Molecular Cloning and Expression of Mn ²⁺ -Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

Abstract

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 shortchain 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.

Host-Inducible Immunogenic Sphingomyelinase-Like Protein, Lk73.5, ofLeptospira interrogans

“…recLk73.3 was only mildly hemolytic for rabbit and horse erythrocytes (Fig. 5B), and this activity was not increased in the presence of well-known phospholipase activators, such as Ca, Mg, and Mn (28,46), at concentrations of 2 to 10 mM (data not shown).…”

“…The central part of Lk73.5 (180 to 462 amino acid residues) corresponding to the exo/endo/phos conservative domain (pfam03372.5) also demonstrated substantial homology to phospholipases from Staphylococcus aureus (49.8%) (39), Bacillus cereus (47.6%) (23), Listeria ivanovii (46.9%) (14), and Pseudomonas spp. (41.9%) (46). Expression of Lk73.5 by cultured leptospiras.…”

The Mucin Box and Signal/Anchor Sequence of Rat Neutral Ceramidase Recruit Bacterial Sphingomyelinase to the Plasma Membrane

“…The molecular mechanism remains to be addressed, but this result is throughly consistent with the previous observation that the C-terminal region of SMase is necessary to hydrolyze cellular SM but not the SM micelles in the presence of detergents. 20) The present study indicates that the mucin box and signal/anchor sequence of a rat neutral ceramidase recruited bacterial SMase to plasma membranes. This method should be applicable in delivering target proteins of eucaryotic or procaryotic origin to plasma membranes in active form and thus should facilitate research in related fields of cell biology as well as cell biotechnology.…”

“…[15][16][17][18][19] We have cloned SMase from an aquatic bacterium, Pseudomonas sp. TK4, 20) and have confirmed its recruitment to the lysosomes of HEK293 cells using the lysosome-sorting motif of a lysosomal acid phosphatase. 21) To determine whether cell-surface SM can be hydrolyzed by bacterial SMase expressed on the cell surface, we tried to recruit SMase to the plasma membranes in the present study.…”

“…20) In this study, MboxSMaseÁC, which lacks 186 C-terminal residues, was also expressed in CHOP cells. It was found to be active in the lysate in the presence of detergents, but no significant degradation of endogenous SM was observed in Mbox-SMaseÁC-expressing transfectants (data not shown).…”