Free Oligosaccharides in the Cytosol of Caenorhabditis elegans Are Generated through Endoplasmic Reticulum-Golgi Trafficking

Abstract

Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-␤-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man 5 GlcNAc 1 , the M5A isomer (Man␣1-3(Man␣1-6)Man␣1-6(Man␣1-3)Man␤1-4GlcNAc), which is different from the corresponding M5B (Man␣1-2Man␣1-2Man␣1-3(Man␣1-6)Man␤1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi ␣-mannosidase I inhibitors revealed decreases in Man 5 GlcNAc 1 and increases in Man 7 GlcNAc 1 . These results suggested that Golgi ␣-mannosidase I-like enzyme is involved in the production of Man 5-6 -GlcNAc 1 , which is unlike in mammals, in which cytosolic ␣-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi ␣-mannosidase II mutant (tm1078) supported this idea, because GlcNAc 1 Man 5 GlcNAc 1 , which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.It is known that N-linked oligosaccharides play important roles in the quality control of glycoproteins. An oligosaccharide composed of 14 sugars (Glc 3 Man 9 GlcNAc 2 ) is transferred en bloc from a dolichol-linked donor to the Asn residue of nascent polypeptide chains by oligosaccharyltransferase (OST), 2 and the oligosaccharides serve as tags indicating the folding state of glycoproteins in the endoplasmic reticulum (ER). Then, properly folded and assembled glycoproteins are transported to their destinations such as the extracellular space, plasma membrane, or inner compartments via the Golgi apparatus, with some accompanying modifications on the oligosaccharides.The presence of free oligosaccharides (FOSs) has been reported in the cytosol of several types of animal cells (1-6). These FOSs were thought to be generated from the ER by the following pathways. First, ER-associated degradation (ERAD) of glycoproteins is involved in generation of FOS. Misfolded or unassembled glycoproteins are recognized by ER degradationenhancing ␣-mannosidase-like protein (EDEM), retro-translocated into the cytosol through the Sec61 translocon (7) or the Hrd1 complex (8), and subjected to ubiquitin-proteasome degradation. The N-glycans on the misfolded glycoproteins are released by peptide:N-glycanase (PNGase) in the cytosol before the degradation by the proteasome (9, 10). Second, FOS...

Generation and Metabolism of Cytosolic Free Oligosaccharides in Caenorhabditis elegans

“…Most of the FOSs in the wild-type strain were FOS-GN1, whereas in the eng-1 null mutant strain, they were FOS-GN2 (Fig. 3) (46). These results indicate that ENGase is responsible for the conversion of FOS-GN2 into FOS-GN1.…”

“…Class 1 a-mannosidase (GH family 47, the portion below the dashed line) includes ER a-mannosidase I, Golgi a-mannosidase I and EDEM. them by high-performance liquid chromatography (HPLC) (46). Table 1 shows the structure of FOSs in the wild-type C. elegans.…”

Enzymes Involved in Generation and Degradation of the Free Oligosaccharides in the Cytosol of Caenorhabditis elegans

“…In our analyses, the M5A isoform [Manα1 3(Manα1 6)Manα1 6(Manα1 3)Manβ1 4GlcNAc] was the major FOS in the wild type C. elegans, 35) whereas in mammals the M5B isoform [Manα1 2Manα1 2Manα1 3(Manα1 6) Manβ1 4GlcNAc] is the major (Table 1). 37 40) The mammalian M5B isoform is generated by the actions of the cytosolic α mannosidase (Man2C1), which is classified as a class 2 α mannosidase.…”

“…5). 35) The genome of C. elegans contains seven putative class 1 α mannosidases: two ER α mannosidases (T03G11.4 and ZC410.3), two Golgi α mannosidases and three EDEMs (ER degradation enhancing α mannosidase like proteins) (C47E12.3, F10C2.5 and ZC506.1) (Fig. 4).…”

“…35) FOS were extracted from worms, PA labeled, and analyzed by HPLC. FOS GN1 and FOS GN2 could be separated by reverse phase HPLC.…”