Structural basis for the specific cleavage of core-fucosylated N-glycans by endo-β-N-acetylglucosaminidase from the fungus Cordyceps militaris

Abstract

N-Linked glycans play important roles in various cellular and immunological events. Endo-β-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75–2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan–binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.

Introduction

N-Linked glycans are oligosaccharides attached to Asn residues of proteins and have key functionalities in various cellular and immunological systems (1, 2). N-Glycans are categorized into three major types, high-mannose, complex, and hybrid types, and there are bi-, tri-, and tetra-antennary glycans with respect to the number of branches (3). Mammalian N-glycans are frequently α-1,6-fucosylated at the Asn-linked GlcNAc of the core N,N′-diacetylchitobiose unit. A high-throughput analysis of the IgG glycome in three isolated human populations revealed that between 91 and 97.7% of N-glycans are core-fucosylated (4). The α-1,6-fucosylation plays important roles in the functionalities of epidermal growth factor receptors, cell adhesion molecules (5), and antibody-dependent cellular toxicity (6), and altered core fucosylation levels have been observed in certain types of diseases (7–9).

Endo-β-N-acetylglucosaminidases (ENGases,² EC 3.2.1.96) hydrolytically cleave the β-1,4-glycosidic bonds within the core N,N′-diacetylchitobiose unit to release N-glycan, leaving a GlcNAc, with or without the core fucose, linked to the Asn residue of proteins (Fig. 1A) (10). The ability of ENGases to chemoenzymatically synthesize homogeneously N-glycosylated proteins (antibodies) has recently attracted significant research attention (11–13). In the Carbohydrate-Active enZyme (CAZy) database (http://www.cazy.org),³ ENGases are classified into glycoside hydrolase (GH) families 18 (GH18), GH73, and GH85 (14). ENGases in GH18 and GH85 catalyze the anomer-retaining type of hydrolysis via the “substrate-assisted” mechanism, in which the N-acetyl group of the substrate GlcNAc participates in the reaction as a nucleophile (15, 16).

Figure 1.

Reaction and diversity of ENGases. A, hydrolytic cleavage site of an N-glycan by Endo-CoM. The best substrate for Endo-CoM (core-fucosylated sialobiantennary) is shown by the Symbol Nomenclature for Glycans (65). The Fuc–GlcNAc–Asn moiety bound to the Endo-CoM structure and the octasaccharide of biantennary complex glycan observed in other ENGase structures are boxed by dashed lines. Subsites of ENGases are indicated by blue characters. B, phylogenetic tree of GH18 ENGases. The fucose-specific ENGases and structure-known ones are selected. Bar, 5% sequence divergence. C, partial amino acid sequence alignment of GH18 ENGases. The catalytic residues and key amino acid residues for the Fuc–GlcNAc recognition of Endo-CoM are indicated above the sequences with red and blue arrows, respectively.

GH18 is one of the best-characterized families in CAZy. Although ∼90 enzymes and proteins in GH18 have three-dimensional structures available in the Protein Data Bank, the majority of them are chitinases (EC 3.2.1.14), which endolytically cleave the β-1,4-glycosidic bonds of the chitin homopolymer. Crystal structures of GH18 ENGases from Elizabethkingia meningoseptica (former name Flavobacterium meningosepticum) (Endo-F1 and Endo-F3) (17, 18), Streptococcus pyogenes (Endo-S and Endo-S2) (19–21), Streptomyces plicatus (Endo-H) (22, 23), Bacteroides thetaiotaomicron (BT3987 and BT1044) (24), and Trichoderma reesei (Endo-T) (25) have been reported. However, most of them are ligand-free (apo) structures. Structures of Endo-F3, Endo-S, and Endo-S2 only contain the octasaccharide of the biantennary complex glycan or high-mannose (Man₇-GlcNAc) glycan as a cleavage product complex (18, 20, 21). These structures provide a structural basis for the enzymatic specificity for the leaving part of the glycan recognized at the “minus” subsites, according to the nomenclature defined in Ref. 26. However, complex structures of both GH18 and GH85 ENGases occupied at the “plus” subsites are not available, and recognition for the protein-side part of the substrates by ENGases is still unclear despite extensive structural studies. Because FUT8 (α1,6-fucosyltransferase) cannot directly attach a core fucose on full-size N-glycans (5, 27), protein engineering to produce ENGases that can efficiently transfer core-fucosylated glycans has been conducted (28, 29). Therefore, there is a high demand for a structural basis for the recognition of the “plus” subsites and the Asn-linked peptide by ENGases.

