Glycobiology Advance Access originally published online on December 27, 2005
Glycobiology 2006 16(4):294-304; doi:10.1093/glycob/cwj074
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Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
1 To whom correspondence should be addressed; e-mail: suhase{at}chem.sci.osaka-u.ac.jp
Received on May 30, 2005; revised on December 15, 2005; accepted on December 26, 2005
It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-ß-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series—series 1: oligosaccharides processed from Man
1-2Man1-6 (Man1-2Man1-3)Man1-6(Man1-2Man1-2Man1-3) Manß1-4GlcNAc (M9A') and Man1-2Man1-6(Man1-3) Man1-6(Man1-2Man1-2Man1-3)Manß1-4GlcNAc (M8A') by digestion with cytosolic -mannosidase; series 2: oligosaccharides processed with Golgi -mannosidases in addition to endoplasmic reticulum (ER) and cytosolic -mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic -mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic -mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Man1-6(Man1-2Man1-2Man1-3)Manß1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic -mannosidase, and Man1-6(Man1- 2Man1-3)Manß1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic -mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi -mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.
Key words: cytosol / endoplasmic reticulum-associated degradation / free oligosaccharides / N-glycosylation / oligosaccharide sequence
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