Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells

Abstract

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.

Basal Autophagy Is Required for the Efficient Catabolism of Sialyloligosaccharides

“…The cytosolic sialidase Neu2 is known to catabolize cytosolic sialyl-fOSs (13). We confirmed by quantitative PCR that Neu2 expression is, if it exists at all, very low in both WT cells and Atg5 Ϫ/Ϫ MEF cells (data not shown).…”

“…There are few reports on the occurrence of sialyl-fOSs in the cytosol of mammalian cells (12,13). Most recent studies, however, have shown that similar sialyl-fOSs are accumulated in human pancreatic (33) or prostate cancers (34) but not in normal epithelial cells, thus suggesting that these glycans can serve as useful tumor markers.…”

“…In this novel pathway, endo-␤-N-acetylglucosaminidase (8,9) and ␣-mannosidase (Man2C1) (10,11) were shown to be involved in the degradation of high mannose-type free oligosaccharides (fOSs), 2 some of which are released by peptide:N-glycanase, in the cytosol of mammalian cells. On the other hand, the precise mechanism by which sialylated, complex-type glycans are catabolized in the cytosol of cells and tissues remains unclear (12,13).…”

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

“…This glycan was previously observed in gastric adenocarcinoma cells, and combined studies using MS/MS and sequential digestions with specific glycosidases confirmed that the Man 1-6 arm was removed to form a characteristic biantennary glycan. 51 Nontypical oligosaccharide peaks which were detected at m/z 1103.4 and 1265.4 (Figure 3a) produced MS/MS fragmentation patterns consistent with Hex 6 PHN and Hex 7 PHN compositions. These neutral oligosaccharides were found in all CEM cell line samples and their presence is suspected to be as a result of culturing cells in medium rather than expression in cancer cells.…”

Introduction to “Glycometabolome”

“…Of course, it should be noted that the scheme presented in Fig. 2 is only for high-mannosetype glycans, and degradation pathway for complex type free glycans, which are also reported in the cytosol of mammalian cells and tissues (37,38), is very poorly understood. Moreover, I frankly wonder if anyone has ever seriously examined whether there is any non-lysosomal degradation pathway for other type of glycans, such as mucintype O-glycans (exception is the recent paper by Dr. Ito's group about theˆnding of neutral b-glycosylceramidase, which could be involved in a novel nonlysosomal catabolic pathway of glucosylceramide (39)).…”