Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2.

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing ß-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

O-Glycan-Dependent Interaction between MUC1 Glycopeptide and MY.1E12 Antibody by NMR, Molecular Dynamics and Docking Simulations.

Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.1E12 is a promising anti-MUC1 antibody with a distinct specificity toward MUC1 modified with an immature O-glycan (NeuAcα(2-3)Galß(1-3)GalNAc) on a specific Thr. However, the structural basis for the interaction between MY.1E12 and MUC1 remains unclear. The aim of this study is to elucidate the mode of interaction between MY.1E12 and MUC1 O-glycopeptide by NMR, molecular dynamics (MD) and docking simulations. NMR titration using MUC1 O-glycopeptides suggests that the epitope is located within the O-linked glycan and near the O-glycosylation site. MD simulations of MUC1 glycopeptide showed that the O-glycosylation significantly limits the flexibility of the peptide backbone and side chain of the O-glycosylated Thr. Docking simulations using modeled MY.1E12 Fv and MUC1 O-glycopeptide, suggest that VH mainly contributes to the recognition of the MUC1 peptide portion while VL mainly binds to the O-glycan part. The VH/VL-shared recognition mode of this antibody may be used as a template for the rational design and development of anti-glycopeptide antibodies.

Sensitive New Assay System for Serum Wisteria floribunda Agglutinin-Reactive Ceruloplasmin That Distinguishes Ovarian Clear Cell Carcinoma from Endometrioma.

Wisteria floribunda agglutinin (WFA)-reactive ceruloplasmin (CP) is a candidate marker for ovarian clear cell carcinoma (CCC) reported in our previous paper. Herein, a new measurement system was developed to investigate its potential as a serum marker for CCC. Site-specific glycome analysis using liquid chromatography/mass spectrometry showed that WFA-CP from CCC binds to WFA via the GalNAcß1,4GlcNAc (LDN) structure. We used mutant recombinant WFA (rWFA), which has a high specificity to the LDN structure, instead of native WFA, to increase the specificity of the serum sample measurement. To improve the sensitivity, we used a surface plasmon field-enhanced fluorescence spectroscopy immunoassay system, which is approximately 100 times more sensitive than the conventional sandwich enzyme-linked immunosorbent assay system. With these two improvements, the specificity and sensitivity of the serum rWFA-CP measurement were dramatically improved, clearly distinguishing CCC from endometrioma, from which CCC originates. This rWFA-CP assay can be used clinically for the serodiagnosis of early-stage CCC, which is difficult to detect with existing serum markers.

Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta, we cloned three putative transcription factor genes, TRM1, TRM2, and MPP1, as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown.

Surface plasmon resonance analysis of complex formation of therapeutic recombinant lysosomal enzymes with domain 9 of human cation-independent mannose 6-phosphate receptor.

The efficacy of enzyme replacement therapy (ERT) for lysosomal storage diseases (LSDs) possibly depends on the cellular uptake of recombinant lysosomal enzymes (LEs), and it is known that cation-independent mannose 6-phosphate receptor (CI-M6PR) on the cell membrane is predominantly involved in the endocytosis of many LEs. To examine the biomolecular interaction between therapeutic LEs and CI-M6PR, we biophysically analyzed the complex formation of four LEs available with domain 9 of human CI-M6PR, a binding site of the receptor, by means of surface plasmon resonance (SPR) biosensor assays. The results revealed that the affinity of the LEs for domain 9 of the receptor increased in the following order: laronidase, agalsidase beta, idursulfase, and alglucosidase alfa; and the high affinity of laronidase for domain 9 of CI-M6PR was due to fast complex formation rather than slow dissociation of the complex. The affinity of the enzymes for domain 9 of CI-M6PR almost coincided with their cellular uptake. The SPR biosensor assay is sensitive and provides important information for the development of effective therapeutic LEs for LSDs.

Functional production of human antibody by the filamentous fungus Aspergillus oryzae.

BACKGROUND: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa. RESULTS: Adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. CONCLUSION: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.

A new strategy for the chemoenzymatic synthesis of glycopeptides by De-O-acetylation with an esterase and glycosylations with glycosyltransferases.

Glycopeptides are fragments of glycoproteins and are important in evaluating the biological roles of carbohydrates in glycoproteins. Fmoc solid-phase peptide synthesis using acetyl-protected glycosylated amino acids is a common strategy for the preparation of glycopeptides, but this approach normally requires chemical de-O-acetylation with a base that ß-eliminates sugar residues and epimerizes the peptide backbone. Here we demonstrate a facile new chemoenzymatic synthetic strategy for glycopeptides, using an esterase for the de-O-acetylation of sugar residues and glycosyltransferases for successive sugar elongations at neutral pH.

Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies.

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galß1-3GalNAc (T-antigen), NeuAcα2-3Galß1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.

Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta also possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of therapeutic glycoproteins. However, genetic methods, which are required for the construction of strains by breeding, have not yet been established in this organism. In this study, we investigated the O. minuta mechanisms of mating and sporulation, which would facilitate genetic analysis in this species. Specifically, we determined DNA sequences around the MAT locus in O. minuta strain NBRC 10746, and found that two MAT loci were flanked by a pair of inverted repeat sequences, as reported in O.polymorpha (Maekawa and Kaneko, PLOS Genet., 10, e1004796, 2014). As in O. polymorpha, mating type in O. minuta appears to be switched by inversion of the chromosomal region between the two MAT loci. We successfully obtained O. minuta diploid cells, which showed vegetative growth on rich medium. The size of the diploid cells was 1.3-fold larger than haploid cells of this species. Diploid cells formed ascospores, which contained 2-4 spores, under nutrient starvation conditions. Phenotypes of the resultant haploid cells exhibited Mendelian segregation, indicating that genetic approaches are applicable to O. minuta.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes.

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans.

Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type N-linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases (MAN1A1 and MAN1A2) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in N-glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I (MAN1B1) in the double KO cells reduced most of the complex-type glycans. In triple KO (MAN1A1, MAN1A2, and MAN1B1) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major N-glycan structures. Therefore, we expressed two lysosomal enzymes, α-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The N-glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type N-glycans.

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity.

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI-anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non-permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well-characterized GPI-anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI-anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells.

Structural modeling and mutagenesis of endo-ß-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Endo-ß-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-ß-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.

Engineering of recombinant Wisteria floribunda agglutinin specifically binding to GalNAcß1,4GlcNAc (LacdiNAc).

Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA (rWFA) was expressed in Escherichia coli and purified. The natural complementary DNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation. We characterized the natural and rWFA by the glycoconjugate microarray and frontal affinity chromatography. rWFA exhibited glycan binding specificity similar to that of natural WFA: both bound to Gal- and N-acetylgalactosamine (GalNAc)-terminated glycans. Moreover, the engineered WFA with an amino acid substitution in Cys-272 yielded a recombinant monomeric lectin with limited binding specificity but wild-type affinity for GalNAc-terminated glycans, specifically GalNAcß1,4GlcNAc. Thus, this engineered lectin may be applied to highly sensitive biomarker detection.

Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones.

A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta.

Development of N- and O-linked oligosaccharide engineered Saccharomyces cerevisiae strain.

Yeast cells have been engineered for the production of glycoproteins as biopharmaceuticals with humanized N-linked oligosaccharides. The suppression of yeast-specific O-mannosylation is important to reduce immune response and to improve heterologous protein productivity in the production of biopharmaceuticals. However, so far, there are few reports of the engineering of both N-linked and O-linked oligosaccharides in yeast cells. In the present study, we describe the generation of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2 N-linked oligosaccharides, an intermediate of mammalian hybrid- and complex-type oligosaccharides, while suppressing O-mannosylation. First, a yeast strain that produces a glycoprotein with Man5GlcNAc2 was isolated by introducing msdS encoding α-1,2-mannosidase into a strain synthesizing Man8GlcNAc2 N-linked oligosaccharides. Next, to suppress O-mannosylation, an O-mannosyltransferase-deficient strain was generated by disrupting PMT1 and PMT2 Although the relative amount of O-linked oligosaccharides in the disruptant was reduced to approximately 40% of that in wild type cells, this strain exhibited growth defects and decreased protein productivity. To overcome the growth defects, we applied a mutagenesis technique that is based on the disparity theory of evolution. Finally, to improve protein productivity of the growth-recovered strain, vacuolar proteases PEP4 and PRB1 were further disrupted. Thus, by combining genetic engineering and disparity mutagenesis, we generated an Saccharomyces cerevisiae strain whose N- and O-linked oligosaccharide synthetic pathways were engineered to effectively produce the heterologous protein.

Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae.

BACKGROUND: Production of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galß1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcß1-3GalNAcα1-O-Ser/Thr) has not been constructed. METHODS: To generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC. RESULTS: Introduction of a codon-optimized UDP-GlcNAc:ßGal ß-1,3-N-acetylglucosaminyltransferase 6 (ß3Gn-T6) gene yielded increases in ß3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of ß3Gn-T6 was observed at Mn(2+) concentrations of 10mM and above. Supplementation of MnCl2 to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl2 to the medium. CONCLUSIONS: Physiological Mn(2+) concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn(2+) supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production. GENERAL SIGNIFICANCE: This study reveals that control of Mn(2+) concentration is important for production of specific mammalian-type glycans in S. cerevisiae.

Comparative study on mannose 6-phosphate residue contents of recombinant lysosomal enzymes.

As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT.