A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans.

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using ß1,2-N-acetylglucosaminyltransferase I (MGAT1)-deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-ß-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR-Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVNLec1. We found that Sf-RVN and Sf-RVNLec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVNLec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVNLec1 cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.

Development of an orally-administrable tumor vasculature-targeting therapeutic using annexin A1-binding D-peptides.

We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.

Overcoming the blood-brain barrier by Annexin A1-binding peptide to target brain tumours.

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

Gallic Acid Inhibits Lipid Accumulation via AMPK Pathway and Suppresses Apoptosis and Macrophage-Mediated Inflammation in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease, sometimes ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Various hits including excessive hepatic steatosis, oxidative stress, apoptosis, and inflammation, contribute to NASH development. Gallic acid (GA), a natural polyphenol, was reported to exert a protective effect on hepatic steatosis in animal models, but the precise molecular mechanisms remain unclear. Here, we examined the effect of GA on hepatic lipid accumulation, apoptosis, and inflammatory response caused by hepatocyte-macrophage crosstalk. We demonstrated that GA attenuated palmitic acid (PA)-induced fat accumulation via the activation of AMP-activated protein kinase (AMPK) in HepG2 cells. GA also ameliorated cell viability and suppressed apoptosis-related gene expression and caspase 3/7 activity induced by PA and H2O2. In a co-culture of lipid-laden Hepa 1-6 hepatocytes and RAW 264 macrophages, GA reduced inflammatory mediator expression and induced antioxidant enzyme expression. These results indicate that GA suppresses hepatic lipid accumulation, apoptosis, and inflammation caused by the interaction between hepatocytes and macrophages. The potential effects of GA observed in our study could be effective in preventing NASH and its complications.

Effect of Cdx2 Polymorphism on the Relationship between Dietary Calcium Intake and Peak Bone Mass in Young Japanese Women.

Studies investigating the effect of the caudal-type homeobox protein 2 (Cdx2) polymorphism in the vitamin D receptor gene and calcium intake on bone mass have shown inconsistent results. This study investigated whether the effect of calcium intake on peak bone mass is affected by Cdx2 polymorphism in young Japanese women. A cross-sectional study of 500 young women was conducted. Dietary intake was assessed by the Food Frequency Questionnaire. The osteo sono-assessment index (OSI), assessed by the qualitative ultrasound method, was used as a bone mass index. The subjects were divided into two groups by the median calcium intake. The OSI was not different among Cdx2 genotypes and between calcium groups (p = 0.960, p = 0.191, respectively). The interaction between calcium and Cdx2 genotypes on the OSI approached significance (GG versus GA and AA genotypes, p = 0.092). The difference in the OSI between calcium groups was significant in the GG genotype (p = 0.028), but not in the GA or AA genotypes (p = 0.501, p = 0.306, respectively). Adjustment for covariates (body mass index and physical activity) did not change the results. In conclusion, the relationship between dietary calcium intake and peak bone mass may vary according to Cdx2 polymorphism.

Daidzein promotes the expression of oxidative phosphorylation- and fatty acid oxidation-related genes via an estrogen-related receptor α pathway to decrease lipid accumulation in muscle cells.

Estrogen-related receptor (ERR)α regulates genes involved in fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) in muscle. The soy isoflavone daidzein was reported to be a putative ERRα activator, but little is known about its effects on gene expression and FA metabolism. This study aimed to clarify whether daidzein affects FAO- and OXPHOS-related genes thereby modulating intracellular FA metabolism in muscle cells. For this purpose, we used the C2C12 murine muscle cell line. ERRα-expressing C2C12 myotubes were treated with 50 µM daidzein, and gene expression was examined. The expression of FAO genes such as pyruvate dehydrogenase kinase 4 (Pdk4) and acyl-coenzyme A dehydrogenase (Acadm) and that of OXPHOS genes such as ATP synthase F1 subunit beta (Atp5b) and cytochrome c (Cycs) was significantly increased by daidzein, and these effects were partially blocked by an ERRα inhibitor. Using a reporter assay, we showed that daidzein enhanced the promoter activity of these genes and that ERRα responsive elements in the promoter region were necessary for the action of daidzein. Finally, daidzein significantly decreased lipid accumulation in C2C12 myotubes associated with increased oxygen consumption. In conclusion, daidzein decreases lipid deposition in muscle cells by regulating the expression of genes related to FAO and OXPHOS via an ERRα-associated pathway at least in part. These results suggest that daidzein would be a beneficial tool to protect against various diseases caused by muscle lipotoxicity.

Associations between nutritional adequacy and insomnia symptoms in Japanese men and women aged 18-69 years: a cross-sectional study.

OBJECTIVES: Sleep and diet are important lifestyle factors for maintaining health. Although previous studies have suggested that sleep quality may be associated with specific nutrient and food intakes, the relationship between nutritional adequacy and sleep quality remains unclear. The purpose of this study was to examine the relationship between sleep quality (insomnia symptoms) and adequate nutrient intake among Japanese adults. DESIGN: Cross-sectional. SETTING: Nationwide population survey conducted in 2013. PARTICIPANTS: 1,997 participants (940 men and 1,057 women) aged 18-69 years. MEASUREMENTS: Insomnia symptoms were assessed using the Athens Insomnia Scale (AIS) and participants were classified into three groups (absent, minor, and moderate-severe) based on the total AIS score. Dietary intake was estimated using a questionnaire and nutrient intake adequacy was evaluated by comparing the self-reported intake with two indices of the Dietary Reference Intakes for Japanese (2015): an estimated average requirement (EAR) and tentative dietary goal for preventing lifestyle-related disease (DG). RESULTS: A total of 205 men (21.8%) and 266 women (25.2%) were categorized as having moderate-severe insomnia symptoms. Among men, moderate-severe symptoms were associated with higher prevalences of inadequate intakes of total dietary fiber, vitamin C, and zinc. However, there was little association between inadequate nutrient intake and insomnia symptoms among women. The number of inadequate nutrients was significantly associated with insomnia symptoms in men (DG, P=0.004; EAR, P=0.003) but not in women. CONCLUSIONS: This study suggested that insomnia symptoms may be associated with nutritional inadequacy in Japanese adults, especially among men.

