Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast

Most therapeutic glycoproteins have been produced in mammalian cell lines. However, the mammalian cell culture system has various disadvantages, i.e., a high culture cost, difficulty in performing a large scale-up because of complicated handling requirements, and the risk of contamination by prion or other unknown pathogenic components through cultivation in the presence of bovine serum. There is thus a growing need for other host cells in which the recombinant glycoproteins can be produced. Recently, we successfully developed a mutant yeast strain engineered in a glycosylation system. The sugar chain produced in the mutant yeast is not immunogenic to the human immuno-surveillance system. In the present study, we selected fibroblast growth factor (FGF) as a model glycoprotein and assessed the bioactivity of FGF produced in yeast in terms of its proliferating activity and tissue distribution in mammalian cells and in the whole body. Structural changes in the sugar chains of FGFs derived from mutant yeast, as compared with those from mammalian cells, did not affect the proliferating activity remarkably. However, the tissue distribution in the mouse differed significantly; a high-mannose type sugar chain was the major determinant of the specific distribution of FGF to the kidney. The mechanism of this phenomenon is still unclear, but our observations suggest that recombinant glycoproteins derived from mutant yeasts producing high-mannose type sugar chains would be applicable for tissue-targeting therapy.     

酵母表达人源化糖蛋白研究进展

与人体天然复杂型糖蛋白相比,使用酵母生产的药用蛋白带有高甘露糖型N-糖链。这一差异在临床应用中产生了许多不良影响。目前,可以通过消除酵母特有的内源糖基化反应,引入哺乳动物细胞中的一系列糖基转移酶及转运蛋白对酵母糖基化路径进行改造,从而使其表达出人源化的复杂型N-聚糖。本文介绍了酵母N-糖基化特点、糖基化不均一性,综述了近年来利用基因工程改造酵母N-糖基化路径获得特定的人源N-连接糖蛋白以及使用内切糖苷酶生产人源糖蛋白的研究进展,并且对存在的问题及今后的发展前景进行了讨论。     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Structures and functional roles of the sugar chains of human erythropoietins.

Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.     

Asn-linked sugar chain structures of recombinant human thrombopoietin produced in Chinese hamster ovary cells

Human thrombopoietin (TPO) that regulates the numbers of megakaryocytes and platelets is a heavily N- and O-glycosylated glycoprotein hormone with partial homology to human erythropoietin (EPO). We prepared recombinant human TPO produced in Chinese hamster ovary (CHO) cells and analyzed the sugar chain structures quantitatively using 2-aminobenzamide labeling, sequential glycosidase digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).We found bi-, tri- and tetraantennary complex-type sugar chains with one or two N-acetyllactosamine repeats, which are common to recombinant human EPO produced in CHO cells. On the other hand, there were triantennary sugar chains with one or two N-acetyllactosamine repeats that were specific to the recombinant human TPO, and their distributions of branch structures were also different. These results suggested that proximal protein structure should determine the branch structure of Asn-linked sugar chains in addition to the glycosyltransferases subset.     

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.     

Monitoring biodistribution of glycoproteins with modified sugar chains

Natural human interferon (hIFN)-gamma has mainly biantennary complex-type sugar chains. Previously, we successfully remodeled its sugar chain structure into: (a) highly branched types; or (b) highly sialylated types, by overexpression of: (a) N-acetylglucosaminyltransferase (GnT)-IV and/or GnT-V; or (b) sialyltransferases, in Chinese hamster ovary (CHO) cells. In addition, we prepared asialo hIFN-gammas by treatment with sialidase in vitro. In the present study, we assessed the bioactivity of remodeled hIFN-gamma in terms of antiviral activity, anticellular activity, and biodistribution. Structural changes to the sugar chains did not have a significant influence on the antiviral and anticellular activities of hIFN-gamma, although the attachment of the sugar chain itself affected both activities. However, the biodistribution differed significantly; the number of exposed galactose residues was the major determinant of the specific distribution to the liver and blood clearance rate of hIFN-gamma. This phenomenon was considered to be mediated by the hepatic asialoglycoprotein receptor (ASGP-R), and we showed a linear, not exponential, enhancement of the distribution to the liver with an increase in the number of exposed galactose residues. We also confirmed this tendency using fibroblast growth factor (FGF). Our observation is not the same as the "glycoside cluster effect." We thus provide important information on the character of modified recombinant glycoproteins.     

Asparagine-linked sugar chains of erythrocyte membrane glycoproteins from Wiskott-Aldrich syndrome are not impaired

In both healthy controls and patients with Wiskott-Aldrich syndrome, the main oligosaccharide in asparagine-linked sugar chains of the membrane glycoproteins of erythrocytes was biantennary sugar chain with bisected G1cNAc (Gal₂-GlcNAc₂-Man₃-GlcNAc-GlcNAc-Fuc-GlcNAc_OT). Biantennary sugar chain with an-fucosyl residue linked at the proximal GlcNAc was seen but biantennary sugar chain without an-fucosyl residue at the proximal GlcNAc was not detected in each subject. There was no difference in quality and quantity of asparagine-linked sugar chains of erythrocyte membrane glycoproteins between healthy controls and the patients. These results suggest that asparagine-linked sugar chains in membrane glycoproteins of hematopoietic cells may not be impaired in Wiskott-Aldrich syndrome.     

Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120

We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH-gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC50 = 3.0 microg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.     

A journey to the world of glycobiology

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB³H₄. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum lgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology.However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Gal1-4Fuc1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N- and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein.It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P₀. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals.These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.     

Glycoform Analysis of N-Glycans Linked to Glycoproteins Expressed in Rice Culture Cells: Predominant Occurrence of Complex Type N-Glycans

Although it has been found that plant endo-β-N-acetylglucosaminidase shows strong activity towards denatured glycoproteins and glycopeptides with high-mannose type N-glycans and free high-mannose type N-glycans bearing the chitobiosyl unit, the endogenous substrates for plant endoglycosidase have not yet been identified. Recently we purified and characterized an endo-β-N-acetylglucosaminidase from rice culture cells and identified the gene encoded (Maeda, M., and Kimura, Y., Trends Glycosci. Glycotech., 17, 205–214 (2005)). Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man_9-5GlcNAc₁) occur at concentration of several micromolar (μM). Hence, in this study we analyzed glycoform of N-glycans linked to glycoproteins expressed in rice culture cells to see whether endogenous glycoproteinous substrate occurs in reasonable amounts. Structural analysis revealed that more than 95% of total N-glycans linked to glycoproteins in the rice cells had the plant complex type structure, including Lewis a epitope-harboring type, although high-mannose type structures account for less than 5% of total N-glycans.     

Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His₆-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His₆-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His₆-tag (designated His₆t-S-CD) was secreted as a monomer. His₆t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His₆t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.     

Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.     

In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.     

Expression of antibodies using single‐open reading frame vector design and polyprotein processing from mammalian cells

We describe a novel polyprotein precursor‐based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single‐open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein‐mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N‐terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

Characterization of Humanized Antibodies Secreted by Aspergillus niger

Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex₆GlcNAc₂ to Hex₁₅GlcNAc₂. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.     

Changes in N-Linked Oligosaccharides during Seed Development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-β-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans.

The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc_2~0Man₃Xyl₁Fuc_1~0GlcNAc₂) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc₂Man₃Xyl₁Fuc₁GlcNAc₂ and GlcNAc₁Man₃Xyl₁Fuc₁GlcNAc₂ decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages.

Concerning the endogenous substrates for plant endo-β-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-β-N-acetylglucosaminidases

Endo-M, endo-β-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-β-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-β-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.