The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. (1/463)

In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.  (+info)

Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases. (2/463)

The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.  (+info)

Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis. (3/463)

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.  (+info)

Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast. (4/463)

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O-glycosylation. Secretion of GAI expressed in the pmt1-disruptant and in the kre2-disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O-linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O-linked oligosaccharides of GAI from the pmt1-disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2-Man4 were reduced in proportion to the reduction of Man1. The O-linked oligosaccharides of GAI from the kre2-disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O-linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.  (+info)

Recombinant human interleukin-12 is the second example of a C-mannosylated protein. (5/463)

The beta-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319beta is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be found.  (+info)

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana. (6/463)

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.  (+info)

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases. (7/463)

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.  (+info)

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure. (8/463)

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.  (+info)