlTOR-RNAi ticks than in the controls. Furthermore, rapamycin inhibited S6K phosphorylation and reduced the expression levels of Vg mRNA and protein in the fat bodies. Vg proteins were not detected in rapamycin-treated fat bodies in the presence of 20-hydroxyecdysone. These results suggest that HlTOR activity is critical for vitellogenesis stimulated by 20-hydroxyecdysone..
13. Kuboki T, Kantawong F, Burchmore R, Dalby MJ, Kidoaki S, 2D-DIGE proteomic analysis of mesenchymal stem cell cultured on the elasticity-tunable hydrogels., Cell Structure and Function, 37, 127-139, 2012.08, The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signaling-related proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and β-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity..
14. Liao M, Boldbaatar D, Gong H, Huang P, Umemiya R, Harnnoi T, Zhou J, Tanaka T, Suzuki H, Xuan X, Fujisaki K, Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks. , Insect Biochem Mol Biol, 38, 3, 285-295, 2008.03, Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods..
15. Gong H, Zhou J, Liao M, Hatta T, Harnnoi T, Umemiya R, Inoue N, Xuan X, Fujisaki K, Characterization of a carboxypeptidase inhibitor from the tick Haemaphysalis longicornis, J Insect Physiol, 53, 10, 1079-1087, 2007.10, A carboxypeptidase inhibitor called HlTCI was isolated from Haemaphysalis longicornis in this study. The full-length cDNA of HlTCI contains an open reading frame (ORF) of 291bp, encoding 96 amino acid residues consisting of a predicted 19-residue signal peptide and a putative mature 77-residue protein. The expected mature protein is cysteine-rich and has 12 cysteine residues assumed to construct six disulfide bridges. The deduced peptide sequence shows 63.9% homology to the carboxypeptidase inhibitor from another ixodid tick, Rhipicephalus bursa. Reverse-transcription PCR (RT-PCR) indicated that HlTCI was specifically expressed in the ovary from partially engorged adult ticks. The recombinant protein of HlTCI (rHlTCI) with glutathione S-transferase (GST) was expressed in Escherichia coli strain BL21 (DE3) and purified by glutathione-Sepharose 4B beads. rHlTCI showed inhibitory activity against digestive metallocarboxypeptidases A and B, but the activity was affected by the increase of the temperature treatment. High concentrations of rHlTCI were shown to significantly accelerate fibrinolysis in vitro. This effect of rHlTCI on clot lysis suggests its promising potential for use in some thrombotic disorders.
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16. Harnnoi T, Sakaguchi T, Nishikawa Y, Xuan X, Fujisaki K, Molecular characterization and comparative study of 6 salivary gland metalloproteases from the hard tick, Haemaphysalis longicornis. , Comp Biochem Physiol B Biochem Mol Biol, 147(1), 1, 93-101, 2007.05, Six genes encoding metalloproteases were identified from the salivary gland of the hard tick, Haemaphysalis longicornis. Comparative analyses have shown the evolutionary distinct and different mRNA expression patterns of each gene during blood feeding. The proteins are synthesized as proenzymes with a prodomain and a metalloprotease/cysteine-rich domain of the reprolysin family. Within the active site, amino acid substitutions were observed. The recombinant Escherichia coli expression of one gene, hlESTMP1, was performed. The immunoblot analysis and indirect fluorescent assay using anti-hlESTMP1 suggested that this protein is mainly expressed in the cytoplasm of the salivary glands and only the mature form of 34 kDa was detectable. The proenzyme expressed by baculovirus was processed into a mature domain, suggesting that proenzyme activation possibly occurs through a pro-protein convertase dependent pathway. The presence of these diverse enzymes might contribute to the greater functional complexity of bioactive molecules in tick saliva to facilitate blood feeding..
17. Jariyapan N, Choochote W, Jitpakdi A, Harnnoi T, Siriyasatein P, Wilkinson MC, Junkum A, Bates PA, Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae), Rev Inst Med Trop Sao Paulo , 49 , 1, 5-10, 2007.01, Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy..
