Norihito Kawasaki

The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation

Misfolded glycoproteins are translocated from the endoplasmic reticulum (ER) into the cytoplasm for proteasome-mediated degradation. OS-9 protein is thought to participate in ER-associated glycoprotein degradation (ERAD). The recombinant biotinylated mannose 6-phosphate receptor homology (MRH) domain of human OS-9 (OS-9(MRH)) together with six kinds of mutated OS-9(MRH) were prepared and mixed with R-phycoerythrin (PE)-labeled streptavidin to form tetramers (OS-9(MRH)-SA). The PE-labeled OS-9(MRH)-SA bound to HeLaS3 cells in a metal ion-independent manner through amino acid residues homologous to those participating in sugar binding of the cation-dependent mannose 6-phosphate receptor, and this binding was greatly increased by swainsonine, deoxymannojirimycin, or kifunensine treatment. N-Acetylglucosaminyltransferase I-deficient Lec1 cells, but not Lec2 or Lec8 cells, were also strongly bound by the tetramer. OS-9(MRH)-SA binding to the cells was strongly inhibited by Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man and Manalpha1,6Man. To further determine the specificity of native ligands for OS-9(MRH), frontal affinity chromatography was performed using a wide variety of 92 different oligosaccharides. We found that several N-glycans containing terminal alpha1,6-linked mannose in the Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man structure were good ligands for OS-9(MRH), having K(a) values of approximately 10(4) M(-1) and that trimming of either an alpha1,6-linked mannose from the C-arm or an alpha1,3-linked mannose from the B-arm abrogated binding to OS-9(MRH). An immunoprecipitation experiment demonstrated that the alpha1-antitrypsin variant null(Hong Kong), but not wild-type alpha1-antitrypsin, selectively interacted with OS-9 in the cells in a sugar-dependent manner. These results suggest that trimming of the outermost alpha1,2-linked mannose on the C-arm is a critical process for misfolded proteins to enter ERAD.

Sugar-binding activity of the MRH domain in the ER α-glucosidase II β subunit is important for efficient glucose trimming

Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GIIalpha) and a tightly associated beta subunit (GIIbeta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIbeta (GIIbeta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIbeta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIbeta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIbeta-MRH binds most strongly to the glycans with the alpha1,2-linked mannobiose structure. GII with the mutant GIIbeta that lost the sugar-binding activity of GIIbeta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIbeta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.

Subcellular localization of ERGIC-53 under endoplasmic reticulum stress condition

Newly synthesized glycoproteins destined for secretion are transported from the endoplasmic reticulum (ER), through the Golgi and toward the cell surface. In this secretion pathway, several intracellular ER- or Golgi-resident transmembrane proteins serve as cargo receptors. ER-Golgi intermediate compartment (ERGIC)-53, VIP36 and VIPL, which have an L-type lectin domain within the luminal portion, participate in the vectorial transport of glycoproteins via sugar-protein interactions. To understand the nature of these receptors, monoclonal antibodies were generated against human ERGIC-53, VIP36 and VIPL using 293T cells expressing these receptors on cell surfaces. These cells were used to immunize rats and for screening antibody-producing clones. Flow cytometric analysis and immunoprecipitation studies showed that the obtained monoclonal antibodies bound specifically to the corresponding cargo receptors. Immunostaining of HeLa cells using the monoclonal antibodies showed that the localization of ERGIC-53 changed from relatively broad distribution in both the ER and the Golgi under normal conditions to a compact distribution in the Golgi under ER stress conditions. This redistribution was also observed by the overexpression of ERGIC-53 and abrogated by co-expression with VIPL but not VIP36. Real-time polymerase chain reaction revealed that ERGIC-53 along with several chaperone proteins was up-regulated after tunicamycin treatment; however, the expression of VIPL was unchanged. Furthermore, ERGIC-53 co-precipitated with VIPL but not VIP36, indicating that ERGIC-53 may interact with VIPL in the ER, which may regulate the localization of ERGIC-53 inside cells. Taken together, these observations provide new insights into the regulation of these cargo receptors and the quality control of glycoproteins within cells.

Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

Detection of weak-binding sugar activity using membrane-based carbohydrates.

