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.

Neuregulin-1 type III knockout mice exhibit delayed migration of Schwann cell precursors.

In an embryonic developmental stage of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations. After birth, they eventually wrap around individual axons to form myelin sheaths, which insulate axons to increase the nerve conduction velocity. Some growth factors and adhesion molecules are known to control these developmental stages from in the fish to in the mammal. Neuregulin-1 (NRG1), which is composed of many alternative splicing variants, is such a growth factor. Among these variants, the type III isoform of NRG1, interacting with ErbB2 and ErbB3 receptors on Schwann cells, plays an essential role in myelination in the fish and the mammal. NRG1 type III is also known to promote migration of fish Schwann cell precursors; however, it still remains to be clarified whether mammalian type III isoform does it. We have therefore generated type III isoform-specific knockout mice in inbred strain. The mice result in delayed migration of the precursors from the dorsal to ventral root via a peripheral ganglion, comparing littermate controls. Similar results are observed in an in vitro migration assay using reaggregated Schwann cell precursors. Furthermore, the knockout mice exhibit reduced myelin thickness, consistent with the established role of NRG1 type III in myelination. These results indicate that in mice, NRG1 type III plays a key role not only in myelination but also in migration.

Arf6 guanine-nucleotide exchange factor cytohesin-2 regulates myelination in nerves.

In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination.

Effects of amino acid substitutions in the sialylmotifs on molecular expression and enzymatic activities of α2,8-sialyltransferases ST8Sia-I and ST8Sia-VI.

Mouse sialyltransferases are grouped into four families according to the type of carbohydrate linkage they synthesize: ß-galactoside α2,3-sialyltransferases (ST3Gal-I-VI), ß-galactoside α2,6-sialyltransferases (ST6Gal-I and ST6Gal-II), N-acetylgalactosamine α2,6-sialyltransferases (ST6GalNAc-I-VI) and α2,8-sialyltransferases (ST8Sia-I-VI). These sialyltransferases feature a type II transmembrane topology and contain highly conserved motifs termed sialylmotifs L, S, III and VS. Sialylmotifs L and S are involved in substrate binding, whereas sialylmotifs III and VS are involved in catalytic activity. In addition to the conventional sialylmotifs, family and subfamily specific sequence motifs have been proposed. In this study, we analyzed the properties and functions of sialylmotifs in characterizing the enzymatic activity of mouse ST8Sia-I and ST8Sia-VI, both of which are α2,8-sialyltransferases involved in the synthesis of either ganglioside GD3 or disialic acid structures on O-glycans, respectively. The ST8Sia-VI-based chimeric enzymes, whose sialylmotif L sequences were replaced with those of ST8Sia-I and ST8Sia-IV (polysialic acid synthetase), were still active toward O-glycans. However, ST8Sia-VI-based chimeric enzymes lost expression or activity when their sialylmotif L sequences were replaced with those of ST3Gal-I and ST6GalNAc-II, suggesting the existence of an ST8Sia family specific motif in the sialylmotif L. The ST8Sia-I- and ST8Sia-VI-based chimeric enzymes lost enzymatic activity when their sialylmotif S sequences were interchanged. Amino acid substitutions in the sialylmotif S of ST8Sia-I and ST8Sia-VI also affected the enzymatic activity in many cases, indicating the crucial and functional importance of the sialylmotif S in substrate binding, which determines the substrate specificity of sialyltransferase.

The adaptor SH2B1 and the phosphatase PTP4A1 regulate the phosphorylation of cytohesin-2 in myelinating Schwann cells in mice.

Mature myelin sheaths insulate axons to increase nerve conduction velocity and protect nerve fibers from stress and physical injury. In the peripheral nervous system, the myelin sheath is produced by Schwann cells. The guanine-nucleotide exchange factor cytohesin-2 activates the protein Arf6 to promote Schwann cell myelination. Here, we investigated the regulation of cytohesin-2 and found that the phosphorylation status of Tyr381 in cytohesin-2 is central to Schwann cell myelination. Knockin mice with a nonphosphorylatable Y381F mutation in cytohesin-2 exhibited reduced myelin thickness and decreased Arf6 activity in sciatic nerve tissue. In HEK293T cells, cytohesin-2 was dephosphorylated at Tyr381 by the protein tyrosine phosphatase PTP4A1, whereas phosphorylation at this site was maintained by interaction with the adaptor protein SH2B1. Schwann cell-specific knockdown of PTP4A1 in mice increased cytohesin-2 phosphorylation and myelin thickness. Conversely, Schwann cell-specific loss of SH2B1 resulted in reduced myelin thickness and decreased cytohesin-2 phosphorylation. Thus, a signaling unit centered on cytohesin-2-with SH2B1 as a positive regulator and PTP4A1 as a negative regulator-controls Schwann cell myelination in the peripheral nervous system.

