Sphingomyelinase of chicken erythrocyte membranes.

Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [³H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg²⁺ and Mn²⁺ activate the enzyme while Ca²⁺ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% ($\text{w}\text{v}$) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.     

Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Structure–activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase–SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH–SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg^2 + and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg^2 + and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus.

The hydrolysis of D-erythro beef brain sphingomyelin and D,L-erythro-N-palmitoylsphingomyelin dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30 degrees C for beef brain sphingomyelin and 41 degrees C for N-palmitoylsphingosine-phosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase-transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus

The hydrolysis of d-erythro beef brain sphingomyelin and $\text{d,l}\text{-erythro}\text{-N-}\text{palmitoylsphingomyelin}$ dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30°C for beef brain sphingomyelin and 41°C for N-palmitoylsphingosinephosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

Biochemical and Structural Information Transduction at the Mesoscopic Level in Biointerfaces Containing Sphingolipids

In this work we describe two aspects of molecular and supramolecular information transduction. The first is the biochemical and structural information content and transduction associated with sphingomyelinase activity. The results disclose a lipid-mediated cross-communication between the sphingomyelinase and phospholipase A₂ pathways. In addition, the two-dimensional degradation of sphingomyelin by sphingomyelinase affects the surface topography and the latter modulates the enzyme activity. The second is the information contained in the compositionally driven lateral organization of whole glial and neuronal membrane interfaces. The myelin monolayer exhibits microheterogeneous topographical structuring and nonhomogeneous lateral thickness of phase separated regions, depending dynamically on the lateral surface pressure. On the other hand, the differential response of functional living cells depends on information contained in the molecular organization of the contacting membrane interface.     

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.     

Entamoeba histolytica: Molecular cloning and characterization of a novel neutral sphingomyelinase

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg²⁺-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn²⁺ and partially inhibited by Zn²⁺. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5 mM Mn²⁺, and abolished by 5 mM Zn²⁺. A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37 kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Human lysosomal sphingomyelinase: substrate efficacy of apolipoprotein/sphingomyelin complexes

Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A-I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by approximately 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino-terminal half of this apoprotein, C-III(1-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 mumol h-1 (mg of protein)-1] at the gel----liquid-crystalline phase transition temperature (Tm) of the complex (40 degrees C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.     

Lipid-dependent regulation of neurotransmitter release from sympathetic nerve endings in mice atria

Neurotransmitter release from sympathetic terminals is a key avenue for heart regulation. Herein, presynaptic exocytotic activity was monitored in mice atrial tissue using a false fluorescent neurotransmitter FFN511, a substrate for monoamine transporters. FFN511 labeling had similarity with tyrosine hydroxylase immunostaining. High [K⁺]_o depolarization caused FFN511 release, which was augmented by reserpine, an inhibitor of neurotransmitter uptake. However, reserpine lost the ability to increase depolarization-induced FFN511 unloading after depletion of ready releasable pool with hyperosmotic sucrose. Cholesterol oxidase and sphingomyelinase modified atrial membranes, changing in opposite manner fluorescence of lipid ordering-sensitive probe. Plasmalemmal cholesterol oxidation increased FFN511 release upon K⁺-depolarization and more markedly potentiated FFN511 unloading in the presence of reserpine. Hydrolysis of plasmalemmal sphingomyelin profoundly enhanced the rate of FFN511 loss due to K⁺-depolarization, but completely prevented potentiating action of reserpine on FFN511 unloading. If cholesterol oxidase or sphingomyelinase got access to membranes of recycling synaptic vesicles, then the enzyme effects were suppressed. Hence, a fast neurotransmitter reuptake dependent on exocytosis of vesicles from ready releasable pool occurs during presynaptic activity. This reuptake can be enhanced or inhibited by plasmalemmal cholesterol oxidation or sphingomyelin hydrolysis, respectively. These modifications of plasmalemmal (but not vesicular) lipids increase the evoked neurotransmitter release.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.     

Niemann Pick disease: presence of the magnesium-dependent sphingomyelinase in brain of the infantile form of the disease.

Abstract— Brain of a 14-month-old patient with the infantile form of Niemann Pick disease was found to be practically devoid of sphingomyelinase activity when assayed at pH 5. When assayed at pH 7.4, in the presence of magnesium ions considerable hydrolysis of sphingomyelin was obtained by brain preparations. The rate of hydrolis of a homogenate of grey matter was about 0.3 μmol. mg protein^?1. H^?1, corresponding to about 15 μmol of sphingomyelin hydrolysed perg brain in 1 h. The possibility is suggested that the presence of the extra-lysosomal, magnesium dependent sphingomyelinase in brain of Niemann Pick patients may be responsible, in part, for the lesser accumulation of sphingomyelin in this tissue relative to other organs, such as liver or spleen.     

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (> 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [³H]oleic acid into cholesterol-[³H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimuated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquinone abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Effect of divalent metal ions on the synthesis of oligosaccharide side chains of Neurospora crassa glycoproteins

Neurospora crassa membrane preparations incorporated mannose from GDP-mannose-[¹⁴C] in the presence of Mg²⁺ into a polyprenol lipid and side chains of protein acceptor(s), which are labile on hydrolysis in weak base. The addition of Mn²⁺ to the reaction mixtures does not affect mannosyl lipid synthesis but it stimulates the transfer of mannose to larger oligosaccharide chains resistant to β-elimination and the transfer of a second mannosyl unit to form an O-glycosidically linked mannobiosyl side chain. Incubation of particulate preparations with polyprenol-mannose-[¹⁴C] in the presence of Mg²⁺ and Mn²⁺ also results in the transfer of a single mannose to the protein. When non-radioactive GDP-mannose is added to this reaction mixture, however, β-elimination yields mannobiose. The mannobiose is labeled in the reducing sugar only. These results indicate that the first mannose of this mannobiosyl side chain is transferred via a lipid intermediate, but the second mannose is transferred directly from GDP-mannose. In the presence of Mg²⁺ and Mn²⁺, mannose apparently is also transferred from polyprenol-mannose-[¹⁴C] to side chains which are resistant to hydrolysis.     

Mn2+ and Mg2+ improved sphingomyelinase production by Lactobacillus rhamnosus FTDC 8313 and binding affinity to sphingomyelin for generation of ceramides

The present research aims to optimize the sphingomyelinase (SMase) activity produced by Lactobacillus rhamnosus FTDC 8313 using divalent metal ions via response surface methodology and to further study the effects of the divalent metal ions on SMase activity using molecular modeling approach. This study also aimed to assess the possibility of increasing ceramide levels in vitro on cultured keratinocytes upon treatment with the extracellular extract of the optimized L. rhamnosus FTDC 8313. Using a central composite design, an optimum point of SMase activity (6.54 mU ml^?1) was produced from a combination of 0.65% (w/v) MnSO₄ and 0.82% (w/v) MgSO₄. 3D response surface indicated that the altered availability of the two ions (Mn²⁺ and Mg²⁺) reduced their effects on SMase activity. In addition, the treatment of the HaCaT cells with optimized extracellular extract of L. rhamnosus FTDC8313 significantly increased (P < 0.05) the conversion of sphingomyelin to ceramide as compared to the control. Molecular docking demonstrated that the addition of Mn²⁺ and Mg²⁺ into the active site of SMase improved the binding affinity between the SMase and sphingomyelin based on its free energy of binding as well as the interaction distances between the important catalytic residues Glu53 and His296.