Pedro A. Romero

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture

1‐Deoxynojirimycin N‐Methyl‐1‐deoxynojirimycin Glucosidase inhibitor Dolichol‐linked oligosaccharide Processing

Short Communication The transmembrane domain of murine α-mannosidase IB is a major determinant of Golgi localization

Summary Murine α1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a “stem” region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine α1,2-mannosidase IB as well as α1,2-mannosidase IB/yeast α1,2-mannosidase and α1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of α1,2-mannosidase IB with that of the homologous yeast α1,2-mannosidase and deleting the “stem” region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the α1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the “stem” region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of α1,2-mannosidase IB is a major determinant of Golgi localization.

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells

The effects of manno‐1‐deoxynojirimycin (ManDJN) and 2,5‐dihydroxymethyl‐3,4‐dihydroxypyrrolidine (DMDP) were compared in IEC‐6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N‐linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H‐sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7–9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7–9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP‐treated cells were non‐glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus‐infected MDCK cells [(1984) J. Biol. Chem. 259, 12409‐12413].

Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with α-1,6 mannosylpolymerase activity

Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 mannose and decorated with alpha-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the alpha-1,6 linked mannose backbone by the M-Pol I complex. Constructs encoding soluble portions of the M-Pol I subunits, Mnn9p and Van1p from Saccharomyces cerevisiae, were expressed in Pichia pastoris. Both subunits had to be expressed in the same strain to obtain the recombinant proteins. Recombinant M-Pol I was made only by the KM71 strain transformed with two vectors: one encoding Mnn9p and the other encoding Van1p. Soluble secreted M-Pol I was purified by sequential chromatography on DEAE-Trisacryl, GDP-Hexanolamine-Sepharose and Superdex 200. Characterization of the purified complex indicates that recombinant M-Pol 1 is a Mnn9p-Van1p heterodimer. Purified M-Pol I was active with alpha-1,6 mannobiose as acceptor and GDP-mannose as donor. HPLC identified five products confirmed to be 3-7 mannose residues long. Digestion with linkage-specific alpha-mannosidases revealed that the linkage formed is exclusively alpha-1,6. No alpha-1,2 mannosyltransferase activity, reported previously for M-Pol I immunoprecipitates from cell extracts was detected. These results provide further information on the role of M-Pol I in mannan biosynthesis.

Effects of tunicamycin, N-methyl-1-deoxynojirimycin, and manno-1-deoxynojirimycin on the biosynthesis of lactosaminoglycans in F9 teratocarcinoma cells

F9 teratocarcinoma cells were incubated with D-[2-3H]mannose or D-[6-3H]galactose, and the labeled glycopeptides obtained after exhaustive digestion by pronase were fractionated on Bio-Gel P-6 before and after treatment by endo-beta-N-acetylglucosaminidase H. Tunicamycin almost completely inhibited the synthesis of lactosaminoglycans found in excluded glycopeptides of large molecular weight. Manno-1-deoxynojirimycin greatly inhibited the incorporation of labeled mannose into both lactosaminoglycan and complex oligosaccharides, while it greatly increased that into Man8GlcNAc and Man9GlcNAc oligosaccharides. In contrast, N-methyl-1-deoxynojirimycin only partially inhibited the incorporation into lactosaminoglycan and complex oligosaccharides, and caused the accumulation of Glc3Man7-9GlcNAc oligosaccharides. These results demonstrate that, in these cells, lactosaminoglycans are N-linked, and suggest that there is transfer of both glucosylated and nonglucosylated oligosaccharides from lipid to protein.

Crystal structure of an alleged mannosylphosphate transferase Ktr6p at 3.06 Å

Ktr6p is an alleged mannosylphosphate transferase from yeast Golgi. It has been implicated in decorating both O-linked and N-lined glycans with mannosylphosphate in vivo. However, based on sequence similarity, Ktr6p belongs to GT15 family of α-1,2mannosyltransferases. To address this disagreement, the soluble portion of Ktr6p was expressed in P. pastoris and purified by liquid chromatography. The purified protein, GDP-mannose and various acceptors were used in a number of direct and indirect activity assays, however, neither manosyltransferase nor mannosylphosphate transferase activity was detected. Ktr6p was crystallized in a number of PEGcontaining conditions, but the crystals resisted all attempts at cryoprotection. Three crystals were used to collect a 3.06 Å resolution dataset on a home source at room temperature. The crystals belong to P 21 21 21 spacegroup with 2 molecules per asymmetric unit. The structure was solved by molecular replacement using a structure of Kre2p, a close homolog from GT15 family (40% sequence identity). The structure was refined to R/Rfree 16.1%/21.2% The overall structure of Ktr6p is very similar to the structure of Kre2p having less than 2 Å overall backbone RMSD. However even at 3 Å resolution the difference in the active site is striking. The guanine moiety binding pocked is occluded by a well-ordered loop making GDP-mannose binding impossible in this conformation. Several aminoacid substitutions in the Mn2+ coordinating environment suggest that Ktr6 does not depend on manganese for its postulated activity. These observations indicate that Ktr6p functions quite differently from Kre2p.