Remarkable transglycosylation activity of glycosynthase mutants of endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae

Abstract

Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae (Endo-D) is an endoglycosidase capable of hydrolyzing the Fc N-glycan of intact IgG antibodies after sequential removal of the sialic acid, galactose, and internal GlcNAc residues in the N-glycan. Endo-D also possesses transglycosylation activity with sugar oxazoline as the donor substrate, but the transglycosylation yield is low due to enzymatic hydrolysis of the donor substrate and the product. We report here our study on the hydrolytic and transglycosylation activity of recombinant Endo-D and its selected mutants. We found that Endo-D preferred core-fucosylated N-glycan for hydrolysis but favored nonfucosylated GlcNAc acceptor for transglycosylation. Several mutants showed significantly enhanced transglycosylation efficiency over the wild type enzyme. Two mutants (N322Q and N322A) were identified as typical glycosynthases that demonstrated remarkable transglycosylation activity with only marginal or no product hydrolysis activity. Kinetic studies revealed that the N322Q [corrected]and N322A glycosynthases had much higher catalytic efficiency for glycosylating the nonfucosylated GlcNAc acceptor. In comparison, the N322Q was much more efficient than N322A for transglycosylation. However, N322Q and N322A [corrected] could not take more complex N-glycan oxazoline as substrate for transglycosylation, indicating their strict substrate specificity. The usefulness of the N322Q glycosynthase was exemplified by its application for efficient glycosylation remodeling of IgG-Fc domain.

FIGURE 1.

Alignment of amino acid sequence of catalytic core of Endo-D, Endo-A, and Endo-M. Residues Asn-322, Glu-324, Tyr-360, and His-371 of Endo-D are highlighted in dark gray.

FIGURE 2.

Hydrolysis rate of Endo-D and its mutants using synthetic substrates. A, reaction scheme. B, hydrolysis rates of fucosylated substrate Fmoc-Asn(Man₃GlcNAc(Fucα1,6)GlcNAc)-OH (1). C, hydrolysis rate of nonfucosylated substrate Fmoc-Asn(Man₃GlcNAc₂)-OH (2). The hydrolysis rates were determined using 2.8 mm of each substrate and quantified by RP-HPLC.

FIGURE 3.

Transglycosylation activity of Endo-D and mutants. A, reaction scheme. B, Fmoc-Asn(Fucα1,6GlcNAc)-OH (3) as the acceptor. C, Fmoc-Asn(GlcNAc)-OH (4) as the acceptor. Square, N322Q; diamond, H371W; triangle, Y360F; star, N322A; cross, E324Q; open square, Endo-D; open diamond, spGH85. The transglycosylation reaction was carried out using 5 mm Man₃GlcNAc-oxazoline as donor and 0.5 mm of each acceptor. The yields were calculated by the ratio of product/acceptor in RP-HPLC.

FIGURE 4.

Glycosylation remodeling of IgG-Fc through transglycosylation with Endo-D mutants.

FIGURE 5.

Analysis of IgG-Fc glycosylation remodeling. The N322Q-catalyzed transglycosylation reaction was carried out using Man₃GlcNAc-oxazoline (5) as donor substrate and (Fucα1,6)GlcNAc-Fc as acceptor substrate. A, SDS-PAGE analysis. Lane 1, native Fc; lane 2, (Fucα1,6)GlcNAc-Fc; lanes 3–8, monitoring of the transglycosylation reaction (15 min–5 h). B, ESI-MS spectrum of the intact native Fc dimer (calculated for nonglycosylated IgG-Fc dimer, M_r = 49,896; found (m/z), 52,763 (G0F homodimer), 52,925 (G0F/G1F heterodimer), 53,084 (G1F homodimer), and 53,400 (G2F homodimer) (deconvoluted data)). C, ESI-MS spectrum of (Fucα1,6)GlcNAc-Fc (calculated, M_r = 50,594; found (m/z), 50,573 (deconvoluted data)). D, ESI-MS spectrum of transglycosylation product Man₃GlcNAc(Fucα1,6)GlcNAc-Fc (calculated, M_r = 51,951; found (m/z), 51,946 (deconvoluted data)).