Lipid moiety of glycosylphosphatidylinositol-anchored proteins contributes to the determination of their final destination in yeast.

Yeasts have two classes of glycosylphosphatidylinositol (GPI)-anchored proteins; one is transferred to the cell wall, whereas the other is retained on the plasma membrane. The lipid moieties of the GPI in Saccharomyces cerevisiae consist of either phosphatidylinositol (PI) or inositolphosphorylceramide (IPC). Cwh43p is involved in the remodeling of lipid from PI to IPC. We found that the GPI lipid moiety of Cwp2p in wild-type cells is PI. To elucidate the physiological role of the lipid remodeling by Cwh43p, we investigated the distribution of Gas1p and Cwp2p by immunoblotting and found that Gas1p with the PI-form GPI lipid moiety in cwh43∆ mutant cells tends to be localized to the cell wall, suggesting that the IPC species in the GPI lipid moiety contributes to the retention of GPI-anchored proteins on the plasma membrane. We also found that CWH43 is genetically related to TED1, which encodes a protein involved in the removal of the ethanolamine phosphate from the second mannose residue in GPI glycan moieties. We propose possible models for the physiological function of Cwh43p and Ted1p in the transfer of GPI-anchored proteins from the plasma membrane to the cell wall.

Identification of Novel Peptidyl Serine α-Galactosyltransferase Gene Family in Plants.

In plants, serine residues in extensin, a cell wall protein, are glycosylated with O-linked galactose. However, the enzyme that is involved in the galactosylation of serine had not yet been identified. To identify the peptidyl serine O-α-galactosyltransferase (SGT), we chose Chlamydomonas reinhardtii as a model. We established an assay system for SGT activity using C. reinhardtii and Arabidopsis thaliana cell extracts. SGT protein was partially purified from cell extracts of C. reinhardtii and analyzed by tandem mass spectrometry to determine its amino acid sequence. The sequence matched the open reading frame XP_001696927 in the C. reinhardtii proteome database, and a corresponding DNA fragment encoding 748 amino acids (BAL63043) was cloned from a C. reinhardtii cDNA library. The 748-amino acid protein (CrSGT1) was produced using a yeast expression system, and the SGT activity was examined. Hydroxylation of proline residues adjacent to a serine in acceptor peptides was required for SGT activity. Genes for proteins containing conserved domains were found in various plant genomes, including A. thaliana and Nicotiana tabacum. The AtSGT1 and NtSGT1 proteins also showed SGT activity when expressed in yeast. In addition, knock-out lines of AtSGT1 and knockdown lines of NtSGT1 showed no or reduced SGT activity. The SGT1 sequence, which contains a conserved DXD motif and a C-terminal membrane spanning region, is the first example of a glycosyltransferase with type I membrane protein topology, and it showed no homology with known glycosyltransferases, indicating that SGT1 belongs to a novel glycosyltransferase gene family existing only in the plant kingdom.

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast.

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI-anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A2 Per1p and O-acyltransferase Gup1p play essential roles in diacylglycerol-type lipid remodelling of GPI-anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphoryl-ceramide by Cwh43p. In this report, we constructed double-mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI-anchored proteins. Based on our analyses of single- and double-mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso-phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag-tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.

Cytotoxic mechanism of selenomethionine in yeast.

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.

Protease-deficient Saccharomyces cerevisiae strains for the synthesis of human-compatible glycoproteins.

Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man5GlcNAc2, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man8GlcNAc2 to Man5GlcNAc2 was observed after integration of Aspergillus saitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-ß by approximately 10-fold.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease.

A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.

Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys.

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

OBJECTIVE: Novel recombinant human lysosomal ß-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. METHODS: A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. RESULTS: The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. INTERPRETATION: This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases.

Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain.

This report describes a novel method for overexpression of (13)C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly (13)C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man(8)GlcNAc(2) oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, (13)C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific (13)C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The (13)C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.

Essential role of YlMPO1, a novel Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN4, in mannosylphosphorylation of N- and O-linked glycans.

