Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

Abstract

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Figure 1. Expression of human mannosidase in mammalian cells

(A) Western blotting analysis of the cytosolic fraction. Lane 1, HEK-293 cells with mock plasmid; lane 2, HEK-293 cells with FLAG–Man2C1 expression plasmid. Stained with anti-FLAG antibody. (B and C) FLAG–Man2C1 was immunostained (green) with an anti-FLAG antibody. In (C), nuclei were stained with DAPI (blue). Bar=10 μm. (D) Subcellular fractionation of FLAG–Man2C1. Lane 1, cytosolic fraction; lanes 2–4, first, second and third wash of the membrane fraction; lane 5, membrane fraction. PDI is a marker for ER luminal protein. (E) Distribution of FLAG–Man2C1. The results were obtained by quantitation of results in (D).

Figure 2. Purification and characterization of FLAG–Man2C1

(A) Silver staining pattern of purified FLAG–Man2C1. Samples, 5 μl (lane 1) or 10 μl (lanes 2–7) were analysed using 7.5% PAGE. Lane M: molecular mass marker; lane 1, cell extract; lane 2, cytosolic fraction; lanes 3–6, first–fourth wash fractions of anti-FLAG antibody beads; lane 7, eluted fraction. (B) Structure of Man₅GlcNAc-PA prepared from RNase B. Man residues with circles are reported to be cleaved by cytoplasmic α-mannosidase only in the presence of Co²⁺. (C) Effect of various mannosidase inhibitors on the activity of purified FLAG–Man2C1.

Figure 3. Suppression of Man2C1 expression by siRNA

(A) Quantitation of mRNA by real-time PCR. Expression levels of Man2A1, Man2B1 and Man2C1 were normalized by the expression of GAPDH. The expression for each gene without siRNA treatment was set to 1 (left column). (B) Mannosidase activity in the cytosolic fraction was assayed using HEK-293 cells with (left-hand column) or without (right-hand column) siRNA treatment.

Figure 4. Structural characterization of Man₅GlcNAc-PA obtained from the cytosol of HEK-293 cells

MALDI–TOF MS analysis of Man5GlcNAc-PA before (A) and after (B) α1,2-mannosidase treatment. Man, ○; GlcNAc, □.

Figure 5. Proposed processing of oligosaccharides in the cytosol

There are two main sources of oligosaccharides that are known to date; one is derived from cytosolic misfolded glycoproteins (by the action of PNGase), whereas the other is from dolichol-linked oligosaccharides, most likely located on the lumenal side [14]. The lumenal oligosaccharides are translocated from the lumen into the cytosol by a putative transporter [–11]. For glycoprotein degradation, oligosaccharides were believed to be Man8 and shorter [2,3]. Once in the cytosol, ENGase [24,25] or chitobiase [18] forms free oligosaccharides with a single GlcNAc at each reducing terminus (Gn1). Free oligosaccharides with a single GlcNAc are now susceptible to the action of a cytosolic α-mannosidase, giving rise to the Man₅GlcNAc structure. This structure is identical with the last dolichol intermediate present on the cytosolic face. The Man₅GlcNAc is transported into the lysosomes by a specific transporter [15,16]. Man, ○; GlcNAc, □; Glc, ●.