Identification of a novel amidase motif in neutral ceramidase

Abstract

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

Figure 1. Time course of induction of human neutral CDase

The neutral CDase was cloned into the pYES2 vector and the construct was transformed into the yeast double-knockout strain Δypc1Δydc1, which lacks the yeast CDases. Gene expression was induced by growth in galactose medium, and at different time points the culture was stopped and homogenates were assayed for CDase activity using D-erythro-C₁₂-NBD-ceramide as substrate. The average values from two independent experiments were plotted. Abbreviation: F. Units, arbitrary fluorescence units.

Figure 2. Biochemical characterization of the human neutral CDase expressed in yeast

(A) The effect of different amounts of yeast protein on CDase activity at different times. (B) Different amounts of human neutral CDase were assayed by incubation at 37 °C for 1 h as described in the Experimental section. For (C) and (D), 60 μg of yeast microsomal protein was used for CDase activity assay and the reaction time was 1 h. (C) Michaelis–Menten representations for CDase activity toward increasing concentrations of NBD-C₁₂-ceramide. (D) pH optimum determination. The buffers used are described in the Experimental section. The results shown are representative of triplicates from at least two independent experiments. Abbreviation: F. Units, arbitrary fluorescence units.

Figure 3. Effects of cations and reducing agents on human neutral CDase expressed in yeast

CDase assays were carried out at 37 °C and for 1 h as described in the Experimental section. (A, B) Effect of increasing concentrations of the indicated cations on CDase activity. (C) Effect of increasing concentrations of reducing agents on CDase activity. Values are the means from two independent experiments. β-ME, 2-ME.

Figure 4. Effects of different protease inhibitors and chemical modifiers on CDase activity

(A) The CDase assay was carried out in the presence of different protease inhibitors at the indicated concentrations for 1 h at 37 °C. (B–E) Chemical-modification reactions were carried out as described in the Experimental section. Control reactions contained no modifiers, but did contain enzyme. (B–C) Purified rat brain CDase enzyme was used for the assays: (B) effect of increasing concentration of DFP upon purified CDase activity; (C) effect of DEPC and NEM upon purified CDase activity. (D–E) Effect of pre-incubation of ceramide on the DFP inhibition of neutral CDase. The human neutral CDase was expressed in yeast and prepared as described in the Experimental section: (D) yeast lysates were preincubated with or without 100 μM DFP in 100 mM phosphate buffer, pH 7.5, at 37 °C for 15 min. (E) Yeast lysates were first preincubated with (500 μM) D-erythro-C₁₂-NBD-ceramide for 30 min on ice, and then incubated with or without 100 μM DFP for another 15 min at 37 °C. The values are expressed as means±S.D. (n=3). The results are representative of triplicates from two independent experiments.

Figure 5. Alignment of neutral CDase from different species

Using the ClustalW algorithm, neutral CDases from different species were aligned and the indicated serine residues were mutated as described in the Experimental section. Identical residues are boxed in black; amino acid numbers of the consensus sequence are shown above the alignment. GenBank accession numbers for neutral CDase are as follows: AF449759 (human), AB057433 (rat), AB037181 (mouse), U82513 (the cellular slime mould Dictyostelium discoideum, Dict. d.), AB112076 (Drosophila melanogaster, Dros. m.), AB016885 [Arabidopsis thaliana, arab. t. (thale cress)].

Figure 6. Identification of the hexaresidue core containing a proposed serine nucleophile in neutral CDases

Mutagenesis of CDase to alter individual residues to other amino acids were performed by PCR of wild-type neutral CDase. The PCR products were sequenced, cloned into pYES2 vector, and then transformed into the yeast double-knockout strain Δypc1Δydc1. Wild-type and mutant neutral CDase proteins were induced by galactose, and yeast microsomal lysates were subjected to CDase activity, reverse CDase activity and Western-blot assays as described in the Experimental section. (A) Effects of mutations in different conserved serines on neutral CDase activity. (B) Effect of mutations of on neutral CDase activity using NBD-C₁₂-ceramide or -C₁₆-ceramide as substrate. (C) Effect of mutations on reverse CDase activity. (D) A representative Western blot showing the expression level of CDase wild-type and mutant proteins. Values are means±S.D. (n=3). results are representative of triplicates from two independent experiments.