Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AglD Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.

Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase.

Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD flippase) is not known. Here we show that MPD-flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO(+) as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD flippase recognizes the dolichol chain of MPD, preferring a saturated alpha-isoprene to unsaturated trans- or cis- alpha-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol flippase that translocates Man(5)GlcNAc(2)-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD flippase.

DPM1, the catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3.

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

In vivo interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-hybrid system.

Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The N-terminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.

Common origin and evolution of glycosyltransferases using Dol-P-monosaccharides as donor substrate.

On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.

Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria.

We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.

Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.

Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5-8.0 in potassium phosphate or Tris/HCl buffers and the enzyme required either Mg2+ or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+ was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.

Activation of GlcNAc-P-P-dolichol synthesis by mannosylphosphoryldolichol is stereospecific and requires a saturated alpha-isoprene unit.

Exogenous mannosylphosphoryldolichol (Man-P-Dol) has previously been shown to stimulate UDP-GlcNAc:dolichyl phosphate N-acetylglucosamine 1-phosphate transferase (GPT1), the enzyme catalyzing the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol). To define the structural specificity of the mannolipid-mediated activation of GPT1, the ability of a variety of mannosylphosphorylisoprenols to stimulate GlcNAc-lipid biosynthesis in microsomal preparations from retinas of the embryonic chick has been tested. For these comparisons several Man-P-isoprenols were synthesized enzymatically and chemically. The catalytic efficiency of activation expressed as the Vmax/Ka ratio was substantially higher for Man-P-Dol95 than for mannosylphosphorylpolyprenol95 (Man-P-Poly95), demonstrating that the saturated alpha-isoprene unit of the dolichyl moiety influences the mannolipid-enzyme interaction. The degree of activation increased with chain length and hydrophobicity of the dolichyl moiety when Man-P-dolichols containing 2, 11, and 19 isoprene units were evaluated. A strict stereospecificity was exhibited as beta-Man-P-Dol95 provided a 100-fold greater stimulation than the corresponding alpha-stereoisomer. The recognition of the saturated alpha-isoprene unit, the influence of chain length, and the strict stereospecificity of the interaction between beta-Man-P-Dol and GPT1 extend the description of the mannolipid-enzyme interaction and provide strong new evidence that Man-P-Dol levels can influence the rate of GlcNAc-P-P-Dol synthesis.

Different fates of the oligosaccharide moieties of lipid intermediates.

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase.

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).