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Amyloid beta (Aβ) 42 peptide accumulated in Alzheimer disease (AD) patients’ brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aβ42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aβ42 cytotoxicity. SH-SY5Y cells exposed to Aβ42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aβ42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained β-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aβ42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aβ42, which may contribute to the reduction of AD risk.Graphical AbstractSketch of benign chaperon activity of polymorphic SERPINA3-R124C. Here, A = Absence of any SERPINA3 (Aβ42 alone); B = Aβ42 + SERPINA3-WT (WT sample); C = Aβ42 + SERPINA3-R124C (R124C sample).

ABSTRACTMany potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitroassay and in vivoprophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.

The original protocol of Red/ET recombination requires 50-bp sequence homology with target vector on both sides of the DNA fragment. To make it more cost effective, we investigated to determine the minimal length of homology required for the system to work. We found that a homology of 9-bp was sufficient to achieve homologous recombination with more than 50% efficiency.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st(-/-)) by embryonic stem cell technology. C6st(-/-) mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st(-/-) mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st(-/-) mice. Further analysis revealed that the number of CD62L(+)CD44(low) T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st(-/-) mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.

Galα1–3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-β-galactosidase C from Clostridium perfringensdestroys the antigenic epitope by cleaving the β-galactosidic linkage in the Galα1–3Galβ1–4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-β-galactosidase from Flavobacterium keratolyticusand β-1,3-glucanases. The enzyme expressed in Escherichia coliremoved the α-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the α-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-β-galactosidase C is a valuable aid in xenotransplantation.

We isolated a cDNA clone encoding mouseN-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcβ1–3Galβ1–4GlcNAc. Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcα2–3Galβ1–4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galβ1–4(Fucα1–3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situhybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

A cDNA encoding a new α2,8-sialyltransferase (ST8Sia III), which exhibits activity toward the Siaα2,3Galβ1,4GlcNAc sequences of N-linked oligosaccharides, was cloned from mouse brain by means of the polymerase chain reaction-based approach. The predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4% identity with those of so far cloned mouse α2,8-sialyltransferases, i.e.GD3synthase (ST8Sia I) and STX (ST8Sia II), respectively. Transfection of the protein A-fused ST8Sia III gene into COS-7 cells led to α2,8-sialyltransferase activity toward sialylated glycoproteins and α2,3-sialylated glycosphingolipids, such as α2,3-sialylparagloboside and GM3. However, the kinetic properties of ST8Sia III revealed that it is much more specific to N-linked oligosaccharides of glycoproteins than glycosphingolipids. The expression pattern of the ST8Sia III gene was clearly different from those of other α2,8-sialyltransferase genes. The expression of the ST8Sia III gene was tissue and stage specific. The ST8Sia III gene was expressed only in brain and testis, and it appeared first in 20 postcoitum embryonal brain and then decreased. Therefore, the new α2,8-sialyltransferase is closely involved in brain development.

We first detected the mRNA expression of follistatin and three subunits of inhibin/activin in rat pancreatic islets by reverse transcription‐polymerase chain reaction (RT‐PCR). Immunohistochemistry using anti‐follistatin serum (against residues 123–134) revealed that follistatin was localized only in insulin‐producing B cells. Although the βA subunit was detectable in the islets, the immunostainable cell types were completely different with two βA antisera, i.e. anti‐βA (1–1O)‐Tyr stained B cells, while anti‐βA (87–99) stained glucagon‐producing A cells. This inconsistent immunoreactivity was probably related to follistatin binding to β subunits of inhibin/activin. This study indicates that follistatin and inhibin/activin in the islet serve as paracrine or autocrine modulators in the endocrine pancreas.

We have detected sialyltransferase activity of recombinant mouse STX, which was cloned from rat brain as a new member of the sialyltransferase family, but sialyltransferase activity of which had not been detected previously [Livingston and Paulson, J. Biol. Chem. (1993) 268, 11504–11507]. The activity of mouse STX was specific toward sialylated glycoproteins. N‐Glycanase treatment and linkage‐specific sialidase treatment of glycoproteins revealed that STX transfers sialic acids through α2,8‐linkages to only N‐linked oligosaccharides of glycoproteins. However, polymerase activity for polysialic acid synthesis was not detected for this sialyltransferase. Since this α2,8‐sialyltransferase gene is highly restricted in fetal and newborn brain, it may be involved in the polysialylation of glycoproteins, especially of N‐CAM.

We have detected sialyltransferase activity of recombinant mouse STX, which was cloned from rat brain as a new member of the sialyltransferase family, but sialyltransferase activity of which had not been detected previously [Livingston and Paulson, J. Biol. Chem. (1993) 268, 11504–11507]. The activity of mouse STX was specific toward sialylated glycoproteins. N-Glycanase treatment and linkage-specific sialidase treatment of glycoproteins revealed that STX transfers sialic acids through α2,8-linkages to only N-linked oligosaccharides of glycoproteins. However, polymerase activity for polysialic acid synthesis was not detected for this sialyltransferase. Since this α2,8-sialyltransferase gene is highly restricted in fetal and newborn brain, it may be involved in the polysialylation of glycoproteins, especially of N-CAM.

