Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Abstract

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.

Fig. 1.

Alignments of amino acid sequences of EGCase I and II and EGALC. The deduced amino acid sequences of EGCase I, EGCase II, and EGALC found in R. equi were aligned using Clustal X. Identical and similar residues are shown by white letters on a black background and black letters in an open box, respectively. Amino acid residues conserved in GH family 5 glycosidases are indicated by open arrowheads. Two glutamates, possibly functioning as an acid/base catalyst and nucleophile, respectively, are indicated by closed arrowheads. The secondary structural elements of EGCase II are shown above the amino acid residues of EGCase I.

Fig. 2.

Substrate specificity of the purified recombinant EGCase I. A: SDS-PAGE showing the purification of the recombinant EGCase I. The purified protein was subjected to SDS-PAGE and visualized with CBB. Lane 1, unbound fraction; lane 2, eluted fraction from a Ni Sepharose 6 Fast Flow column. B: TLC showing the oligosaccharides released from several GSLs by EGCase I and II. Each GSL (2 nmol) was incubated with 100 ng (for GM1a and Fucosyl-GM1a), 200 ng (for Gb3Cer), or 1 μ g (for Gb4Cer) of the recombinant EGCase I (I) or EGCase II (II), or without enzyme (−) at 37°C for 60 min (GM1a, Fucosyl-GM1a, and Gb3Cer) or for 16 h (Gb4Cer). Samples were loaded onto a TLC plate that was developed with chloroform-methanol-0.02% CaCl₂ (5:4:1, v/v/v). GSLs and oligosaccharides were visualized with orcinol-H₂SO₄ reagent. C: The time course for hydrolysis of Fucosy-GM1a and Gb3Cer by EGCase I (closed circle) and EGCase II (open square). Each GSL (2 nmol) was incubated with 0.4 mU (Fucosy-GM1a) or 2.5 mU (Gb3Cer) of the recombinant EGCase I or EGCase II. The extent of hydrolysis was calculated by the method described in EXPERIMENTAL PROCEDURES. Values represent the mean ± SD (n = 3).

Fig. 3.

Characterization of the recombinant EGCase I. A: pH dependency of the recombinant EGCase I for the hydrolysis of GSLs. Fifty millimolar sodium acetate buffer (open square, pH 3.5–6.0) and Tris-HCl buffer (closed square, pH 6.5–8.0) were used. B: Effects of the detergent and organic solvents on the hydrolysis of GM1a by EGCase I. GM1a (2 nmol) was incubated with 0.1 mU of the recombinant EGCase I in 50 mM sodium acetate buffer, pH 5.5, at 37°C for 1 h. C: Transglycosylation activity toward several 1-alkanols by EGCase I. GM1a (2 nmol) was incubated with 1 mU of the recombinant EGCase I in 50 mM sodium acetate buffer, pH 5.5, containing organic solvent at a concentration of 10% (v/v) at 37°C for 30 min. Values represent the mean ± SD (n = 3).

Fig. 4.

Mechanical insights into the difference in reaction efficiency between EGCase I and EGCase II. A: Superposition of the structures of EGCase I (model) and EGCase II (crystal, Protein Data Bank code 2osx). Dotted arrow denotes the EGCase II-specific flexible loop. B: Alignment of amino acid sequences of EGCase I and II and jellyfish EGCase around the flexible loop. C: CBB-staining (left panel) and Western blotting (right panel) of the purified wild-type (WT) and loop-deleted mutant (Δloop) EGCase II. D: TLC showing the fluorescent Cers released from 0.4 nmol of C12-NBD-labeled GM1a and Gb3Cer by WT and Δloop EGCase II. Each GSL was incubated with 50 ng (GM1a) and 400 ng (Gb3Cer) of WT and Δloop EGCase II for 10 min (GM1a) or for 60 min (Gb3Cer). Fluorescent Cer and GSLs were quantified with the fluorescent detector (excitation 475, emission 525 nm). E: Relative activities of WT and Δloop EGCase II toward C12-NBD-GM1a and C12-NBD-Gb3Cer. Values represent the mean ± SD (n = 3).