No abstract

No abstract

We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.

Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcR␥-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcR␥, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to ␣-mannose but not mannan. Thus, Mincle may recognize specific geometry of ␣-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/ chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle ؊/؊ mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle ؊/؊ mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.ITAM ͉ macrophages ͉ signal transduction