Schwann cells respond to cues from axons by transforming their cellular morphology and forming myelin. We demonstrated that the guanine nucleotide exchange factor (GEF) cytohesin-1 promoted myelination by activating the small guanosine triphosphatase (GTPase) Arf6. In mice, ablating cytohesin-1 delayed myelination and diminished the amount of myelin produced. We determined that the Src-family kinase Fyn phosphorylated tyrosine 382 (Y(382)) of cytohesin-1, and we generated transgenic mice that expressed a Schwann cell-specific phosphorylation mutant of cytohesin-1 (Y382F) that could not be targeted by Fyn. During development, these transgenic mice displayed delayed myelination compared to that of wild-type mice, as well as a decrease in the amount of myelin produced, similar to that observed in cytohesin-1⁻/⁻ mice. These findings demonstrate that phosphorylation of cytohesin-1 by Fyn is required for full myelination and suggest that tyrosine phosphorylation of GEFs may be a mechanism to activate small GTPases engaged in cell morphogenesis.
A novel member of the human -galactoside ␣2,6-sialyltransferase (ST6Gal) family, designated ST6Gal II, was identified by BLAST analysis of expressed sequence tags and genomic sequences. The sequence of ST6Gal II encoded a protein of 529 amino acids, and it showed 48.9% amino acid sequence identity with human ST6Gal I. Recombinant ST6Gal II exhibited ␣2,6-sialyltransferase activity toward oligosaccharides that have the Gal1,4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activities toward some glycoproteins and glycolipids, respectively. It is concluded that ST6Gal II is an oligosaccharide-specific enzyme compared with ST6Gal I, which exhibits broad substrate specificities, and is mainly involved in the synthesis of sialyloligosaccharides. The expression of the ST6Gal II gene was significantly detected by reverse transcription PCR in small intestine, colon, and fetal brain, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were much higher than those of the ST6Gal II gene. The ST6Gal I gene was also expressed in all tumors examined, but no expression was observed for the ST6Gal II gene in these tumors. The ST6Gal II gene is located on chromosome 2 (2q11.2-q12.1), and it spans over 85 kb of human genomic DNA consisting of at least eight exons and shares a similar genomic structure with the ST6Gal I gene. In this paper, we have shown that ST6Gal I, which has been known as the sole member of the ST6Gal family, also has the counterpart enzyme (ST6Gal II) like other sialyltransferases.
Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.
Two cDNA clones encoding NeuAc␣2,3Gal1,3GalNAc GalNAc␣2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAc␣2, 3Gal1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Gal1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14 C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[ 14 C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAc␣2,6-sialyltransferase, which requires sialic acid residues linked to Gal1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions, like cell-cell communication, cell-substrate interaction, adhesion, and protein targeting. The transfer of sialic acids from CMP-Sia 1 to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids is catalyzed by a sialyltransferase. Although roles of sialic acids have been proposed in the regulation of many biological phenomena, the purpose of this structural diversity remains largely obscure. To determine the meaning of the diversity of and the regulatory mechanism for the sialylation of glycoconjugates, it is necessary to obtain information on the enzymes themselves and the gene structure of sialyltransferases. Each sialyltransferase exhibits strict specificity for acceptor substrates and linkages (3-6). Although three linkages, Sia␣2,6Gal, Sia␣2,3Gal, and Sia␣2,6GalNAc, are commonly found in glycoproteins (7), and two, Sia␣2,3Gal and Sia␣2,8Sia, occur frequently in gangliosides (8), each of these linkages has been found in both gangliosides and glycoproteins (8 -10).So far, the cloning of three members of the ␣2,6-sialyltransferase family (ST6GalNAc I, II and III) has been reported (11-14). The cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12) and mouse (13,62).2 The overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick ST6GalNAc II is 57.3% ...
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