We previously discovered and characterized GH18 ENGases that are specific for core fucose-containing N-glycans from two fungal species (Beauveria bassiana and Cordyceps militaris) and the bacterium Sphingobacterium sp. strain HMA12 (Endo-SB–ORF1188) (30). C. militaris is the ascomycete fungus that forms sexual fruiting bodies on mycosed pupae (“pupa grass” in China) and is used for traditional oriental medicine (31). The ENGase from C. militaris (hereafter called Endo-CoM) as well as the ENGase from B. bassiana (Endo-BB) and Endo-SB–ORF1188 exhibit activity exclusively on core α-1,6-fucosylated biantennary complex-type oligosaccharides (30). Endo-CoM and Endo-BB were active on fucose-containing glycans on rituximab (IgG) but not on the high-mannose glycans on RNase B. Endo-CoM, Endo-BB, and Endo-SB–ORF1188 form a distinct clade from other GH18 ENGases in a phylogenetic tree (Fig. 1B).

In this study, we report the crystal structures of Endo-CoM in the apo-form and complex forms with l-fucose and fucose-containing ligands occupying the plus subsites. In addition, a mutational study and structural comparison with other ENGases provide structural insights into the core fucose-specific or nonspecific cleavage of N-glycans.

Results and discussion

Crystal structure of Endo-CoM

A recombinant enzyme of the mature Endo-CoM protein with a C-terminal His₆-tag was expressed in Escherichia coli. The purified enzyme migrated in SDS-PAGE as a single band with an estimated molecular mass of ∼30 kDa, in agreement with the theoretical molecular mass of 34,216 Da. The molecular mass of the nondenatured recombinant Endo-CoM protein estimated by calibrated gel-filtration chromatography was ∼20 kDa, suggesting that it is a globular-shaped monomer in solution. The crystal structure of Endo-CoM was solved by the single-wavelength anomalous dispersion (SAD) method using sulfur atoms contained within the native protein (Table 1). Our trials of molecular replacement using the crystal structures of GH18 ENGases as a search model failed due to low homology (<38% amino acid sequence identity). A ligand-free structure and complex structures with Fuc, Fuc-α1,6–GlcNAc, and Fuc-α1,6–GlcNAc–Asn were determined (Table 1). The complex structures with the latter two were determined using a double mutant at the catalytic residues (D154N/E156Q), which were unambiguously identified based on the complete conservation among GH18 enzymes (Fig. 1C). The crystals contained one molecule in the asymmetric unit. A molecular interface analysis using the PISA server predicted that the protein is a monomer in solution (data not shown). The final models contain protein residues 22–315. The four structures of Endo-CoM are virtually the same, and the root mean square deviations (RMSD) for the Cα atoms between them are less than 0.37 Å. Endo-CoM consists of a single (β/α)₈ barrel, which is a typical catalytic domain fold of GH18 enzymes (Fig. 2A). There is an additional N-terminal α-helix, near which a disulfide bond is formed by Cys-24 and Cys-76.

Table 1.

Data collection and refinement statistics

Data set	S-SAD	WT ligand-free	WT fucose	D154N/E156Q Fuc-GlcNAc	D154N/E156Q Fuc-GlcNAc-Asn
Data collectiona
Beamline	BL1A	BL1A	BL5A	NE3A	NW12A
Wavelength (Å)	1.9000	1.1000	1.0000	1.0000	1.0000
Space group	P65	P65	P65	P65	P65
Unit cell (Å)	a = b = 105.86 c = 63.23	a = b = 105.51 c = 63.43	a = b = 105.81 c = 63.93	a = b = 105.73 c = 63.21	a = b = 105.91 c = 62.22
Resolution (Å)	45.84–2.30 (2.38–2.30)	50.00–1.75 (1.78–1.75)	50.0–1.80 (1.83–1.80)	45.78–2.10 (2.16–2.10)	50.00–2.35 (2.39–2.35)
Total reflections	2,855,864	418,677	251,725	120,478	76,166
Unique reflections	18,121 (1,747)	40,703 (2,026)	37,466 (1,851)	23,548 (1,915)	16,268 (776)
Completeness (%)	99.9 (99.5)	100.0 (100.0)	100.0 (100.0)	99.9 (99.4)	99.0 (95.1)
Multiplicity	157.6 (155.0)	10.3 (10.4)	6.7 (6.8)	5.1 (5.2)	4.7 (3.3)
Mean I/σ(I)b	68.7 (46.9)	23.9 (3.0)	16.1 (2.4)	8.0 (1.3)	9.8 (1.7)
Rmerge	0.108 (0.177)	0.120 (0.801)	0.101 (0.841)	0.141 (1.241)	0.134 (0.554)
CC1/2	1.000 (0.999)	0.991 (0.863)	0.990 (0.816)	0.996 (0.484)	0.974 (0.787)
Anomalous completeness (%)	99.9 (99.5)
Anomalous multiplicity	79.1 (77.3)
Refinement
Resolution (Å)		40.59–1.75	45.86–1.80	19.98–2.10	45.90–2.36
No. of reflectionsb		38,543	35,554	22,360	15,471
Rwork/Rfree		0.155/0.184	0.161/0.198	0.198/0.244	0.185/0.236
No. of atoms		2,590	2,593	2,420	2,446
Mol/ASUc		1	1	1	1
Wilson B (Å2)		19.7	25.2	35.4	48.8
Average B (Å2)
Protein		23.7	31.3	44.0	65.7
Ligand			26.8	20.6	93.4
Solvent		35.3	42.1	43.6	63.9
RMSD from ideal values
Bond lengths (Å)		0.013	0.011	0.010	0.008
Bond angles (°)		1.756	1.599	1.613	1.494
Ramachandran plot (%)
Favored		99.3	98.6	96.2	97.6
Allowed		0.7	1.4	3.8	2.4
Outlier		0.0	0.0	0.0	0.0
PDB code		6KPL	6KPM	6KPN	6KPO

^a Values in parentheses are for the highest resolution shell.

