Purification and Characterization of a Novel Ceramidase fromPseudomonas aeruginosa

Abstract

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0 -C18:0 fatty acids and 7-nitrobenz-2-oxa-1,3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K.

Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

“…The mucin box of the mammalian enzymes is highly glycosylated with O-glycans and retains the enzyme at the plasma membrane as a type II integral membrane protein, because a mucin box-deleted mutant CDase and an Ala-replacement mutant enzyme were found to be secreted into the culture medium when expressed in human embryonic kidney 293 cells (20). Furthermore, it was clarified that bacterial and invertebrate neutral CDases, lacking a mucin box, were released into the extracellular milieu (9,18). The present study revealed that znCD possesses a mucin box in which four possible O-glycosylation sites are present (Fig.…”

“…CDase Assay-CDase activity was measured using C12-NBD-Cer as a substrate as described (18,21). Briefly, 400 pmol (for cell lysates) or 2 nmol (for embryo lysates) of C12-NBD-Cer was incubated at 37°C for 1 h with an appropriate amount of enzyme in 40 l of reaction mix (50 mM Tris-HCl buffer, pH 7.5, containing 0.05% Triton X-100).…”

“…Interestingly, neutral CDases of bacteria (18) and Drosophila (9) were solely detached from the cells as a soluble form, whereas those of mammalian origins were mainly recovered in membrane fractions (19). Recently, we clarified the reason for this discrepancy at the molecular level, i.e.…”

Citric acid inhibits a bacterial ceramidase and alleviates atopic dermatitis in an animal model

“…4 Ceramidase is an enzyme that catalyzes the hydrolysis of the N-acyl linkage of ceramide to produce a free fatty acid and a sphingosine base. Okino et al have isolated a ceramidase-producing bacterium, P. aeruginosa AN17, from the skin of patients with atopic dermatitis 5 and cloned the gene encoding its ceramidase. 6 These reports lead to the hypothesis that the ceramidase from colonizing bacteria contributes to the reduction of ceramide in atopic skin lesions and that inhibiting the bacterial ceramidase will prevent the ceramide decrease in atopic skin and improve the disease.…”

“…Citric acid also showed specific mass spectra m/z at 191 [MÀH] À , 213 [M+Na-H] À and 235 [M+2NaÀH] À by ESI MS. We then purchased commercially available citric acid and confirmed its activity against the bacterial ceramidase compared with the purified one. Ceramidase activity was assessed according to the method by Okino et al 5 with minor modifications as described. 7 As shown in Figure 1a, citric acid was found to inhibit the Pseudomonas ceramidase with an IC 50 value of 19.6 mg ml À1 as well as the purified one.…”