Mannose Efflux from the Cells

Abstract

All mammals have 50 -100 M mannose in their blood. However, the source of the dynamic pool of mannose in blood is unknown. Most of it is thought to be derived from glucose in the cells. We studied mannose uptake and release by various cell types. Interestingly, our results show that mannose taken up by the cells through transporters is handled differently from the mannose released within the cells due to glycan processing of protein-bound oligosaccharides. Although more than 95% of incoming mannose is catabolized, most of the mannose released by intracellular processing is expelled from the cells as free mannose predominantly via a nocodazole-sensitive sugar transporter. Under physiological conditions, incoming mannose is more accessible to hexokinase, whereas mannose released within the cells is protected from HK and therefore has a different fate. Our data also suggest that generation of free mannose due to the processing of glycoconjugates composed of glucosederived mannose and its efflux from the cells can account for most of the mannose found in blood and its steady state maintenance.Mannose is an important constituent of N-glycans. The addition of high mannose oligosaccharides to the newly synthesized polypeptide chain and their subsequent processing provide diversity to N-glycan structures. Mannose residues in N-glycans can be derived from either glycogen/glucose or mannose in the blood. Blood has a metabolically active pool of 50 -150 M mannose in steady state; however, mannose homeostasis is not well understood (1). Dietary contribution is insignificant (2), arguing that most of it is probably derived from an endogenous source. Mannose enters the cells via hexose transporters present at the plasma membrane. It is immediately phosphorylated by hexokinase (HK) 2 and then either catabolized via mannose phosphate isomerase (MPI) or diverted toward glycosylation through phosphomannomutase-2 (PMM2) (Scheme 1).Glycosylation precursors, such as mannose 1-phosphate, GDP-mannose, and dolichol phosphate mannose, are used to assemble lipid-linked oligosaccharides (LLO). The glycan portion is then transferred to proteins (3). Mannose, which is incorporated into N-glycans, is trimmed down by mannosidases within the endoplasmic reticulum (ER) and Golgi to generate free mannose. In this study, we show that although more than 95% mannose entering the cell is catabolized, surprisingly, more than 50% of mannose derived from glycan processing and degradation is protected from catabolism and exits the cells as free mannose, most probably via a transporter. This mannose efflux from the cells can account for the majority of mannose found in mammalian blood. EXPERIMENTAL PROCEDURESMaterials-Most of the reagents were purchased from Sigma except BCA protein assay reagent (Pierce). ␣-Minimal essential medium, Dulbecco's modified Eagle's medium (DMEM) with and without glucose, DMEM/F-12 (1:1) medium, and equine serum were from Invitrogen. Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT). Concanavalin A-Sepharo...

Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts

“…5, c and d). [4][5][6][7][8][9][10][11][12][13] C]Mannose incorporation in N-glycans from CDG-Ia fibroblasts increased from 9% (without inhibitor) to 24% (with inhibitor) in the presence of 50 M mannose in CDG-Ia fibroblasts (Fig. 5d).…”

“…CDG-Ib patients can be treated with dietary mannose supplements; however, CDG-Ia patients do not respond to mannose therapy. PMM2 competes with MPI for the common substrate, Man-6-P, the majority of which is catabolized by MPI, leaving only a very small proportion for glycosylation (5). We hypothesized that inhibition of MPI would increase Man-6-P toward glycosylation via residual PMM2 activity in CDG-Ia patients.…”

“…The 13 C/( 12 C ϩ 13 C) ratios were used to calculate isotopic labeling proportion. For labeled hexoses, a fragmentation series of m/z 242 and 314 represents the [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]glucose origin, and m/z 217 and 314 represent the [4-13 C]mannose origin. Glucose contribution to mannose was calculated as an average of spectra intensity ratios at m244/(m242 ϩ m244) and m316/(m314 ϩ m316).…”

“…Because both PMM2 and MPI compete for Man-6-P, we hypothesized that the PMM2/MPI ratio partially determined the amount of mannose toward glycosylation. We labeled nine different cell lines with [6-3 H]glucose and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]mannose and plotted the mannose contribution in N-glycans against the PMM2/MPI ratio ( Fig. 1).…”

“…After 4 h of incubation, the cells were washed thrice with PBS and extracted with chloroform-methanol (2:1), deionized water, and chloroform-methanol-water (10:10:3) solution (5). The cell pellet was dried and resuspended in PBS, 1% SDS, and N-glycans were released by N-glycosidase F. Purified glycans were hydrolyzed by 2 M trifluoroacetic acid at 100°C and dried, and the hydrolysate was derivatized as described in Ref.…”