^b Data cutoff is F > 0σF.

^c Molecules per asymmetric unit are given.

Figure 2.

Crystal structure of Endo-CoM. A, overall structure of D154N/E156Q mutant (ribbon model with rainbow colors) complexed with Fuc–GlcNAc–Asn (white sticks). The side chains of the mutated catalytic residues (Asn-154 and Gln-156) are shown as magenta sticks. A disulfide bond (Cys-24–Cys-76) connecting the additional N-terminal α-helix (blue) is shown as a stick with the sulfur atoms in yellow. B–D, electron density maps of the complex structures. mF_o − F_c omit maps for Fuc (B, 3.0σ), Fuc–GlcNAc (C, 3.0σ), and Fuc–GlcNAc–Asn (D, 1.5σ) are shown. D, Fuc–GlcNAc–FmocAsn was used for the crystal soaking but the Fmoc moiety was disordered. E, comparison of the apo and complex structures at the active site. The apo structure (gray) and the complex structures with Fuc (cyan), Fuc–GlcNAc (yellow), and Fuc–GlcNAc–Asn (green) near subsites +1 and +1′ are superimposed. The ligands and hydrogen bonds are shown with thick sticks and dashed lines (magenta), respectively.

A structural similarity search using the Dali server (32) revealed that Endo-CoM is similar to Endo-S2 and Endo-S (Z score = 35.5; Table 2), which have relatively higher sequence identities (∼38%). Other ENGases showed much lower structural and sequence similarities. Among GH18 chitinases, ChiW from Paenibacillus FPU-7 (33) showed the highest structural similarity (Z score = 21.1) comparable with another fungal ENGase, Endo-T (Z score = 20.3).

Table 2.

Results of the structural similarity search using the Dali server

Other ENGases (BT3987 and Endo-F1) were not found.

Protein	Source organism	PDB (chain)	Zscore	RMSD	Naligna	%seqb
				Å
Endo-S2	S. pyogenes NZ131	6MDV (A)	35.5	1.5	271	38
Endo-S	S. pyogenes MGAS5005	4NUY (A)	35.5	1.7	271	37
BT1044	B. thetaiotaomicron	6Q64 (A)	23.2	2.8	246	22
Endo-F3	E. meningoseptica	1EOM (A)	22.9	2.3	227	22
Chitinase ChiW	Paenibacillus sp. FPU-7	5GZU (A)	21.1	2.7	235	16
Endo-T	T. reesei	4AC1 (X)	20.3	2.8	238	18
Chitinase ChiB	S. marcescens	1E6N (A)	19.6	2.8	240	16
Endo-H	S. plicatus	1C3F (A)	17.9	2.6	210	20

^a Number of aligned residues are shown.

^b Sequence identity is shown.

Complex structures of Endo-CoM

Soaking of the Endo-CoM crystals in a solution containing l-fucose resulted in observation of clear electron density for α-l-fucopyranose (Fig. 2B). The β-anomeric form of the GlcNAc was also clearly observed in the active site when a disaccharide, 2-acetamido-2-deoxy-6-O-(α-l-fucopyranosyl)-d-glucopyranose (Fuc–GlcNAc), was used for crystal soaking (Fig. 2C). We also used N-9-fluorenylmethyloxycarbonyl-Asn (Fuc-α1,6–GlcNAc-β-)-OH (Fuc–GlcNAc–FmocAsn) for a soaking experiment. Although the electron density was relatively ambiguous, we could confidently place atoms of the molecule up to the Asn moiety (Fig. 2D). The Fmoc group attached to the amino group of Asn was disordered, probably because it was exposed to the solvent (Fig. 2A). All of the sugar rings of Fuc and GlcNAc are in the stable chair conformation. The torsion angles of the Fuc-α1,6–GlcNAc glycosidic bond are as follows: ϕ = −83.8°, ψ = 69.8°, and ω = 173.0° for Fuc–GlcNAc, and ϕ = −80.0°, ψ = 77.6°, and ω = 161.1° for Fuc–GlcNAc–Asn with a definition of ϕ (O5-C1–Ox–Cx), ψ (C1–Ox–Cx–Cx+1), and ω (O6–C6–C5–C4). Therefore, the C5–C6–O6 exocyclic group of GlcNAc is in a gt conformation. This conformation was not a prevalent one among the results of molecular dynamic simulations on various forms of fucosylated complex biantennary N-glycans, but high flexibility at this bond was also observed in the same study (34). Protein structures of the three complex forms superimposed very well with no significant movement of the surrounding protein atoms (Fig. 2E). However, the side chain of the catalytic acid/base residue (Glu-156) of the apo (gray in Fig. 2E) and Fuc complexes (cyan) was significantly displaced compared with the complex with Fuc–GlcNAc (yellow) and Fuc–GlcNAc–Asn (green), in which the O4 atom of GlcNAc forms a hydrogen bond with the side chain of Gln-156.

Interactions with Fuc–GlcNAc–Asn and the active-site structure

Fig. 3 shows the active-site structure of Endo-CoM complexed with Fuc–GlcNAc–Asn. The biantennary complex glycan bound to Endo-F3 (Fig. 3A) (18) and the chitin pentamer bound to ChiB (Fig. 3B) (35) are overlaid as blue sticks. The GlcNAc moiety of Fuc–GlcNAc–Asn is bound to the subsite +1 that is located next to the catalytic residue (Glu-156). The O4 atom of the GlcNAc forms a hydrogen bond with the side chain of Gln(Glu)-156, supporting the implication that this residue is the acid/base catalyst. Asn(Asp)-154 is located near the nitrogen atom of the GlcNAc N-acetyl group at the subsite −1 position, confirming that this residue is the stabilizing residue for substrate-assisted catalysis (15).

Figure 3.

Stereoviews of the active site of Endo-CoM complexed with Fuc–GlcNAc–Asn. The protein main chain is shown as a gray ribbon model. The side chains near the ligand, Fuc, and GlcNAc–Asn moieties of the ligand are shown as sticks colored in green, cyan, and yellow, respectively. A, biantennary complex glycan bound to Endo-F3 (PDB ID: 1EOM) is shown as thin blue sticks. B, chitin pentamer bound to ChiB (1E6N) is shown as blue sticks. The N,N′-diacetylchitobiose unit bound at subsites −1 and +1 is highlighted with thick sticks. The side chains of the catalytic residues of Endo-F3 and ChiB are also shown as blue sticks.

The Fuc moiety is located in a side pocket, which we designate as subsite +1′ and is recognized by Endo-CoM with extensive interactions. The O2 and O3 hydroxy groups form hydrogen bonds with the main-chain amide and carboxyl of Tyr-216. Arg-218 and Asn-193 are hydrogen-bonded to the O3 and O4 hydroxy groups and the sugar ring O5 atom, respectively. In addition, the side chain of Trp-253 forms a hydrophobic interaction with the sugar ring of Fuc, the nearest carbon–carbon distance (Trp Cζ3–Fuc C3) being 4.1 Å. The extensive and specific interactions at this site clearly explain the exclusive preference of Endo-CoM for core-fucosylated substrates (30). The residues forming the fucose-binding site (Asn-193, Tyr-216, Arg-218, and Trp-253) are completely conserved in other core fucose-specific ENGases (Endo-BB and Endo-SB–ORF1188) that were identified in our previous study (Fig. 1C) (30).

The Asn moiety is located at a displaced position from the linear homopolymer of the chitin oligosaccharide (subsites +1 to +3 in Fig. 3B), and there is no specific interaction with the protein. The side-chain residues in loop 7 (discussed below) are located near the Asn moiety, but the distances are >3.5 Å and >4.3 Å for Trp-253 and Thr-251, respectively. The Fmoc-linked amino group and the free carboxyl groups of the Asn moiety are exposed to the solvent, suggesting that the ENGase can act on various protein N-glycans without serious steric hindrance.

Mutational analysis

Based on the crystal structure, we performed a mutational analysis on the residues forming the catalytic and fucose-binding sites (Table 3). Mutations at the catalytic residues (E156Q and D154N/E156Q) resulted in significant 2 orders of magnitude loss of activity. Mutations at the fucose-binding site basically reduced the activity on pyridylamino (PA)-fucosyl sialobiantennary, especially for the Y216A and R218A mutants, whereas the W253A mutant retained activity. Unexpectedly, mutation of N193A increased the activity, suggesting that the interaction between Asn-193 and the O5 atom of fucose is not critical for core-fucose specificity. The activities of WT and most mutants (except for Arg-218) toward nonfucosylated PA-sialobiantennary substrate were basically very low (<1%) compared with the fucosylated substrate, consistent with our previous report (30). It was interesting that the R218A mutation slightly increased the activity against the nonfucosylated PA-sialobiantennary substrate (∼2.3-fold). Removal of the long and positively-charged side chain of Arg-218 may reduce the steric assistance at subsite +1′ specificity for Fuc and endowed a broader substrate specificity to the enzyme.

Table 3.

Relative activities of the WT and mutant enzymes of Endo-CoM toward PA substrates

Relative activity (%) compared with the activity of the WT enzyme for PA-fucosyl sialobiantennary (8.03 μmol/min/mg) is shown.

Enzyme	PA-fucosyl sialobiantennary	PA-sialobiantennary
WT	100	0.99
E156Q	1.27	–a
D154N/E156Q	1.20	0.02
N193A	184	0.52
Y216A	3.45	–a
R218A	14.9	2.24
W253A	63.0	0.18

^a – indicates not determined.

Core fucose-binding pocket of ENGases

The low sequence similarity and frequent indels in the potential subsite +1′ region among ENGases (Fig. 1C) have been hampered in facilitating reliable prediction of their core fucose tolerance and specificity. Here, we compared the core fucose-binding site of Endo-CoM (Fig. 4A) with other structure-known ENGases. Endo-F3 exhibits more than 300-fold higher hydrolytic activity to core-fucosylated glycopeptides compared with nonfucosylated ones (36). In the Endo-F3 structure (Fig. 4B), several residues, including Tyr-148, Tyr-152, Asp-190, and Arg-219, surround the putative subsite +1′, and the latter two potentially make hydrogen bonds with Fuc. Molecular surface presentation illustrates that there is a round-shaped pocket for the core fucose in Endo-CoM and Endo-F3 (Fig. 5, A and B).

Figure 4.

Comparison of the fucose-binding site of Endo-CoM (A) with a potential subsite +1′ of Endo-F3 (B) and Endo-S (C). In B (1EOM) and C (6EN3), bound biantennary complex glycan is shown as thin blue sticks, and the Fuc–GlcNAc–Asn ligand of Endo-CoM is overlaid.

Figure 5.

Molecular surfaces of Endo-CoM (A), Endo-F3 (B, 1EOM), Endo-S (C, 6EN3), Endo-S2 (D, 6MDS), Endo-H (E, 1EDT), Endo-F1 (F, 2EBH), BT1044 (G, 6Q64), and Endo-T (H, 4AC1) at the fucose-binding site. The catalytic residues (Asp-154 and Glu-156 in Endo-CoM) and the conserved Tyr residue (Tyr-216 in Endo-CoM), which were used for superimposition of the protein structures, are colored in red and green, respectively. Biantennary complex glycan bound to Endo-F3, Endo-S, and Endo-S2 are shown as thin blue sticks, and the Fuc–GlcNAc–Asn ligand of Endo-CoM is overlaid on the structures of B–H. Residues that potentially form a steric crash with the fucose are indicated by purple characters.

Endo-S can cleave both core-fucosylated and nonfucosylated glycans and thus is described as “tolerant” to core-fucosylation (11, 37). The putative fucose-binding site of Endo-S is relatively open and shallower than those of Endo-CoM and Endo-F3 (Fig. 5C). A side chain conformational change of Gln-308 appears to be possible (Fig. 4C), and it may give slight preference to core-fucosylated glycans. Endo-S2 is also tolerant to core-fucosylated glycans (38, 39) and has an open +1′ subsite similar to Endo-S (Fig. 5D).

A study on Endo-H and Endo-F1 demonstrated that the former exhibited over 50-fold higher activity to core-fucosylated substrates compared with the latter (40). The molecular surface presentation of these ENGases revealed that Endo-H has an open +1′ subsite (Fig. 5E), whereas the side chain of Asp-198 of Endo-F1 exerts severe steric hindrance on the overlaid fucose at its O2 hydroxy (Fig. 5F). Briliūtė et al. (24) reported that BT1044 is tolerant to core-fucosylated glycans. In this study, release of the Fuc–GlcNAc disaccharide from glycans liberated from human serum IgG, human serum IgA, and human colostrum IgA was detected by HPLC and MS, and the activity was not quantitatively described. The molecular surface of BT1044 indicates that it has a very narrow pocket for subsite +1′ and appears to form a small steric clash with the overlaid fucose that can be alleviated by slight movement of the sugar side chain (Fig. 5G). Endo-T was shown to prefer high-mannose–type glycans over complex-type ones (41), but we could not find any literature about its activity on core-fucosylated glycans. The molecular surface presentation indicated that Ser-196, Phe-198, and Leu-171 of Endo-T may form a steric clash with the fucose (Fig. 5H).

Implications for glycan specificity

Structural basis of the specificity for the leaving part of N-glycans has been previously described for Endo-F3, Endo-S, and Endo-S2 (18, 20, 21). Here, we used the loop-displaying system that was used to describe the structural features of Endo-S and Endo-S2, in which the eight loops connecting the β-strands and α-helices of the (β/α)₈-barrel (TIM-barrel) protein scaffold are separately colored (20, 21). A ribbon model of Endo-CoM (Fuc–GlcNAc–Asn complex) colored with this system is shown in Fig. 6A, and the G2 octasaccharide of the biantennary complex glycan bound to Endo-S2 (thin blue sticks; Gal-β1,4-GlcNAc-β1,2-Man-α1,3-[Gal-β1,4-GlcNAc-β1,2-Man-α1,6-]Man-β1,4-GlcNAc) is overlaid on the Endo-CoM structure. The catalytic acid/base residue (Glu-156) is on loop 4 (magenta), and the fucose-binding residues are on loop 5 (sand, Asn-193), loop 6 (red, Tyr-216 and Arg-218), and loop 7 (cyan, Trp-253).

Figure 6.

Glycan-binding clefts of Endo-CoM (A and B), Endo-F3 (C, 1EOM), Endo-S (D, 6EN3), and Endo-S2 (E). The color code for the loops is according to Ref. 20. The biantennary complex glycan bound to the three ENGases (blue sticks) is also schematically shown in a blue box. C, potential binding area for the Gal–GlcNAc-branch of triantennary complex-type glycans is indicated by an orange circle. E, molecular surface of Endo-S2 complexed with the biantennary complex glycan (6MDS) is overlaid with the high-mannose glycan (salmon sticks) bound to the same protein (6MDV). The high-mannose glycan is also schematically shown in a salmon box. A pocket for accommodating the α1,3-branched antenna is indicated by a red circle.

In terms of molecular surface presentation (Fig. 6B), Endo-CoM has a clearly Y-shaped (forked) cleft with a protrusion of loop 2 (yellow) that appears to be suitable for accommodating biantennary glycans. Endo-F3 hydrolyzes both biantennary and triantennary complex-type N-glycans (42). Trastoy et al. (20) predicted a potential binding area for the Gal–GlcNAc-branch of triantennary complex-type glycans on the Endo-F3 structure (orange circle in Fig. 6C) (20). The corresponding area in Endo-CoM (Fig. 6B) as well as in Endo-S (Fig. 6D) and Endo-S2 (Fig. 6E) is blocked by protrusion of loop 7 (cyan). This observation is consistent with the previously determined substrate specificity of Endo-CoM (30); the PA-fucosyl asialotriantennary substrate was not hydrolyzed despite the presence of the core fucose.

Recently, Klontz et al. (21) succeeded in determining the crystal structure of Endo-S2 complexed with an octasaccharide part of a high-mannose glycan (salmon sticks in Fig. 6E; Man₇–GlcNAc). The high-mannose glycan binds to the cleft in a similar manner with the complex biantennary glycan (blue sticks in Fig. 6E). However, a pocket for accommodating the α1,3-branched antenna, which is surrounded by loop 3 (orange) and loop 4 (magenta), was found (red circle in Fig. 6E). Although Endo-F3 and Endo-S do not have a corresponding pocket, Endo-CoM equips a similarly-shaped pocket at a corresponding position (Fig. 6B). Endo-CoM did not exhibit activity against nonfucosylated high-mannose–type PA substrates (M5–M9) (30). Unfortunately, we could not test the activity against core-fucosylated high-mannose–type glycans due to the unavailability of substrates.

Our previous study also demonstrated that the presence of the terminal sialic acids on the biantennary substrate (PA-fucosyl sialobiantennary) increased the activity of Endo-CoM by 2.7–4.6-fold compared with substrates without sialic acids (PA-fucosyl asialobiantennary and PA-fucosyl agalactobiantennary) (30). Interestingly, we found several Arg and Lys residues (Arg-58, Lys-67, Arg-113, and Lys-141) at both nonreducing ends of the Y-shaped cleft (blue in Fig. 6, A and B). These positively-charged residues probably contribute to the high activity with sialo-glycans via electrostatic interactions.

Concluding remarks

In this study, we provided a structural basis for the strict specificity of a GH18 ENGase for core-fucosylated N-glycans that are frequently present in mammal glycoproteins. However, the enzyme used in our study was isolated from a fungus that can infect caterpillars (C. militaris). The CAZy database defines 24 GH18 genes in the genome of C. militaris ATCC 34164, and Zheng et al. (31) reported that the genome of C. militaris strain CM01 contains 20 GH18 genes. The presence of such a large number of GH18 genes has been explained in the context of pathogenicity to infect across the insect cuticle via chitinase activities. In particular, the Endo-CoM gene (locus tag = CCM_08020) was annotated as a chitinase by the genome project (31). According to our survey, the C. militaris genome does not have a fucosyltransferase gene (data not shown), suggesting that Endo-CoM is not dedicated to the degradation or transfer of intrinsic glycoproteins. Cordyceps is a genus of parasitic filamentous fungi whose main target is insects. Because insects have many N-glycans that are core-fucosylated by α1,3-modifications as well as α1,6-modifications (43, 44), efficient degradation of the host glycoproteins accompanied by the action of chitinases may be vital for the invasion and energy intake of the pathogenic fungi. In contrast, another fungal GH18 ENGase, Endo-T, is specific for high-mannose N-glycans, which are observed in fungal and yeast glycoproteins, and apparently it does not have the ability to cleave core-fucosylated N-glycans (41). This is probably because the source organism of Endo-T (T. reesei) mostly relies on the degradation of plant cellulose.

The bacterium E. meningoseptica is a nosocomial human pathogen (45) and has multiple ENGases (Endo-F1, -F2, and -F3) (40). The strong preference of Endo-F3 for core-fucosylated N-glycans has been recognized for a long time, and our study provided a structural explanation for this observation. Interestingly, Endo-CoM and Endo-F3 have a similarly-shaped pocket for fucose (Fig. 5, A and B), but the residues responsible for the recognition are not conserved (Fig. 4, A and B), suggesting that the core-fucose recognition of these ENGases has emerged independently as convergent molecular evolution.

Utilization of ENGases for chemoenzymatic glycosylation engineering is a promising strategy to achieve glycan-defined glycoproteins (46). Therapeutic antibodies with homogeneously-modified glycans produced by this method indeed exhibited enhanced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (47, 48). Recently, Katoh et al. (29) succeeded in producing a mutant GH85 ENGase (Endo-M W251N) that is tolerant to core-fucosylation. In addition, an elegant glycosynthase strategy that enables high-transglycosylation ability has been developed for GH85 enzymes (Asn-175 mutants of Endo-M and Asn-322 mutants of Endo-D) (49–51). These works were performed based on the ligand-free crystal structures of GH85 ENGases (16, 52). Although only 11 ENGases are currently listed as “characterized” enzymes in GH85 of the CAZy database, GH18 contains 22 characterized ENGases. Therefore, our structural information on the core fucose-binding site of GH18 ENGases will support and facilitate future protein engineering of enzymes from wider origins with more precise structural tuning.

The bacterial GH18 ENGase, Endo-S, has been successfully developed as a therapy to inhibit various experimental autoimmune disorders of mammalian systems (53). Endo-CoM was isolated from a eukaryotic origin and has a minimum size of ENGase (∼300 amino acids) that is much smaller than Endo-S (∼1,000 amino acids). Therefore, the new fungal ENGase with a concrete structural basis can be a good alternative for the therapeutic usage to mammalian IgGs because Endo-CoM possibly has an advantage on the recombinant protein production in eukaryotic cells. In the case of GH85, fungal ENGases, such as Endo-M and Endo-CC from Coprinopsis cinerea, are used as commercially available reagents (54, 55). Exploring the usage of fungal GH18 ENGases that has low amino acid sequence identities to bacterial ENGases (<38%) will expand the options for future therapeutic use.

Experimental procedures

Materials

2-Acetamido-2-deoxy-6-O-(α-l-fucopyranosyl)-d-glucopyranose (Fuc–GlcNAc) was purchased from Carbosynth (Compton, Berkshire, UK). N-9-Fluorenylmethyloxycarbonyl-Asn (Fuc-α1,6–GlcNAc-β-)-OH (Fuc–GlcNAc–FmocAsn) was synthesized according to a previously described method (56). 4-Methoxyphenyl α-l-fucopyranosyl-(1→6)-2-acetamide-2-deoxy-β-d-glucopyranoside (OMP-Fuc–GlcNAc) was provided by Dr. A. Ishiwata. l-Fucose was purchased from Wako Pure Chemical Industries (Osaka, Japan). PA-fucosyl sialobiantennary (PA-083) and PA-sialobiantennary (PA-026) were purchased from Masuda Chemical Industries (Takamatsu, Kagawa, Japan). Other chemicals were purchased from Wako Pure Chemical Industries or Sigma unless otherwise stated and were of the highest commercially available grade.

Protein production and purification

The expression plasmid for the mature Endo-CoM protein (residues 19–315 without signal sequence) with a C-terminal His₆-tag was constructed using pET-32b (Novagen, Madison, WI), as described previously (30). Recombinant proteins of the WT Endo-CoM and its mutants were expressed in E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies, Santa Clara, CA). The cells were grown at 37 °C in 1.5 liters of Luria-Bertani medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol until the absorbance reached 1.0 at 600 nm. After cooling the culture in a refrigerator at 4 °C for 40 min, expression was induced using 0.4 mm isopropyl β-d-thiogalactopyranoside and continued at 15 °C for 20 h. The cells were harvested via centrifugation at 8,000 × g for 15 min and suspended in 100 mm Tris-HCl (pH 7.5), 500 mm NaCl, and 0.1 mm phenylmethylsulfonyl fluoride with a concentration of 0.1 g of wet cells per ml. The cells were disrupted via sonication (Branson Sonifier250D; Branson Ultrasonics Division of Emerson Japan, Kanagawa, Japan), and the supernatant was collected via centrifugation at 15,000 × g for 30 min. Fine particles in the supernatant were removed using a syringe filter (Minisart hydrophilic 0.45 μm; Sartorius Stedim Biotech, Göttingen, Germany). The protein was purified to homogeneity using Ni-affinity chromatography (HisTrap FF 5-ml column; GE Healthcare, Buckinghamshire, UK) and gel-filtration chromatography (HiLoad 16/60 Superdex 200 pg column, GE Healthcare). Solutions of 15 and 500 mm imidazole in 50 mm Tris-HCl (pH 7.5) and 150 mm NaCl were used for the wash and elution buffers for Ni-affinity chromatography, respectively. A solution of 20 mm Tris-HCl (pH 7.5) was used for gel-filtration chromatography, and the relative molecular mass of the protein was determined using molecular standards of thyroglobulin (669 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). The protein solution was concentrated using an ultrafiltration centrifugal membrane unit (Vivaspin Turbo 15, MWCO 10,000; Sartorius Stedim Biotech) in a solution of 20 mm Tris-HCl (pH 7.5). The protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) with BSA as a standard. Measurements of the protein concentrations based on absorbance at 280 nm and a theoretical extinction coefficient calculated from the amino acid sequence (68,995 m⁻¹ cm⁻¹), which was calculated using the ProtParam server (https://web.expasy.org/protparam/)³ (57), were also used for purified protein samples to check the consistency.