The mood stabilizer valproic acid improves defective neurite formation caused by Charcot-Marie-Tooth disease-associated mutant Rab7 through the JNK signaling pathway.

Charcot-Marie-Tooth (CMT) disease is the most frequent peripheral neuropathy affecting the Schwann cells and neurons. CMT disease type 2 (CMT2) neuropathies are characterized by peripheral nerve aberrance. Four missense mutations of Rab7, a small GTPase of the Rab family involved in intracellular vesicular trafficking, are associated with the CMT2B phenotype. Despite a growing body of evidence concerning the gene structures responsible for genetically heterogenous CMT2B and other CMT2 neuropathies, little is known about the in vitro neuropathy model and how CMT2B-associated mutation-caused aberrant neuritogenesis is properly reversed. Here, we show that valproic acid (VPA), a classical mood-stabilizing drug, improves defective neurite formation in N1E-115 neuroblastoma cells regardless of which CMT2B-associated Rab7 mutant protein is expressed. The effect is mediated by c-Jun N-terminal kinase (JNK) signaling, but not by deacetylase inhibition activity of VPA itself. Furthermore, VPA has similar effects in dorsal root ganglion (DRG) neurons expressing any of the four mutant Rab7 proteins. Thus, VPA has a previously unknown potential to improve defective neuritogenesis associated with CMT2B in vitro, indicating that JNK should be a potential therapeutic target for treatments aimed at improving neuritogenesis.

Characterization of mouse sialyltransferase genes: their evolution and diversity.

Sialic acids are negatively charged acidic sugars, and sialylglycoconjugates often play important roles in various biological phenomena. Sialyltransferases are involved in the synthesis of sialylglycoconjugates, and 20 members of the mammalian sialyltransferase family have been identified to date. These sialyltransferases are grouped into four families according to the carbohydrate linkages they synthesize: beta-galactoside alpha2,3-sialyltransferases (ST3Gal I-VI), beta-galactoside alpha2,6-sialyltransferases (ST6Gal I and II), GalNAc alpha2,6-sialyltransferases (ST6GalNAc I-VI), and alpha2,8-sialyltransferases (ST8Sia I-VI). Analysis of the amino acid sequence similarities, substrate specificities, and gene structures of mouse sialyltransferases has revealed that they can be further divided into seven subfamilies. The genomic structural resemblance of members of the same subfamily suggests that they arose from a common ancestral gene through gene duplication events. These multiple sialyltransferase genes are needed for fine control of the expression of sialylglycoconjugates, resulting in a variety of developmental stage- and tissue-specific glycosylation patterns.

BIG1/Arfgef1 and Arf1 regulate the initiation of myelination by Schwann cells in mice.

During development of the peripheral nervous system in mammals, Schwann cells wrap their plasma membranes around neuronal axons, forming multiple myelin sheaths. A mature myelin sheath insulates axons and increases nerve conduction velocity while protecting nerve fibers from various stresses such as physical ones. Despite this functional importance, the molecular units that underlie dynamic morphological changes in formation of myelin sheaths are not sufficiently understood. Arf1 is a small guanosine triphosphate-binding protein that plays multiple roles in intracellular trafficking and related signaling, both of which are processes involved in cell morphogenesis. We demonstrate that the Arf1 guanine nucleotide exchange factor, brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1)/Arfgef1, and the effector Arf1 regulate the initiation of myelination of axons by Schwann cells. Schwann cell-specific BIG1 conditional knockout mice, which have been generated here, exhibit reduced myelin thickness and decreased localization of myelin protein zero in the myelin membrane, compared with their littermate controls. BIG1 knockout mouse nerves specifically decrease the amounts of Arf1 in the AP1 clathrin adaptor protein subunits but not the Arf1 binding to GGA1 (Golgi-localized, gamma-adaptin ear-containing, Arf-binding protein 1) transporting proteins. The amounts of Arf1 in the COPI coatomer protein subunits were comparable in the knockout mice and controls. Similar results in myelin thickness are observed in Arf1 conditional knockout mice, which have also been generated here. Thus, the BIG1 and Arf1 unit plays a key role in Schwann cell myelination, newly adding it to the list of molecular units controlling myelination.

Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase 1.

The cellulose-binding domains (CBDs) of fungal cellulases interact with crystalline cellulose through their hydrophobic flat surface formed by three conserved aromatic amino acid residues. To analyze the functional importance of these residues, we constructed CBD mutants of cellobiohydrolase 1 (CBH1) of the thermophilic fungus Humicola grisea, and examined their cellulose-binding ability and enzymatic activities. High activity on crystalline cellulose correlated with high cellulose-binding ability and was dependent on the combination and configuration of the three aromatic residues. Tyrosine works best in the middle of the flat surface, while tryptophan is the best residue in the two outer positions.