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.

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 mucintype sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 ␤1-3 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.glycosylation engineering ͉ mucin-type glycan ͉ podoplanin M ucin-type glycosylation is one of the most abundant posttranslational modifications. The modification is initiated by O-linked N-acetylgalactosamine (GalNAc) to Ser or Thr residues on a peptide backbone and is involved in a variety of important biological processes, such as processing of hormones (1), endocytosis (2), and sorting of apical proteins in the Drosophila embryo (3). Mucin-type glycans are sometimes clustered, forming the ''mucin domain'' found on both membrane-bound (4) and secreted mucins (5), which function as a selective molecular barrier at the epithelial surface (6) and are involved in morphogenetic signal transduction (7). It is also known that changes in expression of mucin and in their glycosylation state are closely associated with the development of cancer and cancer-related processes such as cell growth, differentiation, adhesion, invasion, and immune surveillance (8). Immunohistochemical studies have identified several tumor-associated antigens (TAAs) of adenocarcinoma (9), and most TAAs on mucins were originally found as sialylated mucin-type glycans (10). Podoplanin (also called aggrus) (11, 12), one of mucin-type glycoproteins, acts as a platelet-aggregating factor for cancer cells, a finding that has attracted recent attention because it is well known that the platelet-aggregating activity of cancer cells influences tumor metastasis. In fact, the anti-human podoplanin antibody NZ-1 has an inhibitory effect on lung colonization of human podoplanintransfected cells (13). Additionally, podoplanin is a potential diagnostic marker for many tumors, including testicular tumors, several squamous cell carcinomas, and brain tumors, and may be ...

Human ␤-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of ␣-and ␤-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using ␣-1,6-mannosyltransferase-deficient (och1⌬) yeast as the host. Genes encoding the ␣-and ␤-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (␣␣) and HexB (␤␤). A total of 57 mg of ␤-hexosaminidase isozymes, of which 13 mg was HexA (␣␤), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the ␤-subunit. The purified HexA was treated with ␣-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ؎ 0.1 and 1.7 ؎ 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the ␤-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.

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.

AnimAl models of obesity are mainly categorized as hypothalamic, genetic and diet-induced obesity [1,2]. Hypothalamic obesity was first identified by Hetherington and Ranson [3] in 1940, based on the observation that obesity was produced by electrical destruction of bilateral ventromedial hypothalamic nuclei (VMH) in rats. Subsequently, hypothalamic obesity has been thought of exclusively as obesity induced by VMH lesions. However, it is now recognized that hypothalamic obesity is also produced by the destrucCell proliferation in visceral organs induced by ventromedial hypothalamic (VMH) lesions: Development of electrical VMH lesions in mice and resulting pathophysiological profiles Abstract. We have found that ventromedial hypothalamic (VMH) lesions produced by electrocoagulation induce cell proliferation in visceral organs through vagal hyperactivity, and also stimulate regeneration of partially resected liver in rats. To facilitate identification of proliferative and/or regenerative factors at the gene level, we developed electrical production of VMH lesions in mice, for which more genetic information is available compared to rats, and examined the pathophysiological profiles in these mice. Using ddy mice, we produced VMH lesions with reference to the previously reported method in rats. We then examined the pathophysiological profiles of the VMH-lesioned mice. Electrical VMH lesions in mice were produced using the following coordinates: 1.6 mm posterior to the bregma, anteriorly; 0.5 mm lateral to the midsagittal line, transversely; and 0.2 mm above the base of the skull, vertically, with 1 mA of current intensity and 10 s duration. The VMH-lesioned mice showed similar metabolic characteristics to those of VMH-lesioned rats, including body weight gain, increased food intake, increased percentage body fat, and elevated serum insulin and leptin. However, there were some differences in short period of hyperphagia, and in normal serum lipids compared to those of VMHlesioned rats. The mice showed a similar cell proliferation in visceral organs, including stomach, small intestine, liver, and, exocrine and endocrine pancreas. In conclusion, procedures for development of VMH lesions in mice by electrocoagulation were developed and the VMH-lesioned mice showed pathophysiological profiles similar to those of VMH-lesioned rats, particularly in cell proliferation in visceral organs. These findings have not been observed previously in gold thioglucoseinduced VMH-lesioned mice. This model may be a new tool for identifying factors involved in cell proliferation or regeneration in visceral organs. [5,6], and biochemical changes including hypergastric acid secretion [7,8], hyperlipidemia [1], hyperinsulinemia [1,9], and hyperleptinemia [10,11]. Furthermore, we found cell proliferation in visceral organs (liver, stomach, small intestine, and pancreas) in these rats using [3 H] thymidine uptake [12][13][14]. We also found that cell proliferation in vis-