Crystallography

Crystals were grown at 4 °C using the sitting drop vapor-diffusion method by mixing 1.0 μl of an 8.8 mg/ml protein solution with an equal volume of a reservoir solution containing 0.1 m CHES-NaOH (pH 8.6) and 20% PEG3350. The apo crystals were cryoprotected in the reservoir solution supplemented with 25% PEG300. Complex crystals were prepared by soaking the crystals for 30 min (WT + Fuc) or <1 min (other complex structures) in a reservoir solution supplemented with 25% PEG300 (cryoprotectant) and 100 mm l-fucose, 20 mm Fuc–GlcNAc, 10 mm Fuc–GlcNAc–FmocAsn, or 20 mm OMP–Fuc–GlcNAc. However, no interpretable electron density for OMP–Fuc–GlcNAc was observed. The crystals were flash-cooled by dipping into liquid nitrogen. X-ray diffraction data were collected at 100 K on beamlines at the Photon Factory of the High Energy Accelerator Research Organization KEK (Tsukuba, Japan) and SPring-8 (Hyogo, Japan). The data set was processed using XDS (58) and Aimless (59) for the sulfur-SAD phasing dataset or HKL-2000 (60) for other datasets. Diffraction data for phasing were collected at beamline BL-1A of the Photon Factory, which is designed for long-wavelength experiments (61). Initial phase calculation, phase improvement, and automated model building were performed using PHENIX (62). Manual model building and refinement were carried out using Coot (63) and Refmac5 (64). Molecular graphic images were prepared using PyMOL (Schrödinger, LLC, New York).

Construction of mutants and enzyme assay

Site-directed mutants were constructed using a PrimeSTAR mutagenesis basal kit (TaKaRa Bio, Ohtsu, Japan). The following primers and their complementary primers were used: 5′-GATGTTcagCAGAGCCTGAATGCAAAT-3′ for E156Q; 5′-GATATCaatGTTcagCAGAGCCTGAATGCAAAT-3′ for D154N/E156Q; 5′-CAGAGCgcgGGTCGTAGCATTAGTGGC-3′ for Y216A; 5′-TATGGTgcgAGCATTAGTGGCCTGCAG-3′ for R218A; 5′-GATACCgcgATGGATGGCACCCAGCCG-3′ for N193A; and 5′-ACCAATgcgGGTGATACCACCACCCCG-3′ for W253A (mutated sites shown with lowercase letters).

The activity assay for the WT and mutant enzymes was performed as described previously (30). Either 2 pmol of PA-fucosyl sialobiantennary or PA-sialobiantennary was mixed with a given amount of the protein (3.44 ng of WT, 354 ng of E156Q, 115 ng of D154N/E156Q, 4 ng of N193A and R218A, 36 ng of Y216A, or 11 ng of W253A). The assay was carried out in 10 μl of 100 mm Na-acetate buffer (pH 3.0) at 30 °C for 20 min, after which the reaction was stopped by incubating the mixture at 99 °C for 10 min. The resultant samples were analyzed by HPLC (GL Science, Tokyo, Japan), which was equipped with a Wakosil 5C18 column (Wako Pure Chemicals), set at 40 °C. The HPLC analysis was performed using 50 mm ammonium acetate buffer (pH 4.0) containing 0.15% 1-butanol at a flow rate of 1.5 ml/min. Fluorescence emitted from PA (excitation at 320 nm and emission at 400 nm) was monitored, and the relative hydrolytic activity of the WT enzyme was determined from the peak area of the hydrolyzed PA-fucosyl-GlcNAc or PA-GlcNAc. Experiments for each enzyme were repeated independently at least twice to ensure consistent results.

Author contributions

H. S., Y. Huang, T. A., and S. F. formal analysis; H. S. and Y. Huang investigation; H. S. and S. F. visualization; H. S., Y. Huang, T. A., C. Y., T. K., S. I., Y. Higuchi, K. T., and S. F. writing-review and editing; T. A., T. K., S. I., and Y. Higuchi resources; T. A., Y. Higuchi, K. T., and S. F. data curation; T. A. and S. F. software; T. A., C. Y., and Y. Higuchi methodology; T. A., C. Y., Y. Higuchi, K. T., and S. F. project administration; K. T. and S. F. conceptualization; K. T. and S. F. supervision; K. T. and S. F. funding acquisition; K. T. and S. F. validation; S. F. writing-original draft.