LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

Core fucosylation within the Fc-FcγR degradation pathway promotes enhanced IgG levels via exogenous L-fucose.

α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8+/+), Fut8 heterozygous knockout (Fut8+/-), and Fut8 knockout (Fut8-/-) mice. The IgG levels in serum were lower in Fut8+/- and Fut8-/- mice compared with Fut8+/+ mice. Exogenous L-fucose increased IgG levels in Fut8+/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor Ⅳ (FcγRⅣ), mainly expressed on macrophages and neutrophils, were increased in Fut8+/- mice compared to Fut8+/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRⅣ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRⅣ-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8+/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation.

Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide.

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8-/-) or heterozygous KO (Fut8+/-) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/- mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/- mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/- mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/- mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation.

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model.

Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b.

The N-glycans attached to proteins contain various GlcNAc branches, the aberrant formation of which correlates with various diseases. N-Acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and Gnt-IVb (or MGAT4B) are isoenzymes that catalyze the formation of the ß1,4-GlcNAc branch in N-glycans. However, the functional differences between these isozymes remain unresolved. Here, using cellular and UDP-Glo enzyme assays, we discovered that GnT-IVa and GnT-IVb have distinct glycoprotein preferences both in cells and in vitro. Notably, we show that GnT-IVb acted efficiently on glycoproteins bearing an N-glycan premodified by GnT-IV. To further understand the mechanism of this reaction, we focused on the noncatalytic C-terminal lectin domain, which selectively recognizes the product glycans. Replacement of a nonconserved amino acid in the GnT-IVb lectin domain with the corresponding residue in GnT-IVa altered the glycoprotein preference of GnT-IVb to resemble that of GnT-IVa. Our findings demonstrate that the C-terminal lectin domain regulates differential substrate selectivity of GnT-IVa and GnT-IVb, highlighting a new mechanism by which N-glycan branches are formed on glycoproteins.

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan ß1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non-small-cell lung cancer cells.

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6ß4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.

O-GlcNAcylation regulates ß1,4-GlcNAc-branched N-glycan biosynthesis via the OGT/SLC35A3/GnT-IV axis.

N-Linked glycosylation and O-linked N-acetylglucosamine (O-GlcNAc) are important protein post-translational modifications that are orchestrated by a diverse set of gene products. Thus far, the relationship between these two types of glycosylation has remained elusive, and it is unclear whether one influences the other via UDP-GlcNAc, which is a common donor substrate. Theoretically, a decrease in O-GlcNAcylation may increase the products of GlcNAc-branched N-glycans. In this study, via examination by lectin blotting, HPLC, and mass spectrometry analysis, however, we found that the amounts of GlcNAc-branched tri-antennary N-glycans catalyzed by N-acetylglucosaminyltransferase IV (GnT-IV) and tetra-antennary N-glycans were significantly decreased in O-GlcNAc transferase knockdown cells (OGT-KD) compared with those in wild type cells. We examined this specific alteration by focusing on SLC35A3, which is the main UDP-GlcNAc transporter in mammals that is believed to modulate GnT-IV activation. It is interesting that a deficiency of SLC35A3 specifically leads to a decrease in the amounts of GlcNAc-branched tri- and tetra-antennary N-glycans. Furthermore, co-immunoprecipitation experiments have shown that SLC35A3 interacts with GnT-IV, but not with N-acetylglucosaminyltransferase V. Western blot and chemoenzymatic labeling assay have confirmed that OGT modifies SLC35A3 and that O-GlcNAcylation contributes to its stability. Furthermore, we found that SLC35A3-KO enhances cell spreading and suppresses both cell migration and cell proliferation, which is similar to the phenomena observed in the OGT-KD cells. Taken together, these data are the first to demonstrate that O-GlcNAcylation specifically governs the biosynthesis of tri- and tetra-antennary N-glycans via the OGT-SLC35A3-GnT-IV axis.

Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs), which is a novel mechanism of glycosyltransferase regulation.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation.

Core fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and in vitro and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.

MALDI-TOF mass spectrometry imaging for N-glycans on FFPE tissue sections of mouse NASH liver through Sialic acid Benzylamidation.

Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.

Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan.

Glycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation.

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

Type-I-hypersensitivity to 15 kDa, 28 kDa and 54 kDa proteins in vitellogenin specific to Gadus chalcogrammus roe.

