Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Preparation and biological activities of anti-HER2 monoclonal antibodies with multibranched complex-type N-glycans.

Immunoglobulin G (IgG) has a conserved N-glycosylation site at Asn297 in the fragment crystallizable (Fc) region. Previous studies have shown that N-glycosylation of this site is a critical mediator of the antibody's effector functions, such as antibody-dependent cellular cytotoxicity. While the N-glycan structures attached to the IgG-Fc region are generally heterogenous, IgGs engineered to be homogenously glycosylated with functional N-glycans may improve the efficacy of antibodies. The major glycoforms of the N-glycans on the IgG-Fc region are bi-antennary complex-type N-glycans, while multibranched complex-type N-glycans are not typically found. However, IgGs with tri-antennary complex-type N-glycans have been generated using the N-glycan remodeling technique, suggesting that more branched N-glycans might be artificially attached. At present, little is known about the properties of these IgGs. In this study, IgGs with multibranched N-glycans on the Fc region were prepared by using a combination of the glycosynthase/oxazoline substrate-based N-glycan remodeling technique and successive reactions with glycosyltransferases. Among the IgGs produced by these methods, the largest N-glycan attached was a bisecting N-acetylglucosamine containing a sialylated penta-antennary structure. Concerning the Fc-mediated effector functions, the majority of IgGs with tri- and tetra-antennary N-glycans on their Fc region showed properties similar to IgGs with ordinary bi-antennary N-glycans.

Novel endo-ß-N-acetylglucosaminidases from Tannerella species hydrolyze multibranched complex-type N-glycans with different specificities.

Endo-ß-N-acetylglucosaminidases are enzymes that hydrolyze the N,N'-diacetylchitobiose unit of N-glycans. Many endo-ß-N-acetylglucosaminidases also exhibit transglycosylation activity, which corresponds to the reverse of the hydrolysis reaction. Because of these activities, some of these enzymes have recently been used as powerful tools for glycan remodeling of glycoproteins. Although many endo-ß-N-acetylglucosaminidases have been identified and characterized to date, there are few enzymes that exhibit hydrolysis activity toward multibranched (tetra-antennary or more) complex-type N-glycans on glycoproteins. Therefore, we searched for novel endo-ß-N-acetylglucosaminidases that exhibit hydrolysis activity toward multibranched complex-type N-glycans in this study. From database searches, we selected three candidate enzymes from Tannerella species-Endo-Tsp1006, Endo-Tsp1263 and Endo-Tsp1457-and prepared them as recombinant proteins. We analyzed the hydrolysis activity of these enzymes toward N-glycans on glycoproteins and found that Endo-Tsp1006 and Endo-Tsp1263 exhibited hydrolysis activity toward complex-type N-glycans, including multibranched N-glycans, preferentially, whereas Endo-Tsp1457 exhibited hydrolysis activity toward high-mannose-type N-glycans exclusively. We further analyzed substrate specificities of Endo-Tsp1006 and Endo-Tsp1263 using 18 defined glycopeptides as substrates, each having a different N-glycan structure. We found that Endo-Tsp1006 preferred N-glycans with galactose or α2,6-linked sialic acid residues in their nonreducing ends as substrates, whereas Endo-Tsp1263 preferred N-glycans with N-acetylglucosamine residues in their nonreducing ends as substrates.

Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of Its Conjugated Peptide.

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.

St6gal1 knockdown alters HBV life cycle in HepAD38 cells.

Complex glycans at the cell surface play important roles, and their alteration is known to modulate cellular activity. Previously, we found that HBV replication in HepAD38 altered cell-surface sialylated N-glycan through the upregulation of St6gal1, Mgat2, and Mgat4a expression. Here we studied the effects of knocking them down on HBV replication in HepAD38. Our results showed that St6gal1 knockdown (KD) reduced intracellular HBV rcDNA level by 90%, that Mgat2 KD did not change the intracellular HBV rcDNA level, and that Mgat4 KD increased the intracellular HBV rcDNA level by 19 times compared to Tet(-). The changes in intracellular rcDNA level were followed by the alteration of Pol and HBc expression. Our study suggests that St6gal1 KD contributes more to the HBV life cycle than Mgat2 or Mgat4a KD through the modification of intracellular L, Pol, and HBc expression.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline.

OBJECTIVES: To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities. RESULTS: Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline's stability and Endo-CC's transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 µg GlcNAc-RNase B, 200 µg SG-oxazoline and 3 µg Endo-CCN180H in 20 µl 20 mM Tris/HCl pH 7.5 at 30 °C for 30-60 min. CONCLUSIONS: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

Characterization of novel endo-ß-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

Loss of complex-type N-linked glycans attenuates maximum cell density and susceptibility to human serum of Trypanosoma brucei brucei.

Trypanosoma brucei brucei is a parasitic protist that expresses cell surface proteins modified with complex-type N-linked glycan (NLG), like multicellular organisms. However, little is known about the role of complex-type NLG. In T. b. brucei, it has been shown that either one of the glycosyltransferases, TbGT11 or TbGT15, is sufficient to initiate the synthesis of complex-type NLG. To clarify the role of complex-type NLG, it is necessary to generate cells lacking both enzymes. Therefore, we deleted TbGT11 and TbGT15 from the genome of T. b. brucei for the phenotypic examination. The mutant strain grew in culture, with reduced maximum cell density; showed decreased susceptibility to normal human serum, which contains trypanolytic factors; and lacked uptake of the haptoglobin-hemoglobin complex. These data indicate that protein modification by complex-type NLG is not essential but is required for receptor function.

Chemical and Chemoenzymatic Synthesis of Peptide and Protein Therapeutics Conjugated with Human N-Glycans.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.

Characterizing carbohydrate-protein interactions by nuclear magnetic resonance spectroscopy.

Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate-binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the nonequivalent interactions among oligomeric carbohydrate receptors, have made nuclear magnetic resonance (NMR) an especially powerful tool for studying and defining carbohydrate-protein interactions. Here, we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest.

Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

Humanization of Yeasts for Glycan-Type End-Products.

Yeasts are often considered microorganisms for producing human therapeutic glycosylated end-products at an industrial scale. However, the products with non-humanized glycans limited their usage. Therefore, various methods to develop humanized glycosylated end-products have been widely reported in yeasts. To make full use of these methods, it is necessary to summarize the present research to find effective approaches to producing humanized products. The present research focuses on yeast species selection, glycosyltransferase deletion, expression of endoglycosidase, and expression of proteins with galactosylated and or sialylated glycans. Nevertheless, the yeasts will have growth defects with low bioactivity when the key enzymes are deleted. It is necessary to express the corresponding repairing protein. Compared with N-glycosylation, the function of yeast protein O-glycosylation is not well-understood. Yeast proteins have a wide variety of O-glycans in different species, and it is difficult to predict glycosylation sites, which limits the humanization of O-glycosylated yeast proteins. The future challenges include the following points: there are still many important potential yeasts that have never been tried to produce glycosylated therapeutic products. Their glycosylation pathway and related mechanisms for producing humanized glycosylated proteins have rarely been reported. On the other hand, the amounts of key enzymes on glycan pathways in human beings are significantly more than those in yeasts. Therefore, there is still a challenge to produce a large body of humanized therapeutic end-products in suitable yeast species, especially the protein with complex glycans. CRISPR-Cas9 system may provide a potential approach to address the important issue.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Enteric Glia Regulate Lymphocyte Activation via Autophagy-Mediated MHC-II Expression.

BACKGROUND & AIMS: Enteric glial cells express type II major histocompatibility complex (MHC-II) molecules in Crohn's disease and Chagas disease, but it is unclear whether the expressed molecules are functional. We examined the capabilities of enteric glia to act as an antigen-presenting cell in vivo and whether glial MHC-II has immunomodulatory effects. METHODS: We generated Sox10CreERT2;IABfl/fl mice to ablate MHC-II in enteric glia after exposure to tamoxifen. We measured phagocytic activity and autophagy activation to assess potential peptide sources loaded onto glial MHC-II and measured T- and B-lymphocyte activation and serum and colonic tissue cytokine levels to study enteric glial immunomodulatory capabilities. RESULTS: Enteric glia express MHC-II molecules in response to a subclinical dose of interferon-γ and lipopolysaccharide in vivo. Glial MHC-II expression contributes to effective B-lymphocyte and T-lymphocyte activation with marked effects on T-helper cell (Th)17 and regulatory T cell subtypes. No effect on Th1 or Th2 subtypes was observed. Enteric glial MHC-II does not have a major effect on serum or colonic tissue cytokine levels but may influence local cytokine levels. Glial MHC-II expression requires the activation of autophagy pathways, but activating autophagy alone is not sufficient to drive glial MHC-II expression. CONCLUSIONS: Enteric glia express MHC-II as a mechanism to tune intestinal immune responses. Glial autophagy is triggered in response to proinflammatory stimuli and induces glial antigen presentation, which functions to modulate the activation of T-lymphocyte subsets involved in tolerance. These observations suggest that enteric glia may express MHC-II to maintain immune homeostasis during inflammatory conditions such as Crohn's disease.

Immune responses to injury and their links to eye disease.

The eye is regarded as an immune privileged site. Since the presence of a vasculature would impair vision, the vasculature of the eye is located outside of the central light path. As a result, many regions of the eye evolved mechanisms to deliver immune cells to sites of dysgenesis, injury, or in response to the many age-related pathologies. While the purpose of these immune responses is reparative or protective, cytokines released by immune cells compromise visual acuity by inducing inflammation and fibrosis. The response to traumatic or pathological injury is distinct in different regions of the eye. Age-related diseases impact both the anterior and posterior segment and lead to reduced quality of life and blindness. Here we focus attention on the role that inflammation and fibrosis play in the progression of age-related pathologies of the cornea and the lens as well as in glaucoma, the formation of epiretinal membranes, and in proliferative vitreoretinopathy.

Biochemical characterization and mutational analysis of silkworm Bombyx mori ß-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of ß-1,4-galactosyltransferase family enzymes.

Silkworm Bombyx mori is one of the insect hosts for recombinant protein production at academic and industrial levels. B. mori and other insect cells can produce mammalian proteins with proper posttranslational modifications, such as N-glycosylation, but the structures of N-glycans in B. mori are mainly high mannose- and paucimannose-type, while mammals also produce hybrid- and complex-type glycans. Recently, complex-type N-glycans whose structures are different from mammalian ones have been identified in some insect cell N-glycomes at very low levels compared with levels of high mannose- and paucimannose-type glycans. However, their functions and the enzymes involved in the biosynthesis of insect complex-type N-glycans are not clear, and complex-type N-glycans, except for N-acetylglucosamine-terminated glycans, are still not identified in the B. mori N-glycome. Here, we focused on the ß-1,4-galactosyltransferase family (also known as glycosyltransferase family 7, GT7) that contains mammalian ß-1,4-galactosyltransferase and insect ß-1,4-N-acetylgalactosaminyltransferase. A gene for a GT7 protein (BmGalNAcT) from B. mori was cloned, expressed in a soluble form using a silkworm expression system, and the gene product showed strict ß-1,4-N-acetylgalactosaminyltransferase activity but not ß-1,4-galactosyltransferase activity. A mutation in Ile298 or Ile310, which are predicted to be located in the active site, reduced its glycosyltransferase activity, suggesting that these residues and the corresponding residues are responsible for substrate specificity of GT7. These results suggested that BmGalNAcT may be involved in the complex-type N-glycans, and moreover, bioinformatics analysis revealed that B. mori might have an extra gene for a GT7 enzyme with different specificity in addition to the known insect GT7 glycosyltransferases.

Expression and characterization of silkworm Bombyx mori ß-1,2-N-acetylglucosaminyltransferase II, a key enzyme for complex-type N-glycan biosynthesis.

N-glycans are involved in various physiological functions and their structures diverge among different phyla and kingdoms. Insect cells mainly produce high mannose-type and paucimannose-type glycans but very few mammalian-like complex-type glycans. However, many insects possess genes for proteins homologous to the enzymes involved in complex-type N-glycan synthesis in mammalian cells, and their N-glycosylation pathway is incompletely understood compared with that of mammals. Here, we cloned a candidate gene for ß-1,2-N-acetylglucosaminyltransferase II (GnTII), which is a Golgi-localized enzyme involved in a key step in the conversion to complex-type N-glycans, from silkworm Bombyx mori, and the gene was found to be expressed ubiquitously in the larval and pupal stages. In addition, recombinant B. mori GnTII was expressed as a soluble form using a silkworm-B. mori nucleopolyhedrovirus bacmid expression system. The recombinant enzyme exhibited similar pH and temperature dependency and the same substrate specificity as human GnTII, but deglycosylation with peptide:N-glycanase F did not affect its enzymatic activity. Compared with the structure of human GnTII, the amino acid residues involved in catalytic activity and substrate recognition are almost fully conserved in B. mori GnTII, which is consistent with its enzymatic properties. These results raised the possibility of mammalian-like complex-type N-glycan synthesis using the GnTII ortholog in silkworm.

Improved recombinant protein production in Arabidopsis thaliana.

Production and isolation of recombinant proteins are key steps in modern Molecular Biology. Expression vectors and platforms for various hosts, including both prokaryotic and eukaryotic systems, have been used. In basic plant research, Arabidopsis thaliana is the central model for which a wealth of genetic and genomic resources is available, and enormous knowledge has been accumulated over the past years - especially since elucidation of its genome in 2000. However, until recently an Arabidopsis platform had been lacking for preparative-scale production of homologous recombinant proteins. We recently established an Arabidopsis-based super-expression system, and used it for a structural pilot study of a multi-subunit integral membrane protein complex. This review summarizes the benefits and further potential of the model plant system for protein productions. ABBREVIATIONS: Nb, Nicotiana benthamiana; OT, oligosaccharyltransferase.

Heterologous expression, purification and characterization of human ß-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

ß-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 µg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity.