Yoshifumi Jigami

PRODUCTION OF HUMAN COMPATIBLE HIGH MANNOSE-TYPE (MAN5GLCNAC2) SUGAR CHAINS IN SACCHAROMYCES CEREVISIAE

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for α-1,2-mannosidase with the “HDEL” endoplasmic reticulum retention/retrieval tag was designed and expressed inSaccharomyces cerevisiae. An in vitroα-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an α-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

Mannosylphosphate transfer to yeast mannan

Mannoproteins located in the outermost layer of yeast cell wall determine the walls porosity and thereby regulate leakage of proteins from the periplasmic space and entrance of macromolecules from the environment. In several yeasts, including Saccharomyces cerevisiae, the glycan portion of mannoproteins is composed not only of neutral oligosaccharides containing mannose and N-acetylglucosamine, but also of acidic oligosaccharides containing mannosylphosphate. The mannosylphosphate residues confer a net negative charge on the cell wall, and so change the properties and environment of the cell surface. Progress on mannosylphosphorylation and its regulation in S. cerevisiae is summarized. Two genes required for mannosylphosphate transfer, MNN4 and MNN6, have been cloned, and a functional analysis of these genes suggests a mechanism for mannosylphosphate transfer. Possible functions for mannosylphosphate transfer in yeast are also discussed. These include supply of GMP for sugar nucleotide transport in the Golgi, cross-linking of mannoproteins to beta-glucan, and a cellular stress response to environmental changes. Glycans in pathogenic yeast and protozoa are also modified with mannosylphosphate, and the potential contribution of this modification to the pathogenicity of these organisms is evaluated.

Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion

UDP-l-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-d-glucose to UDP-l-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-d-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, and UDP-4-keto-l-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1–370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371–667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-l-rhamnose via an UDP-4-keto-6-deoxy-d-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His6-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-d-glucose than for dTDP-d-glucose, the preferred substrate for dTDP-d-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-l-rhamnose, UDP-d-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-d-glucose and UDP-l-rhamnose via feedback inhibition by UDP-l-rhamnose and UDP-d-xylose.

YND1, a Homologue of GDA1, Encodes Membrane-bound Apyrase Required for Golgi N- and O-Glycosylation in Saccharomyces cerevisiae*

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in thegda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and ∼50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Δynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Δgda1disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Δynd1Δgda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells.

MNN6, a Member of the KRE2/MNT1 Family, Is the Gene for Mannosylphosphate Transfer in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae theN-linked sugar chain is modified at different positions by the addition of mannosylphosphate. The mnn6 mutant is deficient in the mannosylphosphate transferase activity toward mannotetraose (Karson, E. M., and Ballou, C. E. (1978) J. Biol. Chem. 253, 6484–6492). We have cloned the MNN6gene by complementation. It has encoded a 446-amino acid polypeptide with the characteristics of type II membrane protein. The deduced Mnn6p showed a significant similarity to Kre2p/Mnt1p, a Golgi α-1,2-mannosyltransferase involved in O-glycosylation. The null mutant of MNN6 showed a normal cell growth, less binding to Alcian blue, hypersensitivity to Calcoflour White and hygromycin B, and diminished mannosylphosphate transferase activity toward the endoplasmic reticulum core oligosaccharide acceptors (Man8GlcNAc2-PA and Man5GlcNAc2-PA) in vitro, suggesting the involvement of the MNN6 gene in the endoplasmic reticulum core oligosaccharide phosphorylation. However, no differences were observed in N-linked mannoprotein oligosaccharides between Δoch1 Δmnn1 cells andΔoch1Δmnn1Δmnn6 cells, indicating the existence of redundant genes required for the core oligosaccharide phosphorylation. Based on a dramatic decrease in polymannose outer chain phosphorylation by MNN6 gene disruption and a determination of the mannosylphosphorylation site in the acceptor, it is postulated that theMNN6 gene may be a structural gene encoding a mannosylphosphate transferase, which recognizes any oligosaccharides with at least one α-1,2-linked mannobiose unit.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice

AbstractWe compared two recombinant α-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific α-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h−1 mg protein−1, respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in α-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae

Mannosylphosphorylation is a major oligosaccharide modification that provides negative charge in the Saccharomyces cerevisiae cell wall. Although two genes, MNN6 and MNN4, which encode a mannosylphosphate transferase and its putative positive regulator, respectively, are involved in this modification, the amount of Mnn4p has been found to be a limiting factor for mannosylphosphorylation. The level of mannosylphosphorylation increased at late‐logarithmic and stationary phases of cell growth, and this was correlated to the transcriptional enhancement of MNN4. We also find that mannosylphosphate transfer to mannan is negatively regulated by the protein kinase A pathway, while the presence of 0.5 M potassium chloride enhanced MNN4 transcription. This type of transcriptional regulation is observed in many stress response genes, implying that mannosylphosphate transfer is involved in the cellular response to a variety of stresses.

High level expression of the synthetic human lysozyme gene in Aspergillus oryzae

SummaryAspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed of a higher level compared with the amyB gene because of its multi-copy intergration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

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-β-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 α-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.

GWT1 Gene Is Required for Inositol Acylation of Glycosylphosphatidylinositol Anchors in Yeast

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to β1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029–1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Δgwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Δgwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25–32), is due to the properties of Gwt1p itself and not to other membrane components.