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology.

Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.

Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland.

The silkworm silk glands are powerful secretory organs that can produce and secrete proteins at high levels. As such, it has been suggested that the biosynthetic and secretory power of the silk gland can be harnessed to produce and secrete recombinant proteins in tight or loose association with silk fibers. However, the utility of the silkworm platform is constrained by the fact that it has a relatively primitive protein N-glycosylation pathway, which produces relatively simple insect-type, rather than mammalian-type N-glycans. In this study, we demonstrate for the first time that the silk gland protein N-glycosylation pathway can be glycoengineered. We accomplished this by using a dual piggyBac vector encoding two distinct mammalian glycosyltransferases under the transcriptional control of a posterior silk gland (PSG)-specific promoter. Both mammalian transgenes were expressed and each mammalian N-glycan processing activity was induced in transformed silkworm PSGs. In addition, the transgenic animals produced endogenous glycoproteins containing significant proportions of mammalian-type, terminally galactosylated N-glycans, while the parental animals produced none. This demonstration of the ability to glycoengineer the silkworm extends its potential utility as a recombinant protein production platform.

An Overview and History of Glyco-Engineering in Insect Expression Systems.

Insect systems, including the baculovirus-insect cell and Drosophila S2 cell systems are widely used as recombinant protein production platforms. Historically, however, no insect-based system has been able to produce glycoproteins with human-type glycans, which often influence the clinical efficacy of therapeutic glycoproteins and the overall structures and functions of other recombinant glycoprotein products. In addition, some insect cell systems produce N-glycans with immunogenic epitopes. Over the past 20 years, these problems have been addressed by efforts to glyco-engineer insect-based expression systems. These efforts have focused on introducing the capacity to produce complex-type, terminally sialylated N-glycans and eliminating the capacity to produce immunogenic N-glycans. Various glyco-engineering approaches have included genetically engineering insect cells, baculoviral vectors, and/or insects with heterologous genes encoding the enzymes required to produce various glycosyltransferases, sugars, nucleotide sugars, and nucleotide sugar transporters, as well as an enzyme that can deplete GDP-fucose. In this chapter, we present an overview and history of glyco-engineering in insect expression systems as a prelude to subsequent chapters, which will highlight various methods used for this purpose.

Engineering ß1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells.

ß1,4-galactosyltransferase I (B4GALT1) is a Golgi-resident enzyme that elongates glycoprotein glycans, but a subpopulation of this enzyme is secreted following proteolytic cleavage in its stem domain. We hypothesized that engineering B4GALT1 to block cleavage and secretion would enhance its retention and, therefore, its function. To test this hypothesis, we replaced the cytoplasmic/transmembrane/stem (CTS) domains of B4GALT1 with those from human α1,3-fucosyltransferase 7 (FUT7), which is not cleaved and secreted. Expression of FUT7-CTS-B4GALT1 in insect cells produced lower levels of secreted and higher levels of intracellular B4GALT1 activity than the native enzyme. We also noted that the B4GALT1 used in our study had a leucine at position 282, whereas all other animal B4GALT1 sequences have an aromatic amino acid at this position. Thus, we examined the combined impact of changing the CTS domains and the amino acid at position 282 on intracellular B4GALT1 activity levels and N-glycan processing in insect cells. The results demonstrated a correlation between the levels of intracellular B4GALT1 activity and terminally galactosylated N-glycans, N-glycan branching, the appearance of hybrid structures, and reduced core fucosylation. Thus, engineering B4GALT1 to reduce its cleavage and secretion is an approach that can be used to enhance N-glycan elongation in insect cells.

Identification and characterization of receptors for ion transport peptide (ITP) and ITP-like (ITPL) in the silkworm Bombyx mori.

Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1-2.6 × 10(-8) M, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori.

A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells.

Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.

Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway.

The inability to produce recombinant glycoproteins with authentic N-glycans is a limitation of many heterologous protein expression systems. In the baculovirus-insect cell system, this limitation has been addressed by glycoengineering insect cell lines with mammalian genes encoding protein N-glycosylation functions ("glycogenes") under the transcriptional control of constitutive promoters. However, a potential problem with this approach is that the metabolic load imposed by the expression of multiple transgenes could adversely impact the growth and/or stability of glycoengineered insect cell lines. Thus, we created a new transgenic insect cell line (SfSWT-5) with an inducibly mammalianized protein N-glycosylation pathway. Expression of all six glycogenes was induced when uninfected SfSWT-5 cells were cultured in growth medium containing doxycycline. Higher levels of expression and induction were observed when SfSWT-5 cells were cultured with doxycycline and infected with a baculovirus. Interestingly, there were no major differences in the short-term growth properties of SfSWT-5 cells cultured with or without doxycycline. Furthermore, there were no major differences in the phenotypic stability of these cells after continuous culture for over 300 passages with or without doxycycline. Baculovirus-infected Sf9 and SfSWT-5 cells produced about the same amounts of a model recombinant glycoprotein, but only the latter sialylated this product and sialylation was more pronounced when the cells were treated with doxycycline. In summary, this is the first report of a lower eukaryotic system with an inducibly mammalianized protein N-glycosylation pathway and the first to examine how the presumed metabolic load imposed by multiple transgene expression impacts insect cell growth and stability.