Shigeki Inumaru

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-typeN-glycans, the most frequent structure of insectN-glycan is the paucimannosidic type, Man3GlcNAc2(±Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated β-N-acetylglucosaminidase (GlcNAcase)-mediated removal ofN-acetylglucosamine residues from the precursorN-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion ofN-acetylglucosamine residues from intermediateN-glycans is critical for the assembly of paucimannosidicN-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus.

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 microg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 microg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.

Production of biologically active, heterodimeric porcine interleukin-12 using a monocistronic baculoviral expression system.

A baculoviral expression system for the production of biologically active, heterodimeric interleukin (IL)-12 was developed by utilizing foot-and-mouth disease virus (FMDV) self-cleaving peptide, 2A. Recombinant porcine IL-12 (rpoIL-12) was produced by insect cells after infection with recombinant baculoviruses expressing the gene encoding a fusion protein of p35 and p40 subunits of IL-12 connected with 2A. By reducing and non-reducing SDS-PAGE analyses, it was demonstrated that rpoIL-12 had a heterodimeric structure which was resulted from 2A-dependent cleavage of the precursor fusion protein. In contrast, uncleaved, monomeric rpoIL-12 was produced by infection with baculoviruses expressing the gene lacking the 2A sequence. To assess the biological activities of these recombinants, we performed the proliferation assays of PHA-activated human PBMCs. The heterodimeric rpoIL-12 induced proliferation in a dose-dependent manner, whereas the uncleaved rpoIL-12 did not. Moreover, such biological activity was specifically inhibited by addition of anti-IL-12 antibodies or rpoIL-12 p40. These observations suggest that FMDV 2A can exert its self-cleaving activity even in a heterologous system, and that biologically active, heterodimeric rpoIL-12 can be generated by monocistronic expression of the p35/p40 fusion gene in combination with the 2A sequence.

Efficient Production of Biologically Active Porcine Interleukin-18 by Coexpression with Porcine Caspase-1 Using a Baculovirus Expression System

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.

Molecular cloning and expression profile analysis of a novel β‐D‐N‐acetylhexosaminidase of domestic silkworm (Bombyx mori)

Lepidoptera such as the domestic silkworm (Bombyx mori) produce proteins modified with unsialylated, mannose‐rich moieties known as ‘high mannose‐type’N‐glycans. However, we observed that, under intrinsic acetylglucosaminidase (GlcNAcase)‐inhibited conditions, moth cells tend to synthesize different types of glycoform with sialic acid modification. To identify molecules essential to assemble Lepidoptera‐specific N‐glycans, we performed BLAST analysis on the silkworm genetic database and isolated the entire coding sequence of novel Bombyx GlcNAcase, BmGlcNAcase 2. This enzyme showed weak homology to currently known, lysosome‐associated eukaryotic hexosaminidases, but it revealed remarkable similarity with recently reported glycosyl hydrolases of Spodoptera and Bombyx. Interestingly, BmGlcNAcase 2 was found to be expressed in embryos and in certain tissues of molting larvae (i.e. ovary, fat bodies, mid‐intestine, skin), but not in pupae, suggesting its unique function in the carbohydrate metabolism of juvenile silkworm.

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda.

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.

Production of a monoclonal antibody that recognizes bovine stem cell factor (SCF) and its use in the detection and quantitation of native soluble bovine SCF in fetal bovine serum

Stem cell factor (SCF) is a pluritropic hematopoietic cytokine that acts predominantly on the proliferation and differentiation of hematopoietic progenitor cells. SCF has long been thought to be present in fetal bovine serum (FBS) as an endogenous factor that stimulates the growth of hematopoietic progenitor cells in FBS-supplemented cultures. To detect and quantitate bovine SCF in FBS, we produced a monoclonal antibody (mAb) by immunizing mice with recombinant soluble bovine SCF protein, which was expressed in insect cells by using a baculovirus system. Using the mAb, we purified native soluble bovine SCF from FBS by immunoaffinity chromatography. Western blot analysis revealed that the purified SCF protein had a molecular weight of 33 kDa. In addition, an enzyme-linked immunosorbent assay (ELISA) incorporating the mAb revealed that the levels of native soluble SCF in commercially available FBS were likely to be <100 pg/ml. These results suggest that the concentration of native soluble bovine SCF present in FBS may be insufficient to promote additive biologic effects on the growth of hematopoietic progenitor cells in FBS-supplemented cultures.

Effect of a single intrauterine administration of recombinant bovine interferon-τ on day 7 of the estrous cycle on the luteal phase length and blood profile in dairy cows

This study tested the effect of recombinant bovine interferon-tau (rboIFN-τ) on the length of estrous cycle, luteal lifespan and side effects of rboIFN-τ in the cow. A normal estrous cycle in six non-lactating cycling Holstein cows was observed (non-treated cycle), and either 2.0 mg of liposomalized rboIFN-τ (treated cycle) or bovine serum albumin (BSA; placebo cycle) was infused in the uterus on day 7 of the estrous cycle (day 0=day of ovulation). Rectal temperature, heart rate and respiratory rate were recorded and blood samples were collected before and after the treatments. The length of the estrous cycle and corpus luteum lifespan in rboIFN-τ treated cycles were not significantly different from those of the non-treated and placebo cycles. In contrast, the rboIFN-τ treatment caused a transient increase in rectal temperature and a decrease in the number of peripheral lymphocytes and neutrophils after the treatment.

Cloning of porcine interleukin (IL)-12 receptor β2 (IL-12Rβ2) gene and its application to a rapid biological assay for human/porcine IL-12

Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.

Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins

The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system.