18. Harnnoi T, Watchabunsook S, Sakaguchi T, Xuan X, Fujisaki K, Characterization of Haemaphysalis longicornis recombinant cement-like antigens and preliminary study of their vaccination effects., J Vet Med Sc, 68, 12, 1289-1295, 2006.12, Two genes encoding immunodominant antigens, hlim2 and hlim3, were obtained from a salivary gland cDNA library of the hard tick, Haemaphysalis longicornis. The recombinant proteins were expressed in Escherichia coli as the GST fusion protein and used for immunization. We observed that the attachment rate of nymphal ticks fed on mice immunized with GST-hlim3 was significantly lower than that in the control group during the initial days of feeding. However, immunization with GST-hlim3 did not affect the engorgement rate of the ticks. In sharp contrast, GST-hlim2 did not influence the attachment rate and feeding period of ticks but had a significant reduction in the engorgement body weight. These data highlight the suitability of the 2 recombinant cement-like proteins for use in a cocktail vaccine..
19. Thasaneeya Harnnoi, Songwut Watchabunsook, Takeshi Sakaguchi, Xuenan Xuan, Kozo Fujisaki, Characterization of Haemaphysalis longicornis recombinant cement-like antigens and preliminary study of their vaccination effects, Journal of Veterinary Medical Science, 68, 12, 1289-95, 2006.12, Two genes encoding immunodominant antigens, hlim2 and hlim3, were obtained from a salivary gland cDNA library of the hard tick, Haemaphysalis longicornis. The recombinant proteins were expressed in Escherichia coli as the GST fusion protein and used for immunization. We observed that the attachment rate of nymphal ticks fed on mice immunized with GST-hlim3 was significantly lower than that in the control group during the initial days of feeding. However, immunization with GST-hlim3 did not affect the engorgement rate of the ticks. In sharp contrast, GST-hlim2 did not influence the attachment rate and feeding period of ticks but had a significant reduction in the engorgement body weight. These data highlight the suitability of the 2 recombinant cement-like proteins for use in a cocktail vaccine..
20. Harnnoi T, Sakaguchi T, Xuan X, Fujisaki K, Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis. , J Vet Med Sci, 68, 11, 1155-1160, 2006.11, A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine..
21. Narissara Jariyapan, Wej Choochote, Atchariya Jitpakdi, Thasaneeya Harnnoi, Padet Siriyasatein, Mark C. Wilkinson, Paul A. Bates, A glycine- and glutamate-rich protein is female salivary gland-specific and abundant in the malaria vector Anopheles dirus B (Diptera
Culicidae), Journal of Medical Entomology, 10.1603/0022-2585(2006)43[867:AGAGPI]2.0.CO;2, 43, 5, 867-874, 2006.11, Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton & Harrison (=An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of ≈4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding..
22. Thasaneeya Harnnoi, Takeshi Sakaguchi, Xuenan Xuan, Kozo Fujisaki, Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis, Journal of Veterinary Medical Science, 68, 11, 1155-60, 2006.11, A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine..
23. Narissara Jariyapan, Wej Choochote, Atchariya Jitpakdi, Thasaneeya Harnnoi, Padet Siriyasatein, Mark C Wilkinson, Paul A Bates, A glycine- and glutamate-rich protein is female salivary gland-specific and abundant in the malaria vector Anopheles dirus B (Diptera
Culicidae), Journal of Medical Entomology, 43, 5, 867-74, 2006.09, Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton and Harrison (= An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of approximately 4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding..
24. Jantana Wongsantichon, Thasaneeya Harnnoi, Albert J. Ketterman, A sensitive core region in the structure of glutathione S-transferases, Biochemical Journal, 10.1042/BJ20030394, 373, 3, 759-765, 2003.08, A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, sitedirected mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25°C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater Km towards GSH and a differing Km towards CDNB. The Vmax ranged from 44.7 to 87.0 μmol/min per mg (wild-type, 44.7 μmol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit-subunit interface. Finally, the data suggest that Leu103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability..
25. Aaron J. Oakley, Thasaneeya Harnnoi, Rungrutai Udomsinprasert, Kanya Jirajaroenrat, Albert J. Ketterman, Matthew C.J. Wilce, The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B, Protein Science, 10.1110/ps.21201, 10, 11, 2176-2185, 2001.11, Glutathione S-transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1-3 (complexed with glutathione) and AdGST1-4 (apo form), at 1.75 and 2.45 Å resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C-terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione-binding helix α2 and flanking residues are disordered in the AdGST1-4 (apo) structure, yet ordered in the AdGST1-3 (GSH-bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi- and human pi-class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1-4 and AdGST1-3 have different conformations..
26. P. Uparanukraw, N. Morakote, T. Harnnoi, A. Dantrakool, Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum, Parasitology Research, 10.1007/s004360100440, 87, 9, 751-757, 2001.08, The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33-39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory-secretory products of aL3..