Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2

Author: Yanagida Kanta   Natsuka Shunji   Hase Sumihiro  

Publisher: Oxford University Press

ISSN: 1460-2423

Source: Glycobiology, Vol.16, Iss.4, 2006-04, pp. : 294-304

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

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--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 Manα1-2Manα1-6 (Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3) Man1-4GlcNAc (M9A′) and Manα1-2Manα1-6(Manα1-3) Manα1-6(Manα1-2Manα1-2Manα1-3)Man1-4GlcNAc (M8A′) by digestion with cytosolic α-mannosidase; series 2: oligosaccharides processed with Golgi α-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic α-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic α-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 α-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 Manα1-6(Manα1-2Manα1-2Manα1-3)Man1-4GlcNAc (M5B′), the end product from M9A′ by digestion with cytosolic α-mannosidase, and Manα1-6(Manα1- 2Manα1-3)Man1-4GlcNAc, derived from series “2” oligosaccharides by digestion with cytosolic α-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi α-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.

Related content