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Published online before print July 20, 2006

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1005559

Received for publication October 4, 2005.
Revised June 19, 2006.
Accepted for publication June 20, 2006.

Article

Probing the cis interactions of the inhibitory receptor Siglec-7 with 2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of 2,8-sialyltransferase gene expression

Tony Avril ^*, Simon J. North , Stuart M. Haslam , Hugh J. Willison , and Paul R. Crocker ^*@

*Division of Cell Biology and Immunology, Wellcome Trust Biocentre, University of Dundee, United Kingdom; Division of Molecular Biosciences, Imperial College London, South Kensington Campus, United Kingdom; and University Department of Neurology, Southern General Hospital, Glasgow, United Kingdom

^@ To whom correspondence should be addressed. E-mail: p.r.crocker{at}dundee.ac.uk.

	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 2,8-linked disialic acids, a motif found in "b-series" gangliosides and some glycoproteins. To investigate the presence of 2,8-linked disialic acids on NK cells, T cells, monocytes, and B cells, we first analyzed their expression of all known 2,8-sialyltransferase genes by quantitative PCR. Unlike T cells, B cells, and monocytes, NK cells consistently expressed mRNA, encoding ST8Sia VI, which creates 2,8-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 2,8-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 2,8-disialylated glycan. Together, these results suggest that NK cells are decorated with 2,8-disialic acid structures implicated in regulation of cellular activation via interactions with Siglec-7.

Key Words: NK cells • sialic acid • MALDI-TOF

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