Karen E. Pace

Cutting Edge: CD7 Delivers a Pro-Apoptotic Signal During Galectin-1-Induced T Cell Death

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.

CD45 Modulates Galectin-1-Induced T Cell Death: Regulation by Expression of Core 2 O-Glycans

Galectin-1 induces death of immature thymocytes and activated T cells. Galectin-1 binds to T cell-surface glycoproteins CD45, CD43, and CD7, although the precise roles of each receptor in cell death are unknown. We have determined that CD45 can positively and negatively regulate galectin-1-induced T cell death, depending on the glycosylation status of the cells. CD45+ BW5147 T cells lacking the core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) were resistant to galectin-1 death. The inhibitory effect of CD45 in C2GnT− cells appeared to require the CD45 cytoplasmic domain, because Rev1.1 cells expressing only CD45 transmembrane and extracellular domains were susceptible to galectin-1 death. Moreover, treatment with the phosphotyrosine-phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) enhanced galectin-1 susceptibility of CD45+ T cell lines, but had no effect on the death of CD45− T cells, indicating that the CD45 inhibitory effect involved the phosphatase domain. Expression of the C2GnT in CD45+ T cell lines rendered the cells susceptible to galectin-1, while expression of the C2GnT in CD45− cells had no effect on galectin-1 susceptibility. When CD45+ T cells bound to galectin-1 on murine thymic stromal cells, only C2GnT+ T cells underwent death. On C2GnT+ cells, CD45 and galectin-1 co-localized in patches on membrane blebs while no segregation of CD45 was seen on C2GnT− T cells, suggesting that oligosaccharide-mediated clustering of CD45 facilitated galectin-1-induced cell death.

Novel Innate Immune Functions for Galectin-1: Galectin-1 Inhibits Cell Fusion by Nipah Virus Envelope Glycoproteins and Augments Dendritic Cell Secretion of Proinflammatory Cytokines

Galectin-1 (gal-1), an endogenous lectin secreted by a variety of cell types, has pleiotropic immunomodulatory functions, including regulation of lymphocyte survival and cytokine secretion in autoimmune, transplant disease, and parasitic infection models. However, the role of gal-1 in viral infections is unknown. Nipah virus (NiV) is an emerging pathogen that causes severe, often fatal, febrile encephalitis. The primary targets of NiV are endothelial cells. NiV infection of endothelial cells results in cell-cell fusion and syncytia formation triggered by the fusion (F) and attachment (G) envelope glycoproteins of NiV that bear glycan structures recognized by gal-1. In the present study, we report that NiV envelope-mediated cell-cell fusion is blocked by gal-1. This inhibition is specific to the Paramyxoviridae family because gal-1 did not inhibit fusion triggered by envelope glycoproteins of other viruses, including two retroviruses and a pox virus, but inhibited fusion triggered by envelope glycoproteins of the related Hendra virus and another paramyxovirus. The physiologic dimeric form of gal-1 is required for fusion inhibition because a monomeric gal-1 mutant had no inhibitory effect on cell fusion. gal-1 binds to specific N-glycans on NiV glycoproteins and aberrantly oligomerizes NiV-F and NiV-G, indicating a mechanism for fusion inhibition. gal-1 also increases dendritic cell production of proinflammatory cytokines such as IL-6, known to be protective in the setting of other viral diseases such as Ebola infections. Thus, gal-1 may have direct antiviral effects and may also augment the innate immune response against this emerging pathogen.

O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

Preparation of recombinant human galectin-1 and use in T-cell death assays.

Publisher Summary This chapter describes the procedure of preparation of recombinant human galectin-1, and discusses methods used to detect galectin-1-induced death in T cells. Galectin-1 is a member of an evolutionarily conserved family of lectins that share structural similarities within their carbohydrate-recognition domain. The abundant expression of galectin-1 in thymus and peripheral lymphoid organs prompted the examination of the effects of galectin-1 on thymocytes and T cells. While adhesive properties for galectin-1 had been proposed, it is found that galectin-1 induced apoptotic death of immature thymocytes and activated the T cells. The T-cell subsets that were susceptible to galectin-1 were also the T-cell subsets that undergo physiologic cell death during T-cell development in the thymus or during the resolution of the immune response in the periphery. To understand the potential immunomodulatory effects of galectin-1, the mechanism of galectin-1-induced T-cell death is elucidated. The chapter also discusses the purification of recombinant galectin-1, the preparation of β-lactosyl sepharose, use of galectin-1 in T-cell death assays, concentration of galectin-1 in T-cell death assays, cell shrinkage and blebbing, and light-scattering properties of the cells.

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding β-galactoside sugars. Confirming its identity as a galectin family member, theDrosophila galectin bound β-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophilagalectin was very abundant in embryonic, larval, and adultDrosophila. During embryogenesis, Drosophilagalectin had a unique and specific tissue distribution.Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.

Insect galectins: Roles in immunity and development

As evidenced by the reviews in this special issue of Glycoconjugate Journal, much research is focused on determining functions for mammalian galectins. However, the identification of precise functions for mammalian galectins may be complicated by redundancy in tissue expression and in target cell recognition of the many mammalian galectins. Therefore, lower organisms may be useful in deciphering precise functions for galectins. Unfortunately, some genetically manipulable model systems such as Caenorhabditis elegans may have more galectins than mammals.Recently, galectins were identified in two well-studied insect systems, Drosophila melanogaster and Anopheles gambiae. In addition to the powerful genetic manipulation available in these insect models, there is a sophisticated understanding of many biological processes in these organisms that can be directly compared and applied to mammalian systems. Understanding the roles of galectins in insects may provide insight into precise functions of galectins in mammals. Published in 2004.