Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Abstract

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

Keywords: Endo-M; N-linked glycosylation; core fucose; endo-β-N-acetylglucosaminidase; glycoconjugate; glycoprotein; glycosidase; glycoside hydrolase; transglycosylation.

FIGURE 1.

Comparison of the protein structures of GH85 enzymes. A, the protein surface structure around the catalytic core of Endo-A complexed with a trimannosyl thiazoline (Protein Data Bank accession number 3FHQ). B, the protein surface structure around the catalytic core of Endo-D complexed with a thiazoline (Protein Data Bank accession number 2W92). C, superimposition of the Endo-A and Endo-D structures (stereo view). The four amino acids of interest are indicated. D, alignment of the amino acid sequences around the proposed catalytic core of Endo-M, Endo-A, and Endo-D. Residues Gly-125, Gln-128, Trp-228, and Trp-251 of Endo-M are highlighted in light gray. Residues Asn-175 and Glu-177, the putative catalytic residues, are highlighted in pink.

FIGURE 2.

Evaluation of the hydrolytic activity of Endo-M mutants using core fucosylated synthetic substrate. A, schematic of the reaction of trimannosyl biotinylated synthetic substrates with and without core fucose, catalyzed by Endo-M. M, d-mannose; GN, d-GlcNAc; F, l-fucose. B, TLC analysis of the hydrolysis of each substrate by Endo-M variants. C, MALDI-TOF/MS analysis of the reaction mixtures of the W251N mutant enzyme with a core fucosylated synthetic substrate, Man₃GlcNAc₂Fuc₁-biotin. Top, before adding the enzyme; bottom, after incubation with the enzyme. Schematics of the estimated structures represent the following: d-GlcNAc (blue square), d-mannose (green circle), and l-fucose (red triangle).

FIGURE 3.

Relative hydrolytic activities of Endo-M Trp-251 mutants on synthetic substrates. Endo-M Trp-251 mutants were obtained by saturation mutagenesis, except the W251C mutant. The relative hydrolysis rates for both synthetic substrates (Man₃GlcNAc₂-biotin; open bars; Man₃GlcNAc₂Fuc₁-biotin; filled bars) were measured, and the highest values were taken as 100%. Data were obtained using a single assay.

FIGURE 4.

MALDI-TOF/MS analyses of the glycans from rituximab glycopeptides, released by treatment with various enzymes. Glycopeptides of rituximab were prepared and treated with PNGase F (A), wild-type Endo-M (B), W251N variant (C), and Endo-S (positive control) (D). The released oligosaccharides were collected, permethylated, and analyzed by MALDI-TOF/MS, as described under “Experimental Procedures.” Schematics of the estimated structures represent the following: Neu5Ac (purple diamond), d-galactose (yellow circle), d-GlcNAc (blue square), d-mannose (green circle), and l-fucose (red triangle).

FIGURE 5.

Hydrolysis of the N-glycans of human lactoferrin by the W251N variant. A, MALDI-TOF/MS spectrum of permethylated N-glycans released from the tryptic glycopeptides of human lactoferrin by PNGase F. B, hLF (10 μg) was treated with either Endo-S (100 units), wild-type Endo-M (2 μg), or Endo-M W251N mutant (2 μg), and then the aliquots were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The singly glycosylated hLF (top deglycosylated hLF (DG-hLF) band) was observed upon treatment with Endo-S and wild-type Endo-M, as indicated. However, the non-glycosylated hLF (bottom deglycosylated hLF band) as well as singly glycosylated hLF were detected upon treatment with the Endo-M W251N variant. C, MALDI-TOF/MS spectra of permethylated glycans released from hLF samples by wild-type Endo-M (left) and the W251N variant (right). The estimated glycan structures based on the m/z values of precursor ion mass and MS/MS fragment (data not shown) are depicted in the figures. Schematics of the estimated structures represent the following: Neu5Ac (purple diamond), d-galactose (yellow circle), d-GlcNAc (blue square), d-mannose (green circle), and l-fucose (red triangle). Asterisks indicate the hexose polymer peaks.

FIGURE 6.

Transglycosylation by the W251N mutant enzyme. A, a reversed-phase HPLC profile of the transglycosylation reaction of N175Q/W251N mutant enzyme with Fucα1–6GlcNAc-biotin as the acceptor substrate and SG-oxazoline as the donor substrate after a 1-h incubation. B, MALDI-TOF/TOF-MS spectra of the transglycosylation products. A precursor MS peak at m/z 2719, Neu5Ac₂Hex₅HexNAc₄dHex₁-biotin, [M + 3Na]⁺, was subjected to MALDI-TOF/TOF-MS analysis. The fragment ion peaks in the MS/MS spectrum of m/z 2719 correspond to the signature ions for sialylated glycans: loss of a terminal Neu5Ac plus a sodium ion (m/z 2407); loss of two pairs of terminal Neu5Ac plus a sodium ion (m/z 2093); loss of a Neu5Ac plus a sodium ion and a Neu5Ac-Hex-HexNAc moiety plus a sodium ion (m/z 1727); a sodium adduct of dHex-HexNAc-biotin (m/z 695); and a sodium adduct of Neu5Ac (m/z 335). Schematics of the structures represent the following: Neu5Ac (diamond), galactose (filled circle), GlcNAc (filled square), mannose (open circle), l-fucose (triangle). C and D, yields of the transglycosylation product in the reaction of Endo-M mutants with either Fucα1–6GlcNAc-biotin (C) or GlcNAc-biotin (D) as the acceptor substrate and SG-oxazoline as the donor substrate. Data represent the average of three independent reactions with mean ± S.D. (error bars). E, transglycosylation product yields from the reaction of the Endo-M N175Q/W251N double mutant with increased amounts of SG-oxazoline donor substrate in optimized reaction conditions.