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The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wild-type and beta1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.

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␤1,3-N-acetylglucosaminyltransferase 2 (␤3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated ␤3GnT2-deficient (␤3GnT2 ؊/؊ ) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in ␤3GnT2 ؊/؊ mice. ␤3GnT2 ؊/؊ T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3 /CD28 and proliferated more strongly than T cells from WT mice. ␤3GnT2 ؊/؊ B cells also showed hyperproliferation on BCR stimulation. Macrophages from ␤3GnT2 ؊/؊ mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on Nglycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.␤-1,3-N-acetylglucosaminyltransferase ͉ glycosyltransferase ͉ hyperactivation ͉ immune response

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