A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

Morokuma, Daisuke,Hino, Masato,Tsuchioka, Miho,Masuda, Akitsu,Mon, Hiroaki,Fujiyama, Kazuhito,Kajiura, Hiroyuki,Kusakabe, Takahiro,Lee, Jae Man Korean Society of Sericultural Science 2018 International Journal of Industrial Entomology Vol.36 No.1

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.

ONIOM and Its Applications to Material Chemistry and Catalyses

Morokuma, Keiji Korean Chemical Society 2003 Bulletin of the Korean Chemical Society Vol.24 No.6

One of the largest challenges for quantum chemistry today is to obtain accurate results for large complex molecular systems, and a variety of approaches have been proposed recently toward this goal. We have developed the ONIOM method, an onion skin-like multi-level method, combining different levels of quantum chemical methods as well as molecular mechanics method. We have been applying the method to many different large systems, including thermochemistry, homogeneous catalysis, stereoselectivity in organic synthesis, solution chemistry, fullerenes and nanochemistry, and biomolecular systems. The method has recently been combined with the polarizable continuum model (ONIOM-PCM), and was also extended for molecular dynamics simulation of solution (ONIOM-XS). In the present article the recent progress in various applications of ONIOM and other electronic structure methods to problems of homogeneous catalyses and nanochemistry is reviewed. Topics include 1. bond energies in large molecular systems, 2. organometallic reactions and homogeneous catalysis, 3. structure, reactivity and bond energies of large organic molecules including fullerenes and nanotubes, and 4. biomolecular structure and enzymatic reaction mechanisms.

Neurological outcomes and their correlation to fetal behavior patterns

( Seiichi Morokuma ) 대한산부인과학회 2019 대한산부인과학회 학술대회 Vol.105 No.-

According to Konrad Lorenz (1935), it is feasible to make valid assumptions about brain function from observed behavior. The development of fetal behavior correlates with the developmental process of the anatomical neural network. During the last three decades, real-time ultrasound has facilitated in vivo observations of human fetal movements. In line with Lorenz’s viewpoint, we aimed at describing the ontogeny of fetal behavior. This was accomplished not only by observing the developmental process of each movement along with the progress in gestation but also through the investigation of various movements in relation to or concurrent with one another, to gradually become integrated into more complex movements. Thus, one could, through direct observation of fetal movements, predict whether the nervous system is developing properly. Several investigators have suggested that anatomical defects manifest as functional defects. To identify fetuses at high risk for poor neurological outcomes, we first assessed a novel ultrasound evaluation system based on our findings of fetal behavior. This system screened for decrease or lack of fetal movements, abnormal patterns of fetal heart rate, congenital CNS malformations, polyhydramnios of unknown cause, and ultrasound evaluation of fetal brain functions, including movement of extremities, breathing movements, ultradian rhythm, and REM and NREM sleep periods. We then assessed the correlations between fetal brain functions and neurological outcomes in infancy (mental retardation, cerebral palsy, and low developmental quotient). During screening, we prospectively evaluated 4978 fetuses receiving prenatal and intrapartum management in our hospital that were delivered between 32 and 41 weeks’ gestation and identified 93 cases with suspected impairment. Of the 93 fetuses, 26 underwent the second step of ultrasound examination at 35-40 weeks’ gestation. Our findings revealed that this method was adequately sensitive (80%) and specific (88%) in identifying neurological impairment. We concluded that this method was mainly useful in a clinical setting for establishing fetal CNS examination of the initial signs for functional impairment, rendering it suitable for clinical applications.

A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

( Daisuke Morokuma ),( Masato Hino ),( Miho Tsuchioka ),( Akitsu Masuda ),( Hiroaki Mon ),( Kazuhito Fujiyama ),( Hiroyuki Kajiura ),( Takahiro Kusakabe ),( Jae Man Lee ) 한국잠사학회 2018 International Journal of Industrial Entomology Vol.36 No.1

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.

Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system

Daisuke Morokuma,Jian Xu,Hiroaki Mon,Kazuma Hirata,Masato Hino,Shoko Kuboe,Mami Yamashita,Takahiro Kusakabe,이재만 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.2

Glycosylation is an important post-translational modification that confers various biological activities, structural stability, and inter-molecular interactions to proteins. Baculovirus expression vector system (BEVS) is widely used to produce recombinant glycoproteins, which may not be suitable for clinical use due to differences in the N-linked glycan structure between insects and mammals. It is necessary to develop an appropriate model protein-base platform for glycoanalysis to engineer the insect-type N-glycosylation pathway into human type efficiently. In this study,we employed human plasma protein alpha 1-acid glycoprotein (α1AGP). Itwas highly secreted from cultured silkworm cells and larvae when using the BEVS and glycosylated with insect type N-linked glycans. Interestingly, when separated on SDS-PAGE, the purified recombinant α1AGP secreted into silkworm haemolymph generated six distinct products from three alternative translates, suggesting that α1AGP has variations for the recognition or choice of glycosylation sites.

Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm–baculovirus protein expression system

Atsushi Masuda,Jian Xu,TakumiMitsudome,Daisuke Morokuma,Hiroaki Mon,Yutaka Banno,Takahiro Kusakabe,이재만 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.2

Endo-β-N-acetylglucosaminidase H (Endo H) catalyzes cleavage between the GlcNAc residues of the chitobiose core of N-linked glycans, leaving one GlcNAc residues attached to asparagine. Endo H cleaves high mannose and hybrid, but not complex, N-linked oligosaccharides on glycoproteins. Because of its unique specificity, Endo H is widely used for the structural and functional analyses of glycoproteins. In our previous study, the recombinant Endo H was produced as a secreted protein using silkworm–baculovirus expression system, but the yield was low (30 μg Endo H/10 ml larval hemolymph) compared to that of Escherichia coli. In this study, we purified active recombinant Endo H as an intracellular protein from fat body of silkworm infected with the recombinant baculovirus expressing Endo H without the exogenous signal peptide. Remarkably, the yield (9.3mgfrom20 silkwormlarvae)was about 310-fold higher than that secreted into larval hemolymph as reported previously. In addition,we screened the silkwormstrains maintained in Kyushu University and identified n17 as a high-level expression strain for Endo H.

High-level expression and purification of biologically active human IL-2 using silkworm-baculovirus expression vector system

Masato Hino,Takuji Kawanami,Jian Xu,Daisuke Morokuma,Kazuma Hirata,Mami Yamashita,Noriko Karasaki,Tuneyuki Tatsuke,Hiroaki Mon,Kazuhiro Iiyama,Noriho Kamiya,Yutaka Banno,Takahiro Kusakabe,이재민 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.2

Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4+ T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 ismore suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli-derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2.

THEORY–EXPERIMENT RELATIONSHIP OF THE "SHRINKING HOT GIANT" ROAD OF DYNAMIC FULLERENE SELF-ASSEMBLY IN HOT CARBON VAPOR

Z. WANG,S. IRLE,G. ZHENG,K. MOROKUMA 성균관대학교(자연과학캠퍼스) 성균나노과학기술원 2007 NANO Vol.2 No.1

Though subject to intensive studies, the formation mechanism of buckminsterfullerene C60 and related higher fullerenes has long evaded discovery. To elucidate their atomistic self-assembly mechanism, we have performed high-temperature quantum chemical molecular dynamics simulations on carbon vapor model systems initially consisting of C2 molecules. Our simulations reveal a coherent mechanism how highly ordered fullerene cages naturally self-assemble under nonequilibrium conditions, following a series of irreversible processes from the polymerization of C2 molecules to vibrationally excited giant fullerenes, which then shrink by C2 evaporation down to the smallest spherical, isolated pentagon rule obeying species C70 and C60 as the smallest and kinetically most stable species of the shrinking process. We show that the potential energy surface associated with giant fullerene cage growth, measured by an average cluster curvature, is downhill all the way, and in agreement with high-level energetics from density functional theory. This fullerene formation mechanism is a good example of dynamic self-assembly leading to dissipative structures far from thermodynamic equilibrium, and the "shrinking hot giant" road provides a natural explanation for the observed cage size distributions in a random optimization process consistent with several important experimental observations.

Expression, purification, and characterization of highly active endo-α-Nacetylgalactosaminidases expressed by silkworm-baculovirus expression system

Akihiro Morio,Jian Xu,Akitsu Masuda,Yurie Kinoshita,Masato Hino,Daisuke Morokuma,Hatsumi M. Goda,Nozomu Okino,Makoto Ito,Hiroaki Mon,Ryosuke Fujita,Takahiro Kusakabe,이재만 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2

The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial Oglycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.

Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system

Takumi Yano,이재만,Jian Xu,Yoshiki Morifuji,Akitsu Masuda,Masato Hino,Daisuke Morokuma,Ryosuke Fujita,Masateru Takahashi,Takahiro Kusakabe,Hiroaki Mon 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2

Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that Cterminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase.