Shigeru Tsuboi

Core 2 Oligosaccharide Biosynthesis Distinguishes between Selectin Ligands Essential for Leukocyte Homing and Inflammation

Mammalian serine/threonine-linked oligosaccharides (O-glycans) are commonly synthesized with the Golgi enzyme core 2 beta-1,6-N-acetylglucosaminyltransferase (C2 GlcNAcT). Core 2 O-glycans have been hypothesized to be essential for mucin production and selectin ligand biosynthesis. We report that mice lacking C2 GlcNAcT exhibit a restricted phenotype with neutrophilia and a partial deficiency of selectin ligands. Loss of core 2 oligosaccharides reduces neutrophil rolling on substrata bearing E-, L-, and P-selectins and neutrophil recruitment to sites of inflammation. However, the diminished presence of L-selectin ligands on lymph node high endothelial venules does not affect lymphocyte homing. These studies indicate that core 2 oligosaccharide biosynthesis segregates the physiologic roles of selectins and reveal a function for the C2 GlcNAcT in myeloid homeostasis and inflammation.

A Novel, High Endothelial Venule–Specific Sulfotransferase Expresses 6-Sulfo Sialyl Lewisx, an L-Selectin Ligand Displayed by CD34

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells

Aberrant expression of cell surface carbohydrates such as sialyl Lewis X is associated with tumor formation and metastasis. In order to determine the roles of sialyl Lewis X in tumor metastasis, mouse melanoma B16‐F1 cells were stably transfected with α1,3‐fucosyltransferase III to express sialyl Lewis X structures. The transfected B16‐F1 cells, B16‐FTIII, were separated by cell sorting into three different groups based on the expression levels of sialyl Lewis X. When these transfected cells were injected into tail veins of C57BL/6 mice, B16‐FTIII·M cells expressing moderate amounts of sialyl Lewis X in poly‐N‐acetyllactosamines produced large numbers of lung tumor nodules. Surprisingly, B16‐FTIII·H cells expressing the highest amount of sialyl Lewis X in shorter N‐glycans died in lung blood vessels, producing as few lung nodules as B16‐FTIII·N cells which lack sialyl Lewis X. In contrast, B16‐FIII·H cells formed more tumors in beige mice and NK cell‐depleted C57BL/6 mice than did B16‐FTIII·M cells. B16‐FTIII·H cells bound to E‐selectin better than did B16‐FTIII·M cells, but both cells grew at the same rate. These results indicate that excessive expression of sialyl Lewis X in tumor cells leads to rejection by NK cells rather than tumor formation facilitated by attachment to endothelial cells.

6′-Sulfo Sialyl Lex but Not 6-Sulfo Sialyl Lex Expressed on the Cell Surface Supports L-selectin-mediated Adhesion

In order to determine if a sulfated oligosaccharide on the cell surface can function as an L-selectin ligand, a novel approach for in vitro transfer of oligosaccharides was utilized (Srivastava, G., Kaun, K. J., Hindsgaul, O., and Palcic, M. M. (1992) J. Biol. Chem. 267, 22356-22361). CHO cells were incubated with synthetic 6′-sulfo sialyl Lex, NeuNAcα2→3(sulfate-6)Galβ1→4(Fucα1→3)GlcNAc or 6-sulfo sialyl Lex, NeuNAcα2→3Galβ1→4[(Fucα1→3)sulfate→6GlcNAc] oligosaccharide linked to C-6 of a fucose residue in GDP-fucose and a milk fucosyltransferase. The resultant CHO cells expressing 6′-sulfo sialyl Lex or 6-sulfo sialyl Lex on their cell surface were tested for adhesion to E-selectin and L-selectin chimeric proteins coated on plates. The results indicate that 6′-sulfo sialyl Lex supports L-selectin-mediated adhesion much better than sialyl Lex similarly tagged on the cell surface. In contrast, 6-sulfo sialyl Lex containing a sulfate group on the N-acetylglucosamine residue did not support adhesion with either selectin. These combined results suggest that 6′-sulfo sialyl Lex is a much better ligand than sialyl Lex oligosaccharide for L-selectin.

Branched O-linked oligosaccharides ectopically expressed in transgenic mice reduce primary T-cell immune responses.

Core 2 β‐1,6‐N‐acetylglucosaminyltransferase, C2GnT, is a key enzyme in O‐linked oligosaccharide (O‐glycan) biosynthesis and the resultant core 2 branch serves as a backbone for additional glycosylation to form oligosaccharide ligands such as sialyl Lex. Since the expression of C2GnT is highly regulated during T‐cell development and increases in pathological conditions such as the Wiskott–Aldrich syndrome, we have generated transgenic mice overexpressing C2GnT in the T‐cell lineage. Surprisingly, T lymphocytes in the transgenic mice develop normally, but they exhibit a reduced immune response when assayed by delayed‐type hypersensitivity, proliferation upon stimulation and cytokine production. Moreover, T lymphocytes from the transgenic mice adhere much less efficiently to ICAM‐1 and fibronectin than do T lymphocytes from non‐transgenic mice. These results indicate that overexpression of the core 2 branched O‐glycans in T lymphocytes results in reduced immune responses due to impaired cell–cell interaction. Such an impaired immune response may be one of the causes for immunodeficiency in the Wiskott–Aldrich syndrome.

