Professor, Department of Pathology and Laboratory Medicine
Why study Glycobiology?
Sugar molecules attached to proteins expressed at the cell surface are increasingly recognized as playing an important role in the control of cell-cell interaction. Specific oligosaccharides can be recognized by sugar binding proteins (so called lectins) and this interaction has the potential to control how a cell interacts with other cells. We are studying the enzymes, "glycosyltransferases" that allow formation of oligosaccharide groups made from sugars such as sialic acid, fucose, galactose and N-acetylglucosamine to form ligands for selectins [see diagram 2]. Expression of selectin ligands for instance on cells of the immune system is required for these cells to migrate to a site of inflammation, leave the blood vessel and migrate into the tissue where they then participate in the immune response to e.g. a pathogen. Understanding the mechanisms that control the activities of glycosyltransferases that lead to formation of selectin ligands will thus lead to a better understanding of processes that control migration of cells of the immune system to sites of inflammation.
A brief introduction to O-glycan formation: A major fraction of the outer cell surface is covered by carbohydrate attached to lipids or proteins. These carbohydrate moieties are thought to play an important role in cell-cell interaction or modulation of biological processes. Carbohydrates can be attached to proteins in two major ways resulting in so called N-glycans and O-glycans. N-glycans have N-acetylglucosamine (GlcNAc) linked to the amide group of asparagine residues while carbohydrate of O-glycans is attached to hydroxyl groups of certain amino acids (mainly serine and threonine). O-glycans can be further divided into multiple subgroups depending on the nature of the amino acid residue and sugar group involved in the carbohydrate-protein linkage. i) in mucin-type O-glycoproteins N-acetylgalactosamine (GalNAc) is linked to serine or threonine; ii) for intracellular glycoproteins N-acetylglucosamine is linked to serine or threonine and iii) for proteoglycans xylose is linked to serine or threonine. Members of the first of these O-glycan subgroups are the focus of interest of our laboratory.
Core carbohydrate structures of mucin type O-glycans: Mucin type O-glycosylation is initiated by enzymatic addition of GalNAc to serine or threonine by the UDP-GalNAcT:polypeptide N-acetyl-galactosaminyltransferase family of enzymes in the Golgi. Depending on which saccharide groups are subsequently attached to this first GalNAc residue, mucin O-glycans are divided into four major subtypes [see diagram 1]. 1) The core 1 structure is formed by addition of galactose to form Gal b1-3GalNAc-aSer/Thr. 2) The core 2 structure requires the core 1 structure as substrate and is formed by addition of GlcNAc to form Galb1-3 (GlcNAcb1-6) GalNAc-aSer/Thr. 3) The core 3 structure is formed by the addition of GlcNAc to form GlcNAc b1-3GalNAc-aSer/Thr. 4) The core 4 structure requires the core 3 structure as substrate and is formed by addition of GlcNAc to form GlcNacb1-3 (GlcNAcb1-6) GalNAc-aSer/Thr. Other modifications to the core GalNAc structure have also been found but appear to be uncommon. Of the four main core O-glycan structures the core 1 and 2 structures are widely distributed while the core 3 and core 4 structures are less common and expression has been mostly associated with mucin producing tissue of the digestive tract. Commonly the core 2 and the core 4 branches are elongated with one or multiple lactosamine structures (Galb1-4GlcNAc).
The core 2 b-1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) enzyme family:C2GlcNAcT isoenzymes create the core 2 O-glycan branch by adding GlcNAc to the Gal b1-3 GalNAc core 1 structure (see above) expressed on Ser or Thr residues. So far three separate C2GlcNAcT enzymes, termed C2GlcNAcT-I, C2GlcNAcT-II and C2GlcNAcT-III have been described and regulation of C2GlcNAcT enzyme activity is thought to be important because addition of lactosamine structures and subsequent modification with fucose and sialic acid results in the formation of Lex and sialyl-Lex sugar groups [see diagram 2] that constitute ligands of selectins. C2GlcNAcT-I, the most studied of the three isoenzymes, is widely expressed and its contribution to the biosynthesis of selectin ligands has been well established. C2GlcNAcT-II is expressed in mucous epithelial cells, where it participates in mucin production and C2GlcNAcT-III is highly expressed in the thymus, possibly reflecting a unique role in T cell development. Mice with deficiencies in C2GlcNAcT-II or C2GlcNAcT-III have not yet been reported, whereas analyses of C2GlcNAcT-Inull mice have shown that it is essential for P-selectin ligand formation on myeloid cells and activated T cells. Interestingly C2GlcNAcT-Inull mice have a relatively mild phenotypee. They have neutrophilia and show reduced binding of peripheral blood neutrophils to selectins, indicating a role of C2GlcNAcT in myeloid homeostasis and inflammation. C2GlcNAcT-III has been shown in an in vivo model to contribute to P-selectin ligand formation in activated CD8 T cells, whether C2GlcNAcT-II contributes under physiological conditions to selectin ligand formation and cell trafficking has not yet been shown.