Mannosylphosphorylation of N- and O-glycans, which confers negative charges on the surfaces of cells, requires the functions of both MNN4 and MNN6 in Saccharomyces cerevisiae. To identify genes relevant to mannosylphosphorylation in the dimorphic yeast Yarrowia lipolytica, the molecular functions of five Y. lipolytica genes showing significant sequence homology with S. cerevisiae MNN4 and MNN6 were investigated. A set of mutant strains in which Y. lipolytica MNN4 and MNN6 homologues were deleted underwent glycan structure analysis. In contrast to S. cerevisiae MNN4 (ScMNN4), the Y. lipolytica MNN4 homologue, MPO1 (YlMPO1), encodes a protein that lacks the long KKKKEEEE repeat domain at its C terminus. Moreover, just a single disruption of YlMPO1 resulted in complete disappearance of the acidic sugar moiety in both the N- and O-linked glycan profiles. In contrast, even quadruple disruption of all ScMNN6 homologues, designated YlKTR1, YlKTR2, YlKTR3, and YlKTR4, resulted in no apparent reduction in acidic sugar moieties. These findings strongly indicate that YlMpo1p performs a significant role in mannosylphosphorylation in Y. lipolytica with no involvement of the Mnn6p homologues. Mutant strains harboring the YlMPO1 gene disruption may serve as useful platforms for engineering Y. lipolytica glycosylation pathways for humanized glycans without any yeast-specific acidic modifications.

Characterization of endoplasmic reticulum-localized UDP-D-galactose: hydroxyproline O-galactosyltransferase using synthetic peptide substrates in Arabidopsis.

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35 degrees C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.

An NDPase links ADAM protease glycosylation with organ morphogenesis in C. elegans.

In the nematode Caenorhabditis elegans, the gonad acquires two U-shaped arms through the directed migration of its distal tip cells (DTCs), which are located at the tip of the growing gonad arms. A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, regulates directional migration of DTCs: MIG-17 is synthesized and secreted from the muscle cells of the body wall, and diffuses to the gonad where it is required for DTC migration. The mig-23 mutation causes defective migration of DTCs and interacts genetically with mig-17. Here, we report that mig-23 encodes a membrane-bound nucleoside diphosphatase (NDPase) required for glycosylation and proper localization of MIG-17. Our findings indicate that an NDPase affects organ morphogenesis through glycosylation of the MIG-17 ADAM protease.

Engineering of mucin-type human glycoproteins in yeast cells.

Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucin-type sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.

Mutation of high-affinity methionine permease contributes to selenomethionyl protein production in Saccharomyces cerevisiae.

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

Saccharomyces cerevisiae CWH43 is involved in the remodeling of the lipid moiety of GPI anchors to ceramides.

The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities.

Fatty acid remodeling of GPI-anchored proteins is required for their raft association.

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3. The surface GPI-APs isolated from the PGAP2 and -3 double-mutant Chinese hamster ovary (CHO) cells had unsaturated chains, such as oleic, arachidonic, and docosatetraenoic acids in the sn-2 position, whereas those from wild-type CHO cells had exclusively stearic acid, a saturated chain, indicating that the sn-2 chain is exchanged to a saturated chain. We then assessed the association of GPI-APs with lipid rafts. Recovery of unremodeled GPI-APs from the double-mutant cells in the detergent-resistant membrane fraction was very low, indicating that GPI-APs become competent to be incorporated into lipid rafts by PGAP3- and PGAP2-mediated fatty acid remodeling. We also show that the remodeling requires the preceding PGAP1-mediated deacylation from inositol of GPI-APs in the endoplasmic reticulum.

Lipid remodeling of GPI-anchored proteins and its function.

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.

Production of human beta-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene.

Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human beta-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although the therapeutic effect was observed, in order to obtain the higher therapeutic effect with a little dose as possible, increased phosphorylation of recombinant beta-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, the overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a beta-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that the overexpression of MNN4 caused a 3-fold increase in phosphorylated N-glycans of recombinant beta-hexosaminidase A. The recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that beta-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.