Roles of O‐linked oligosaccharides in immune responses

Many functional glycoproteins are expressed on the lymphocyte cell surface. Some of them carry O‐linked oligosaccharides (O‐glycans), which are conjugated through serine or threonine residues. During various biological processes, including T‐cell activation, a tetrasaccharide on the T‐cell surface is dramatically converted to a branched hexasaccharide, called core2 O‐glycan. The same structural change in O‐glycans is also found on the lymphocytes from patients with immunodeficiency conditions such as Wiskott‐Aldrich syndrome and AIDS. Several studies revealing the roles of core2 O‐glycans in immune responses show that this is a biologically significant change. In particular, core2 O‐glycans expressed on the cell surface reduce cell–cell interactions, thereby regulating immune responses. Furthermore, core2 O‐glycan is a key backbone structure in forming selectin ligands. Thus, O‐linked oligosaccharides, in particular those containing core2 branches, play vital roles in immune responses and may play dual roles in certain situations. This review will summarize the results obtained from various studies investigating the roles of O‐glycans in immunological processes. BioEssays 23:46–53, 2001.

MUCIN-TYPE O-GLYCANS AND LEUKOSIALIN

Mucin-type O-glycans on leukocytes acquire functions once they contain core 2 branches, which can be synthesized by core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT). Recently, understanding the roles of mucin-type O-glycans has been significantly advanced by generating transgenic mice overexpressing C2GnT or knockout mice defective in C2GnT. This review article summarizes previous results implicating the roles of mucin-type O-glycans and the most recent studies to test such a hypothesis. These results, taken together, demonstrate that mucin-type O-glycans either facilitate or attenuate cell adhesion depending on the structures of non-reducing termini.

Requirement for a Complex of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein in Podosome Formation in Macrophages

Chemotactic migration of macrophages is critical for the recruitment of leukocytes to inflamed tissues. Macrophages use a specialized adhesive structure called a podosome to migrate. Podosome formation requires the Wiskott-Aldrich syndrome protein (WASP), which is a product of the gene defective in an X-linked inherited immunodeficiency disorder, the Wiskott-Aldrich syndrome. Macrophages from WASP-deficient Wiskott-Aldrich syndrome patients lack podosomes, resulting in defective chemotactic migration. However, the molecular basis for podosome formation is not fully understood. I have shown that the WASP interacting protein (WIP), a binding partner of WASP, plays an important role in podosome formation in macrophages. I showed that WASP bound WIP to form a complex at podosomes and that the knockdown of WIP impairs podosome formation. When WASP binding to WIP was blocked, podosome formation was also impaired. When WASP expression was reduced by small interfering RNA transfection, the amount of the complex of WASP with WIP decreased, resulting in reduced podosome formation. Podosomes were restored by reconstitution of the WASP-WIP complex in WASP knockdown cells. These results indicate that the WASP-WIP complex is required for podosome formation in macrophages. When podosome formation was reduced by blocking WASP binding to WIP, transendothelial migration of macrophages, the most crucial process in macrophage trafficking, was impaired. These results suggest that a complex of WASP with WIP plays a critical role in podosome formation, thereby mediating efficient transendothelial migration of macrophages.

Overexpression of Branched O-Linked Oligosaccharides on T Cell Surface Glycoproteins Impairs Humoral Immune Responses in Transgenic Mice

The aberrant expression of core 2O-glycans on T cell surface glycoproteins has been associated with various immunodeficient syndromes such as Wiskott-Aldrich syndrome and AIDS. To determine the effect of this aberrant expression of core 2 O-glycans on immune responses, we previously generated transgenic mice overexpressing core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) in T cells, and demonstrated that T cell primary immune responses mediated through interaction between T cells and antigen-presenting cells are impaired in the transgenic mice (Tsuboi, S., and Fukuda, M. (1997)EMBO J. 16, 6364–6373). In this study, we determined whether overexpression of core 2 oligosaccharides on T cells leads to impaired humoral immune responses by B cells using the same transgenic mice. When T cells were activated, both T and B cells from the transgenic and control mice expressed an equivalent amount of CD40L and CD40, which are, respectively, the receptor and counter-receptor for the interaction between T and B cells. However, activated T cells from the transgenic mice induced B cell proliferation less efficiently than those from control mice, regardless of whether B cells were isolated from control or the transgenic mice. This suggests that overexpression of core 2 O-glycans on T cell surface glycoproteins renders T cell-B cell interaction inefficient. Moreover, in the transgenic mice both immunoglobulin isotype switching and germinal center formation were also impaired. Taken together, these results indicate that aberrant expression of core 2 O-glycans on T cell surface glycoproteins results in impaired humoral immune responses due to an impaired interaction between T and B cells.

A Complex of Wiskott-Aldrich Syndrome Protein with Mammalian Verprolins Plays an Important Role in Monocyte Chemotaxis

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder, Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells, and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis, resulting in recurrent infection. However, the molecular basis of such chemotactic defects is not understood. Recently, the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP, WIP, and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked, chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition, our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis.