title: Probing the cis interactions of the inhibitory receptor Siglec-7 with alpha2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of alpha2,8-sialyltransferase gene expression.
abstract: Siglec-7 is a CD33-related sialic acid-binding Ig-like lectin expressed strongly on NK cells, where it can function as an inhibitory receptor. Its sialic acid-binding activity on NK cells is masked by cis interactions with sialylated glycans, which are likely to be important for regulating the inhibitory function of Siglec-7, which exhibits an unusual preference for alpha2,8-linked disialic acids, a motif found in "b-series" gangliosides and some glycoproteins. To investigate the presence of alpha2,8-linked disialic acids on NK cells, T cells, monocytes, and B cells, we first analyzed their expression of all known alpha2,8-sialyltransferase genes by quantitative PCR. Unlike T cells, B cells, and monocytes, NK cells consistently expressed mRNA encoding ST8Sia VI, which creates alpha2,8-linked disialic acids on O-linked glycans of glycoproteins. All blood leukocytes expressed ST8Sia IV, implicated in polysialic acid synthesis, and NK cells variably expressed high levels of ST8Sia V mRNA required for GT3 expression. Two human IgM antibodies, Ha1 and Pi1, with specificity for the alpha2,8-disialyl motif reacted strongly with NK cells in a sialic acid-dependent manner and less strongly with T cells and monocytes. Antibody-induced clustering of Siglec-7 on NK cells resulted in partial colocalization with anti-Ha1. Finally, MALDI-TOF mass spectrometric analysis of isolated NK cell O-glycans revealed the presence of a peak at mass-to-charge ratio of 1619.4 mass units, corresponding to a putative alpha2,8-disialylated glycan. Together, these results suggest that NK cells are decorated with alpha2,8-disialic acid structures implicated in regulation of cellular activation via interactions with Siglec-7.
authors.affiliation: Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Avril
authors.forename: Simon J
authors.initials: SJ
authors.lastname: North
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Hugh J
authors.initials: HJ
authors.lastname: Willison
authors.forename: Paul R
authors.initials: PR
authors.lastname: Crocker
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D037102:Lectins / Q000276:immunology* / Q000378:metabolism; D007962:Leukocytes / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000201:enzymology / Q000276:immunology; D008024:Ligands; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D012799:Sialyltransferases / Q000096:biosynthesis / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D015214:Antigens, Differentiation, Myelomonocytic; D037102:Lectins; D008024:Ligands; D011134:Polysaccharides; D012333:RNA, Messenger; C402206:SIGLEC7 protein, human; D012794:Sialic Acids; D012799:Sialyltransferases; C464440:alpha2,8-sialyltransferase VI