Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.