Katsuhiro Kita

A Novel Enzyme That Cleaves the N-Acyl Linkage of Ceramides in Various Glycosphingolipids as Well as Sphingomyelin to Produce Their Lyso Forms

We describe a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids. The enzyme was purified about 300-fold with 5% recovery from the culture filtrate of a newly isolated bacterium (Pseudomonas sp. TK4) by ammonium sulfate precipitation followed by several steps of high performance liquid chromatography. The purified enzyme preparation was completely free of exoglycosidases, sphingomyelinase, and proteases, and showed a single protein band corresponding to a molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis after staining with Coomassie Brilliant Blue. The enzyme shows quite wide specificity, i.e. it hydrolyzes both neutral and acidic glycosphingolipids, and simple glycosphingolipid cerebrosides to polysialogangliosides such as GQ1b. Furthermore the enzyme also hydrolyzes sphingomyelin to produce the respective lyso form. However, the enzyme shows hardly any activity on ceramides, indicating that it is completely different from the ceramidase (EC 3.5.1.23) reported previously. This enzyme, which is tentatively named sphingolipid ceramide N-deacylase, should greatly facilitate the further study of sphingolipids as well as lysosphingolipids.

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.

Activation of bacterial ceramidase by anionic glycerophospholipids: possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase

We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) [Okino, Tani, Imayama and Ito (1998) J. Biol. Chem. 273, 14368--14373]. In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents. Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective. Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific omega-hydroxyacyl Cer, by the enzyme in the absence of detergents. It was confirmed that several strains of P. aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S. aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol. Since both P. aeruginosa and S. aureus are suspected of being present in microflora of atopic skin, we speculate that S. aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase.

Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8.

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.

Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.

Enzymatic N-deacylation of sphingolipids

Publisher Summary Lysosphingolipids, sphingolipids with an N-deacylated ceramide moiety, are present at low levels in normal tissues, but accumulate abnormally in cells in various lysosomal storage diseases. For example, in Krabbes disease, caused by a deficiency of β -galactosylceramidase, abnormal accumulation of galactosylceramide as well as its lyso form is observed. Lyso-GM2 and lysosphingomyelin have been detected in the brain of patients with Tay-Sachs and Niemann-Pick type-A disease, respectively, whereas they are barely detectable in the normal brain. Lysosphingolipids inhibit protein kinase C, which could be responsible for the pathogenesis of sphingolipidoses. Several lines of evidence have suggested the biological significance of lysosphingolipids in various cell activities. Lysosphingolipids are useful for preparing sphingolipid derivatives containing appropriately labeled fatty acids and can be coupled with either appropriate proteins or gel matrix for affinity columns utilizing the amino groups newly generated in lysosphingolipids. This chapter describes a novel enzyme, tentatively designated sphingolipid ceramide N-deacylase (SCDase), which is capable of cleaving the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. To date, the preparation of lysosphingolipids has been performed using purely chemical procedures, which are somewhat troublesome, time-consuming, and give a low yield. Using the SCDase we were able to obtain easily the lyso forms of all species of glycosphingolipids and sphingomyelin without any alternation of their polar portions and sphingoid moieties.

[52] Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and ω-aminoceramide

Publisher Summary This chapter isolates a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids and tentatively names it “sphingolipid ceramide N-deacylase” (SCDase). SCDase is found to catalyze the hydrolysis and its reverse reaction as well, under different conditions. Under acidic conditions including a high concentration of Triton X-100, SCDase catalyzes the hydrolysis reaction efficiently. In contrast, at neutral pH with a low concentration of the detergent, the reverse reaction tends to be favored. It is possible therefore to utilize the desired reaction of SCDase if the conditions are changed. This chapter describes the synthesis of [ 14 C]ceramide, [ 14 C]glycosphingolipids, and ω-aminoceramide by the reverse hydrolysis (condensation) reaction of SCDase. Because the specificity of the enzyme is very specific to the amino group of sphingosine bases, no by-products are generated during the reaction. The fact that a wide range of sphingolipid species and sample amounts (from the nanomole to millimole level) can be used is significantly advantageous.