We first detected the mRNA expression of follistatin and three subunits of inhibin/activin in rat pancreatic islets by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry using anti-follistatin serum (against residues 123–134) revealed that follistatin was localized only in insulin-producing B cells. Although the βA subunit was detectable in the islets, the immunostainable cell types were completely different with two βA antisera, i.e. anti-βA (1–1O)-Tyr stained B cells, while anti-βA (87–99) stained glucagon-producing A cells. This inconsistent immunoreactivity was probably related to follistatin binding to β subunits of inhibin/activin. This study indicates that follistatin and inhibin/activin in the islet serve as paracrine or autocrine modulators in the endocrine pancreas.

The mouse ST8Sia II (mST8Sia II/STX) gene encodes a neural cell adhesion molecule-specific polysialic acid synthase whose expression is regulated during the developmental stages of mouse brain. To elucidate the molecular mechanism by which the expression is tissue-specifically and developmentally regulated, we isolated the complete genomic DNA and characterized the promoter of the gene for mST8Sia II. The gene encoding mST8Sia II was found to span about 80 kilobases and to be composed of six exons. Primer extension and S1 nuclease protection analyses revealed that the transcription started from 167 nucleotides upstream of the translational initiation site. Promoter analyses of the 5′-flanking region of the mST8Sia II gene using a luciferase gene reporter system revealed strong promoter activity in retinoic acid-induced differentiated P19 cells, which highly express the mST8Sia II gene. Deletion analyses demonstrated that the minimal promoter activity detected for the proximal region 325 base pairs upstream from the translational initiation codon (−158 to +167) could be modulated by various sequences within the 9.5-kilobase 5′-flanking region. The minimal promoter was embedded in a GC-rich domain (74%, GC content), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. The positive regulatory region located between −159 and −659 contained two additional Sp1 binding motifs and a long pyrimidine-rich region. We also found that the minimal promoter region of the mST8Sia II gene was sufficient for expression of a reporter gene in mST8Sia II gene-expressing neural differentiated P19 cells but not in nonexpressing ones. Thus the TATA-less GC-rich minimal promoter region of mST8Sia II probably controls the cell type-specific expression of the mST8Sia II gene.

The mouse Siaα2,3Galα1,4GalNAc α2,8-sialyltransferase (ST8Sia III) genomic gene, whose transcripts are only expressed in fetal and newborn brain and testis, was isolated and its 5′-flanking region was analyzed. The gene was found to span about 8 kb and to be composed of only four exons. The genomic ST8Sia III gene is much smaller and its organization much simpler than other sialyltransferase genes so far reported, which span more than 25 kb and comprise seven or more exons. In particular, the sialyl motif L of ST8Sia III, which is a highly conserved region in all cloned sialyltransferases, was in one exon. In contrast, this motif is encoded by discrete exons in the other sialyltransferases. The ST8Sia III gene was highly expressed in the mouse brain and gave rise to at least three transcripts (2.1 kb, 2.4 kb, and 6.5 kb), which differed in the length of their 3′-untranslated regions through the alternative use of different polyadenylation sites. Primer extension and S1 nuclease protection analyses of mRNA prepared from newborn brain revealed that ST8Sia III gene expression started from a unique site at 382 nt upstream of ATG. Although the promoter region lacked an apparent TATA or CCAAT box and potential regulatory motifs, a transfection experiment involving neuroblastoma cells expressing ST8Sia III demonstrated the minimal promoter activity exhibited by the proximal region 418 bp upstream from the ATG codon, which suggests the presence of tissue-specific enhancer elements.

It was reported recently by our group that the transfection of GD3 synthase cDNA into Neuro2a cells, a neuroblastoma cell line, caused cell differentiation with neurite sprouting (Kojima et al, 1994; J. Biol Chem., 269, 30451–30456). To further explore this phenomenon in detail, we applied tet-racycline-regulated system to control the expression of GD3 synthase cDNA in Neuro2a cells. Under this system, the process of Neuro2a cell differentiation was rather slow, about 3 weeks of cell culturing in the absence of tetracy-cline was required for most cells to extend the neurite-like structures. The RNase protection assay indicated that the mRNA of GD3 synthase gene was first detected between 4 h and 8 h after the gene was activated and kept at approximately the same level through the process. Furthermore, time-course analysis of total ganglioside expressions has shown that GD3 and GT1b gangliosides appeared on the cell surface early in the process and reached the maximum level around day 6. We also found that the amounts of GD3 and GT1b on the cell surface started to decrease after day 6 and returned gradually to the basal values after 3 weeks. On the other hand, GQ1b and GD1b were started to be synthesized at early stage and the amounts were continuously to increase through the whole Neuro2a morphological change process. In addition, time-course analysis by flow cytometry method for GD3 and GQ1b suggested that the conversions of simple gangliosides to more complex gangliosides may be required to induce the Neuro2a differentiation. Our results indicated that the combination of cDNA transfection and regulated gene expression is a powerful tool to study the function of glycolipids and should have a general application to the glycobiology field.