Identification and characterization of a core fucosidase from the bacterium Elizabethkingia meningoseptica

Abstract

All reported α-l-fucosidases catalyze the removal of nonreducing terminal l-fucoses from oligosaccharides or their conjugates, while having no capacity to hydrolyze core fucoses in glycoproteins directly. Here, we identified an α-fucosidase from the bacterium Elizabethkingia meningoseptica with catalytic activity against core α-1,3-fucosylated substrates, and we named it core fucosidase I (cFase I). Using site-specific mutational analysis, we found that three acidic residues (Asp-242, Glu-302, and Glu-315) in the predicted active pocket are critical for cFase I activity, with Asp-242 and Glu-315 acting as a pair of classic nucleophile and acid/base residues and Glu-302 acting in an as yet undefined role. These findings suggest a catalytic mechanism for cFase I that is different from known α-fucosidase catalytic models. In summary, cFase I exhibits glycosidase activity that removes core α-1,3-fucoses from substrates, suggesting cFase I as a new tool for glycobiology, especially for studies of proteins with core fucosylation.

Keywords: allergy; chemical rescue; core fucosidase; core α-1,3 fucosylation; core α-1,3-fucose; glycobiology; glycoprotein; mass spectrometry (MS); mutagenesis; oligosaccharide.

Figure 1.

Phylogenetic tree of cFase I and characterized α-l-fucosidases. The amino acid sequences of all enzymes were obtained from GenBank^TM. Sequence alignment was performed using ClustalW with the MEGA 5.0 program. The tree was constructed using a neighbor-joining method. GenBank^TM accession numbers of these proteins are as follows: Drosophila melanogaster DmFuca (AAM50292.1); Homo sapiens FucA2 (NP_114409.2); Canis lupus familiaris ClFucA1 (CAA63362.1); H. sapiens FucA1 (AAA52481.1); Dictyostelium discoideum DdFuca (AAO51149.1); T. maritima TM0306 (AAD35394.1); S. solfataricus SsFucA1 (AY887105); F. graminearum FgFuca (AFR68935.1); B. thetaiotaomicron BT2970 (AAO78076.1); Lactobacillus casei AlfB (CAQ67877.1); AlfC (CAQ67984.1); Elizabethkingia meningoseptica cFase I (WP_047034007.1); B. bifidum BbAfcB (BAH80310.1); Streptomyces sp. SsFuc1 (AAD10477.1); Bifidobacterium longum BiAfcB (ACJ53394.1); Clostridium perfringens Afc2 (ABG83106.1); B. thetaiotaomicron BT2192 (AAO77299.1); Arabidopsis thaliana AtFUC1 (NP_180377.2); A. thaliana Axy8 (CAB36703.1); Lilium longiflorum LlFuc (BAF85832.1); Aspergillus nidulans AN8149.2 (EAA59171.1); B. bifidum BbAfcA (AAQ72464.1); C. perfringens Afc3 (ABG82552.1); B. longum Blon 2335 (ACJ53393.1).

Figure 2.

Enzymatic characterization of purified recombinant cFase I. The enzymatic activity and stability of cFase I at various pH values (a and b) and temperatures (c and d) are shown. The concentration-dependent inhibition of cFase I activity by Cu²⁺ ions (e) and deoxyfuconojirimycin (f) is shown. All reactions were triplicated for statistical evaluations. Vertical error bars indicate standard deviation.

Figure 3.

Hydrolysis by recombinant cFase I of fucosylated oligosaccharides on 3-FL and Lewis X. 3-FL (a) and Lewis X (b) were incubated in the absence (a1 and b1) and presence (a2 and b2) of purified cFase I, and the reaction mixtures were subjected to ESI-MS analysis. The molecular ion peaks at m/z 511.13 (a1) and 552.17 (b1) correspond to sodium adducts of 3-FL and Lewis X, respectively. The molecular ion peaks at m/z 365.08 and 406.12 are sodium adducts of lactose (calculated, 365.1) and lacto-N-biose (calculated, 406.1), respectively. The peak at m/z 187.0 is a sodium adduct of fucose monosaccharide.