An excellent textbook to introduce you to the field is: "Essentials of Glycobiology" Edited By Ajit Varki et al.
Research Interests in the Ziltener Lab:
CD43: A long-term interest of our research group has been the study of CD43, a member of the leukocyte mucin family of glycoproteins expressed by all hemopoietic cells with the exception of erythrocytes and plasma cells. CD43 is considered to be the most abundant cell surface molecule expressed on lymph-hemopoietic cells and is thought to paradoxically exhibit both anti-adhesive and pro-adhesive activities. Reports on CD43 function are somewhat controversial with different laboratories reporting contradictory findings. Mice deficient in CD43 have a surprisingly mild phenotype displaying increased T cell adhesiveness and T cell hyper-responsiveness to mitogens and alloantigens. We investigated whether T cell development was perturbed in these mice. Analysis of T cell development in mice that carried the CD43null mutation and a male antigen specific T cell receptor transgene (HY male antigen) revealed that neither positive T cell selection in female mice nor negative T cell selection in male mice were affected by loss of CD43. These observations were surprising in light of the reported hyper-responsiveness of CD43null T cells and we re-examined T cell responsiveness in CD43null T cells. We found that CD43+ and CD43null littermates on the C57Bl/6 background exhibited no differences in response to mitogen. The previous reports of a hyper-responsive phenotype of CD43null mice is likely due to the mixed 129xC57Bl/6 genetic origin of these mice.
We and others have more recently been able to show that CD43 can express core 2 dependent E-selectin ligands. However, in contrast to reports from other laboratories, competitive in vivo recruitment assays carried out in our laboratory using a cutaneous inflammation model failed to demonstrate a physiological role for this interaction. Similarly there are conflicting reports on CD43 involvement in T cell homing to lymph nodes, while some report CD43 to be a positive regulator of T cell homing others report the opposite. CD43 is also implicated in intracellular signaling and in regulation of apoptosis. Work currently ongoing in our laboratory is focused on the functional significance of CD43 signaling and CD43 shedding.
Development of monoclonal antibodies as tools to study glycosyltransferase activities: In past work we have established the use of anti-CD43 antibodies mAb's 1B11, developed in our laboratory and mAb S7, developed by John Kemp at the University of Iowa College of Medicine, as tools for the analysis of murine CD43 glycoforms expressed on T cells. On activated T cells mAb 1B11 recognizes CD43 130kDa carrying core 2 O-glycan branches while on resting T cells mAb S7 binds to CD43 115kDa that carries core 1 branches. Differential binding of mAb 1B11 and mAb S7 to CD43 offers a convenient way to asses the core 2 activity status of individual T cells. Work by us and others also showed that C2GlcNAcT can furthermore modulate the glycosylation of PSGL-1, CD44, CD45 and the phosphatase RPTP1a and it is now generally accepted that C2GlcNAcT alters O-glycan branching in general.
When hemopoietic cells from CD43null mice were stained with 1B11, CD43-independent binding of 1B11 was observed on peripheral CD8 T cells, while no binding was detected on CD4 T cells, B cells, or bone marrow cells. We have identified the novel 1B11 target as a hyposialylated form of CD45RB exclusively expressed on resting, peripheral CD8 T cells, but not on CD4 T cells or any other cell type. Interestingly, recognition of CD43 and CD45RB by 1B11 is differentially affected by core 2 O-glycan branching and sialic acid. Whereas 1B11 recognition of CD43 on activated T cells required both core 2 O-glycan branching and sialic acid, 1B11 recognition of CD45 only occurred in the absence of core 2 glycosylation and absence of sialic acid. For people using mAb 1B11 we have established some [Rules for the interpretation of 1B11 reactivity].
Cytokine regulation of selectin binding sites: Work on CD43 has allowed our laboratory to develop model systems to study core 2 O-glycan branch formation now recognized to be essential for formation of selectin binding sites recognized by P-selectin [see diagram2] important in the control of recruitment of effector cells to areas of inflammation. Next to C2GlcNAcT enzymes there are other glycosyltransferases that also contribute essential components of the P-selectin ligand structure including fucosyltransferases VII (FucT VII), sialyltransferases and tyrosinesulfotransferases. Cytokines have been implicated in the regulation of the activities of some of these enzymes threby regulating formation of functional selectin binding sites. Earlier studies have shown that FucT VII can be induced by IL-12 and TGFb and that IL-4 can inhibit this glycosyltransferase. We have shown using an in vitro approach that IL-2, IL-15 also could upregulate C2GlcNAcT-I and FucT VII and support formation of functional P-selectin binding epitopes. Interestingly while the activities of several key glycosyltransferases, required for selectin ligand formation in activated T cells, can be controlled by cytokines we subsequently found that in vivo P-selectin ligand formation proceeded as effectively in absence of any of these cytokines pointing to other, as yet undefined, signals that control formation of this important ligand.
