PMC

LLO profiles in Normal and Mpi^−/− fibroblasts in response to mannose. Cells were incubated in the presence of the indicted amount of mannose in medium containing 5.5 mM glucose and 20% FBS for 24 hours and LLO glycans prepared and analyzed in the Experimental Procedures. Identity of the glycans was determined by comparison with authentic standards. Representative experiments are shown Panels: (a) control mouse fibroblasts, 0μM mannose; (b), control mouse fibroblasts, 500μM mannose; (c), Mpi^−/− mouse fibroblasts 0μM mannose; (d), Mpi^−/− mouse fibroblasts 500μM mannose; (e), Mpi^−/− mouse fibroblasts 0μM mannose; (f), Mpi^−/− mouse fibroblasts 20μM mannose; (g), Mpi^−/−mouse fibroblasts 100μM mannose; (h), Mpi^−/− mouse fibroblasts 500μM mannose.

(A) Effect of mannose on weight loss rate. Normal mice (Control); Mice treated with 2,000 mg/kg mannose (Man); Mice treated with DSS (DSS); Mice treated with DSS and 1,000 mg/kg mannose (DSS + L Man); Mice treated with DSS and 2,000 mg/kg mannose (DSS + H Man). (B) Effect of mannose on DAI. (C) Representative photos and the length of colon. (D) Representative H&E staining of the colon from each group of mice. (E) Effect of mannose on oxidative stress (GSH, MPO, and LPO). (F) Effect of mannose on the intestinal barrier. Data represent the mean ± standard deviation (n = 10). DSS, dextran sodium sulfate; DAI, disease activity index; GSH, reduced glutathione; MPO, myeloperoxidase; LPO, lipid peroxide. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the DSS group.

Combination treatment with mannose diet and PMI suppression exerts antileukemia activity ex vivo and in vivo in a leukemia mouse model. (A) Schematic outline of the blast crisis CML and MLL‐AF9 mouse model. Blast crisis CML cells with shScr or shPmi were transplanted into sublethally irradiated mice. (B) Evaluation of glycolytic activity by mannose or fructose of normal whole bone marrow cells (WBM), c‐Kit⁻ fractions from normal bone marrow, blast crisis CML cells, and MLL‐AF9 leukemia cells. (C, D) Leukemia cells infected with shScr or shPmi were cultured in methylcellulose medium with or without mannose. Graph shows the number of colonies and cell counts of blast crisis CML (C) and MLL‐AF9 (D). Addition of mannose decreased cell proliferation, especially in shPmi. (E) Secondary recipients of 10⁵ blast crisis CML cells transduced with shScr or shPmi were fed a normal or mannose diet. Recipient mice fed mannose lived significantly longer, and survival was significantly extended when the mannose diet was combined with shPmi (shScr, normal diet, n = 7; shPmi, normal diet, n = 7; shScr, mannose diet, n = 8; shPmi, mannose diet, n = 8). (F) Mannose diet significantly decreased the total numbers of GFP⁺ leukemia cells (n = 5). (G) Secondary recipient mice of 10⁴ MLL‐AF9 cells were fed a normal or mannose diet. Recipient mice fed mannose lived significantly longer (normal diet, n = 9, mannose diet, n = 8). The significance of differences in survival between cohorts was assessed by a log‐rank test. *P < .05; **P < .01; ***P < .001

(A) Glycan modificatons of Bl-Eng2 identified included N-linked (indicated by square) and O-linked glycans (indicated by circle) with (shown as red with P) and without phosphorylation. (B) Glycosylation sites and glycoform annotation of rBl-Eng2. The O-linked mannose residues at the N-terminus, including the catalytic GH16 domain and surrounding sequence, are short and range from 1 to 3 mannoses. The number in the circle indicates the number of mannose residues. Green circle means mannose, red circle means phosphorylated mannose, with the number in red circle indicating the position of mannose being phosphorylated. As shown, the O-linked glycans at the C-terminus are generally longer and have size ranging between 2 and 21 mannose residues. Glycosylation of the Serine/Threonine rich region was not profiled due to lack of enzyme cleavage sites. (C) Annotated electron transfer/higher-energy collision dissociation (EThcD) tandem mass spectrum of a representative O-linked glycopeptide (QKLIS_{Hex(7)phospho}EE) of rBl-Eng2, Hex = Mannose. Singly charged Y-ions (peptide plus glycan remnant) are annotated along the top. Blue asterisks (*) represent the deamination peaks. A series of sequence-specific b (labeled in blue) and y (labeled in red) type of fragment ions enable derivation of amino acid sequence of the peptide. A series of sequential loss of mannose peaks (-162 Da, indicated by green circles between two dashed lines above the blue double arrowhead) suggests a total of 7 mannose residues modified at the serine residue. Two adjacent peaks with identical number of mannose residues with 80 Da mass difference (shown as–P above the yellow double arrowhead) suggest the loss of phosphorylation, which modifies the 2^nd mannose residue attached to the serine residue of the O-glycopeptide. The oxonium ions generated from glycan cleavage by HCD suggest the presence of various glycan moieties, annotated in green, in the low m/z region. (D) Glycan linkage information revealed by GCMS analysis. 1,2-linked mannose and terminal mannose are the major linkages of the glycans, with trace amounts of 1,4-linked Mannose, 1,6-linked mannose, 2,3-linked mannose and 2,6-linked mannose. Relative percentages of these glycans are shown on the right side of the panel.