Protein-sugar interactions underlie many biological events. Although protein-sugar interactions are weak, they are regulated in physiological conditions including clustering, association with other proteins, pH condition, and so on. The elucidation of the precise specificities of sugar-binding proteins is essential for understanding their biological functions. To detect the weak-binding activity of carbohydrate-binding proteins to sugar ligands, we studied lectin tetramer binding to cell-surface carbohydrates by flow cytometry. Tetramerization of lectins enhanced their avidity for sugar ligands, and sugar chains displayed on the cell surfaces were easily accessible to such soluble lectins. In this chapter, we describe methods to (1) prepare biotinylated soluble lectin, (2) obtain R-phycoerythrin-labeled lectin tetramer, and (3) measure tetramer binding to various lectin-resistant cell lines or cells treated with sugar-processing inhibitors. This approach enabled us to detect the weak sugar-binding activity of lectins (K(a) approximately 10(4)M(-1)), especially those from animals, and also to elucidate their specificity for sugar ligands.

LC3-Associated Phagocytosis Is Required for Dendritic Cell Inflammatory Cytokine Response to Gut Commensal Yeast Saccharomyces cerevisiae

The human fungal microbiota known as mycobiota is increasingly recognized as a critical factor in human gut health and disease. Non-pathogenic commensal yeasts such as Saccharomyces cerevisiae promote homeostasis in the gut, whereas dysbiosis of the gut mycobiota is associated with inflammation. Glycan-binding receptors (lectins) are key host factors in host–mycobiota interaction in the gut. They are expressed on immune cells such as dendritic cells (DCs) and recognize fungal polysaccharides. This interaction is imperative to mount appropriate immune responses for immune homeostasis in the gut as well as clearance of fungal pathogens. Recent studies demonstrate that microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis (LAP) is involved in lectin–fungi interactions. Yet, the biological impact of LAP on the lectin function remains largely elusive. In this report, we demonstrate that in mouse LAP is linked to dendritic cell-associated lectin 2 (Dectin-2), a C-type lectin specific to fungal α-mannan polysaccharide. We found that mouse Dectin-2 recognizes commensal yeast S. cerevisiae and Kazachstania unispora. Mouse bone marrow-derived DCs (BMDCs) produced inflammatory cytokines TNFα and IL-1β in response to the yeasts in a Dectin-2 and spleen tyrosine kinase (Syk)-dependent manner. We found that S. cerevisiae and K.u2009unispora induced LAP in mouse BMDCs upon internalization. Furthermore, LC3 was activated by stimulation of BMDCs with the yeasts in a Dectin-2 and Syk-dependent manner. To address the biological impact of LAP on Dectin-2 yeast interaction, we established a knock-in mouse strain (Atg16L1E230, thereafter called E230), which BMDCs exhibit autophagy-active and LAP-negative phenotypes. When stimulated with yeasts, E230 BMDCs produced significantly less amounts of TNFα and IL-1β. Taken together, we revealed a novel link between Dectin-2 and LAP that enables host immune cells to respond to mycobiota.

DCIR3 and DCIR4 are co-expressed on inflammatory and patrolling monocytes

Dendritic cell inhibitory receptor 3 (DCIR3) is a member of dendritic immuno-receptor family, of which protein expression has been unknown. We established a specific monoclonal antibody against mouse DCIR3 and investigated the expression of DCIR3 on immune cells of various immune organs. We found that DCIR3 was expressed on monocytes, but not on eosinophils and neutrophils. We also found the existence of a dichotomy in the levels of the expression of DCIR3 on monocytes in bone marrow, blood and spleen. Further investigation of the expression of several cell surface markers on DCIR3High cells and DCIR3Low cells revealed that DCIR3High cells were Ly-6C- CD43High CD11c+ CD80+ NK1.1+ patrolling monocytes and that DCIR3Low cells were Ly-6C+ CD43Low CD11c- CD80- NK1.1- inflammatory monocytes. These results and our previous finding that DCIR4 is expressed at high level in patrolling monocytes and at a low level in inflammatory monocytes (Kameda etxa0al., 2016) suggest that DCIR3 and DCIR4 are simultaneously expressed on monocytes. Indeed, DCIR4+ CD11b+ monocytes from various immune organs expressed DCIR3. We also found that DCIR1 was expressed on DCIR4Low inflammatory monocytes but not on DCIR4HIgh patrolling monocytes. The anti- DCIR3 antibody established in this study, together with the previously established anti-DCIR1 and anti-DCIR4 antibodies, would be a valuable tool to investigate biology and pathophysiology of monocytes.