Role of PSGL-1 in T cell development and T cell recirculation: The C2GlcNAcT-I glycosyltransferase is constitutively expressed in myeloid cells including hemopoietic stem cells. In order to explore a possible function of branched O-glycans on myeloid development we used C2GlcNAcT-I overexpression strategies and found that in vivo overexpression of C2GlcNAcT-I blocks myeloid but not T cell development, indicating a differential role core 2 O-glycans in myeloid and lymphoid cell development. To further elucidate the significance of C2GlcNAcT-I and its relevant substrate PSGL-1 in lymphohemopoiesis we have, in collaboration with the laboratory of Fabio Rossi, employed the parabiotic animal model to study trafficking of bone marrow stem cells to the thymus. Our work showed that C2GlcNAcT-I dependent interaction of PSGL-1 expressed on thymic progenitor cells with P-selectin expressed on thymic endothelial cells is an important component of the thymic progenitor homing process. P-selectin appears to function as a gatekeeper that controls progenitor importation and the level of thymic P-selectin expression is subject to a feedback loop that is controlled by thymic progenitor content. Our data are the first to highlight a role for P-selectin-PSGL-1 interaction in a non-inflammatory setting, as PSGL-1 adhesive interactions were previously believed to be central and exclusive to the control of effector cell recruitment to sites of inflammation.
Analysis of T cell subset distribution in parabiosis experiments involving PSGL-1 deficient mice provided a second unexpected discovery that PSGL-1 may be required for efficient homing of naïve T cells into lymph nodes. Close examination of the phenomenon uncovered a hitherto unknown chemotaxis enhancing function for PSGL-1. Our data show that the secondary lymphoid chemokines CCL21 and CC19, but not SDF-1, bind PSGL-1 on naïve T cells. This chemokine binding to PSGL-1 is associated with an approximate 100% increase in chemotactic response of resting T cells to CCL21 and CCL19, resulting in a significant enhanced homing efficiency into secondary lymphoid organs. The chemotaxis enhancing effect of PSGL-1 was not observed for B cells and the effect is lost on activated T cells in a C2GlcNAcT-I dependent mechanism. This C2GlcAcT-I dependent loss of enhanced chemotactic response of activated T cells to CCL21 and CCL19 parallels loss of L-selectin shedding after T cell activation and we speculate that both these mechanisms are working together to reduce the potential for activated T cells to re-enter secondary lymphoid organs and direct them to the sites of inflammation. Our discovery of the bi-functional nature of PSGL-1 significantly expands the functional scope of this molecule and suggests reconsideration of previous analyses and conclusions of experiments using PSGL-1 knockout mice or PSGL-1 inhibition experiments.
Seo Wooseok and Hermann J. Ziltener CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis. Blood 114:3567-3577, 2009
Carlow, D.A., M.R. Gold, and H.J. Ziltener. Lymphocytes in the peritoneum home to the omentum and are activated by resident dendritic cells. Journal of Immunology 183:1155-1165 (2009).
Klaus Gossens, Silvia Naus, Stephane Y. Corbel, Shujun Lin, Fabio M. V. Rossi, Jürgen Kast, & Hermann J. Ziltener. Thymic progenitor homing and lymphocyte homeostasis are linked via S1P controlled expression of thymic P-selectin/CCL25. Journal of Experimental Medicine 206:761-778 (2009).
Veerman, Krystle, Michael J. Williams, Kenji Uchimura, Mark S. Singer, Jasmeen S. Merzaban, Douglas A. Carlow, Philip Owen, Jesús Rivera-Nieves, Steven D. Rosen and Hermann J. Ziltener.. Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs. Nature Immunology 8: 532 - 539 (2007).
Carlow DA, Ziltener HJ. CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency. Journal of Immunology 177(9):6450-9 (2006).
Rossi FM, Corbel SY, Merzaban JS, Carlow DA, Gossens K, Duenas J, So L, Yi L, Ziltener HJ. Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1. Nature Immunology 6:626-634 (2005).
Randhawa AK, Ziltener HJ, Merzaban JS, Stokes RW. CD43 is required for optimal growth inhibition of Mycobacterium tuberculosis in macrophages and in mice. Journal of Immunology 175:1805-1812 (2005).
Carlow, D, Williams, M., and Ziltener. H Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo. Journal of Immunology 174(7): 3959-66 (2005).
Tomlinson-Jones, A., Federsppiel, B., Ellies, L.G., Williams M.J., Burgener R., Duronio, V.,Smith, C.A. Takei F. and Ziltener, H.J. Characterization of the activation form of CD43 on murine T lymphocytes. Journal of Immunology. 153: 3426-3439 (1994).