Figure 4.

Sequence analysis of cFase I. Structure-based sequence alignment of some GH29 family members. The 3D structures of T. maritima α-fucosidase (TM0306, Protein Data Bank code 1HL8), B. thetaiotaomicron α-fucosidases (BT2970 and BT2192, Protein Data Bank codes 2WVV and 3EYP, respectively), B. longum α-fucosidase (BiAfcB, Protein Data Bank code 3UES), and E. meningoseptica α-fucosidase (cFase I) were aligned using PROMALS3D, and the figure was produced using ESPript. The secondary structures of TM0306 and BiAfcB are indicated above and below the sequences, respectively. Identical residues are shown in white letters on a red background, and similar residues are shown in red letters on a white background. The residues (in TM0306) that are important for Fuc binding are indicated by magenta circles. The purple star denotes the conserved nucleophile. Green stars indicate experimentally confirmed acid/base residues and/or acid/base candidates in cFase I. A ligand-induced mobile loop (173–182 in BiAfcB) is boxed in yellow, and the corresponding extended loop of cFase I is highlighted with yellow squares.

Figure 5.

Chemical rescue of the mutants (D242A, E302A, and E315A) of cFase I. The effect of varying concentrations of sodium azide (a) and sodium formate (b) on the rate of enzyme-catalyzed cleavage is shown. Reactions were performed at 37 °C, pH 4.6, with pNP-fucoside as the substrate.

Figure 6.

Defining cFase I activity by MALDI-TOF and other analyses. Core defucosylation of PLA₂ (a and c) and HRP (b and d) by cFase I is shown. Enzyme-digested glycoprotein was subjected to CBB staining (top), Western blot (WB) analysis with rabbit polyclonal anti-HRP antibodies (middle), and lectin blot (LB) analysis with biotinylated AAL (bottom). Control (lane 1), only substrate; cFase I (lane 2), substrate treated with purified cFase I; PNGF (lane 3), substrate treated with PNGase F; cFase I +PNGF (lane 4), substrate sequentially treated with purified cFase I and PNGase F; PNGF-II (lane 5), substrate treated with PNGase F-II, used as a positive control. N-Glycan profile of PLA₂ (c) and HRP (d) treated without (top) and with (bottom) cFase I, N-glycans of untreated or treated substrate were released by PNGase F-II. All the peaks were annotated with GlycoMod server and referenced to the previous study; all molecular ions are present in sodiated form ([M + Na]⁺). The peak of a potassium adduct ([M + K]⁺) was evident beside that of a sodiated form, with ∼16 Da addition that was not labeled in the graph. The glycan structure of peak m/z 1485.46 was verified to contain an outside chain fucose by MS/MS.

Figure 7.

Core defucosylation of PLA₂ and HRP by cFase I mutants. Enzyme-digested glycoprotein was subjected to CBB staining and, Western blot (WB) analysis with rabbit polyclonal anti-HRP antibodies. Control, only substrate; D242A, substrate treated with D242A mutant of cFase I; E302A, E315A, and 249Δ265, substrate treated with E302A, E315A, and 249Δ265 mutants, respectively. WT, substrate treated with wildtype cFase I.

Figure 8.

Potential impacts of cFase I treatment on binding to PLA₂ by IgE in allergic patient sera. PLA₂ and cFase I-treated PLA₂ were exposed to serum samples collected from allergic patients and assayed for IgE-mediated reactivities, respectively. a, eight samples demonstrated a positive IgE binding to PLA₂ by ELISA, and the binding was significantly attenuated by cFase I treatment (p = 0.0369). The sera collected from healthy people were used as a control (serumH). Statistical analysis comparing IgE binding with PLA₂ and to defucosylated PLA₂ was assessed by parried t test. b, four of eight samples positive in ELISA demonstrated a positive detection by immunoblotting (sera 21, 32, 31, and 27). After cFase I treatment, the band intensity decreased.