BACKGROUND: Fish roe allergy is a common health problem in countries where sea food is a major part of the diet, such as Japan. ß'-component (ß'-c) in fish roe has been identified as a major antigen for patients who show hypersensitivity to various fish roes. However, little is known about causative antigens for patients reactive to fish roe of specific species. METHODS: Serum and basophils were obtained from patients who had reactivity to roes of Gadus chalcogrammus (GC) and/or other fish species. GC roe specific antigens were analyzed by immunoblotting, histamine release assay (HRA) and mass spectrometry. Recombinant-fragments of vitellogenin (Vg) were obtained by the Escherichia coli expression system. RESULTS: Serum IgE of a patient with specific reactions to GC roe bound to 15, 28, 40 and 70 kDa-proteins in GC roe extract. Mass spectrometry analysis revealed that proteins in these bands contained fragments corresponding to Vg. Immunoblotting of Vg immunoprecipitated by rabbit anti-Vg antiserum from the extract revealed 15, 28 and 54 kDa fragments bound by the patient's IgE. These bindings were inhibited by the pretreatment of recombinant phosvitin (rPv) and ß'-c (rß'-c). Fractions obtained by native gel electrophoresis containing 15, 28 and 54 kDa proteins, but not the other fractions, induced significant histamine release from the patient's basophils. Sera of the other patients with GC roe specific-IgE showed IgE binding to rPv and/or rß'-c. CONCLUSIONS: The 15, 28 and 54 kDa-fragments of Vg which include structures of Pv and ß'-c, could be antigens for GC roe specific type-I-hypersensitivity.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Sialylation of extracellular superoxide dismutase (EC-SOD) enhances furin-mediated cleavage and secretion.

Extracellular superoxide dismutase (EC-SOD, SOD3) protects tissues against oxidative damage by detoxifying superoxide anions, particularly in the lungs and cardiovascular system. EC-SOD undergoes several posttranslational modifications including N-glycosylation and proteolytic cleavage. While the roles of proteolytic cleavage have been well studied, the structure and function of EC-SOD N-glycans are poorly understood. Here we analyzed glycan structures on native EC-SOD purified from human sera, and identified sialylated biantennary structures. Using glycan maturation-defective CHO mutant cells, we further revealed that the presence of terminal sialic acids in the N-glycans of EC-SOD enhanced both the secretion and furin-mediated C-terminal cleavage of EC-SOD. These results provide new insights into how the posttranslational modifications of EC-SOD control its functions.

Bisecting GlcNAc modification stabilizes BACE1 protein under oxidative stress conditions.

ß-Site amyloid precursor protein-cleaving enzyme-1 (BACE1) is a protease essential for amyloid-ß (Aß) production in Alzheimer's disease (AD). BACE1 protein is known to be up-regulated by oxidative stress-inducing stimuli but the mechanism for this up-regulation still needs to be clarified. We have recently found that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc) by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene) and that GnT-III deficiency reduces Aß-plaque formation in the brain by accelerating lysosomal degradation of BACE1. Therefore, we hypothesized that bisecting GlcNAc would stabilize BACE1 protein on oxidative stress. In the present study, we first show that Aß deposition in the mouse brain induces oxidative stress, together with an increase in levels of BACE1 and bisecting GlcNAc. Furthermore, prooxidant treatment induces expression of BACE1 protein in wild-type mouse embryonic fibroblasts (MEFs), whereas it reduces BACE1 protein in GnT-III (Mgat3) knock-out MEFs by accelerating lysosomal degradation of BACE1. We purified BACE1 from Neuro2A cells and performed LC/ESI/MS analysis for BACE1-derived glycopeptides and mapped bisecting GlcNAc-modified sites on BACE1. Point mutations at two N-glycosylation sites (Asn(153) and Asn(223)) abolish the bisecting GlcNAc modification on BACE1. These mutations almost cancelled the enhanced BACE1 degradation seen in Mgat3(-/-) MEFs, indicating that bisecting GlcNAc on BACE1 indeed regulates its degradation. Finally, we show that traumatic brain injury-induced BACE1 up-regulation is significantly suppressed in the Mgat3(-/-) brain. These results highlight the role of bisecting GlcNAc in oxidative stress-induced BACE1 expression and offer a novel glycan-targeted strategy for suppressing Aß generation.