Inhibition of Acidic Sphingomyelinase by Xanthone Compounds Isolated from Garcinia Speciosa

Sphingomyelinase is considered to be involved in the regulation of apoptosis and cell growth. In the course of our screening for acidic sphingomyelinase inhibitors we isolated three xanthone compounds, α-mangostin, cowanin, and cowanol, from the bark of Garcinia speciosa. These compounds competitively inhibited bovine brain-derived acidic sphingomyelinase with IC₅₀ values of 14.1, 19.2, and 10.9 μM, respectively and inhibited the acidic sphingomyelinase more effectively than the neutral sphingomyelinase of bovine brain. α-Mangostin inhibited the acidic sphingomyelinase in the most selective manner. α-Mangostin was chemically modified and its structure-activity relationships are discussed.     

Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-alpha (TNF-alpha) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-alpha (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of N(G)-nitro-L-arginine methyl ester, TNF-alpha produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-alpha. Pretreatment of the arteries with desipramine (10 microM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-alpha on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-alpha activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-alpha inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.     

Acidic extracellular pH increases calcium influx-triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase-9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4-6.5) induced matrix metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase 1/2 and p38 mitogen-activated protein kinases and nuclear factor-kappaB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L-type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase-9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expression. BAPTA-AM reduced acidic extracellular pH-induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor-kappaB activity. These data suggest that the calcium influx-triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase-9 expression, at least in part, through nuclear factor-kappaB activation.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Synthesis and evaluation of a difluoromethylene analogue of sphingomyelin as an inhibitor of sphingomyelinase

A sphingomyelin analogue 2, in which the long alkenyl chain and the phosphodiester moiety of sphingomyelin were replaced by a phenyl and an isosteric difluoromethylenephosphonic acid, was prepared to evaluate its inhibitory potency to sphingomyelinase. The analogue non-competitively inhibited the neutral sphingomyelinase in bovine brain microsomes with an IC50 of 400 microM. The compound had the ability to suppress tumor necrosis factor alpha-induced apoptosis of PC-12 neurons at a low concentration of 0.1 microM.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases

Fas/CD95/Apo-I has been shown to stimulate a variety of molecules including several members of the caspase family and the acidic sphingomyelinase (Martin and Green 1995; Gulbins et al, 1995). Here, we demonstrate that Fas receptor-triggered activation of the acidic sphingomyelinase, consumption of sphingomyelin, release of ceramide, and subsequent activation of JNK and p38-K are regulated by caspases. Inhibition of caspases by Ac-YVAD-chloromethylketone or transient CrmA transfection prevented stimulation of acidic sphingomyelinase, release of ceramide and activation of JNK and p38-K upon Fas-receptor crosslinking. Likewise, Fas triggered apoptosis was almost completely blocked by Ac-YVAD-chloromethylketone or CrmA mediated inhibition of caspases. The results suggest a new signalling cascade from the Fas receptor via caspases to acidic sphingomyelinase, ceramide and JNK/p38-K.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

Amyloid-beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-beta peptide (Abeta) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Abeta induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Abeta-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Abeta and ceramide induced OLG death. In addition, Abeta activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Abeta cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Abeta-induced OLG death. Glutathione (GSH) precursors inhibited Abeta activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Abeta-induced death. These results suggest that Abeta induces OLG death by activating the nSMase-ceramide cascade via an oxidative mechanism.     

A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin.

Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.     

A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase

Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.     

Neutral Magnesium-Dependent Sphingomyelinase from Liver Plasma Membrane: Purification and Inhibition by Ubiquinol

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than alpha-tocopherol, alpha-tocopherylquinone, alpha-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol- 10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.     

Biological activities of alpha-mangostin derivatives against acidic sphingomyelinase

Deprenyl and benzofenone-type congeners of alpha-mangostin 1 have been synthesized to understand their role for the inhibitory activity against sphingomyelinase (SMase). While removal of the prenyl group of the right side (11 and 12) caused loss of the selectivity between ASMase (acidic sphingomyelinase) and NSMase (neutral sphingomyelinase), the prenyl group of the left side appeared to increase the inhibitory activities (16 and 17).     

Renal sodium-D-glucose cotransport system. Involvement of tyrosine residues in sodium-transporter interaction

The papain inhibitor from human spleen was purified by extraction in isotonic sucrose, acetone fractionation, papain-Sepharose affinity chromatography and gel filtration on Sephadex G-50. The purified inhibitor was fractionated by electrofocusing into four major isoelectric variants with pI values of 4.7, 5.0, 6.0 and 6.5. These variants can be classified into two groups: the acidic type, comprising the variants with pI 4.7 and 5.0, and the neutral type, comprising the variants with pI 6.0 and 6.5. The following properties distinguish the two types: 1. Immunological properties: antibodies raised against either of the neutral variants precipitated both of these, but not the acidic variants. The antiserum against the human epidermal cysteineproteinase inhibitor precipitated the acidic variants, but not the neutral variants. 2. Molecular size: two-dimensional electrophoresis of the purified inhibitor gave molecular weights of 11400 for the acidic variants and 12000 for the neutral variants. The pI 6.0 variant contained two compounds with molecular weights of 12000 and 12800. 3. Enzyme spectrum: human cathepsin B was inhibited by the acidic type, while the neutral type was a poor inhibitor. Both types inhibited cathepsin H, papain, ficin and bromelain, although the inhibition of bromelain did not exceed 70%. Human cathepsin D, bovine trypsin and chymotrypsin and porcine elastase were not inhibited by either type.     

A new Zn2+-stimulated sphingomyelinase in fetal bovine serum

Fetal bovine serum contains a Zn2+-dependent sphingomyelinase with optimal activity at pH 5.5 in vitro. Activity could be demonstrated with a liposomal sphingomyelin substrate suspension but was stimulated up to 15-fold by Triton X-100. Under a variety of conditions tested, phosphatidylcholine, lysophosphatidylcholine, glycerophosphocholine, and p-nitrophenyl phosphate were not substrates for this activity. Several inhibitors of serum alkaline and acid phosphatases had no effect on the activity. The enzyme resembles the acid lysosomal sphingomyelinase in pH optimum and inhibition by AMP but differs in inhibition by EDTA, stimulation by Zn2+, and heat lability at 55 degrees C. It resembles the neutral, Mg2+-stimulated enzyme in inhibition by EDTA and heat lability but differs in metal ion requirement and pH optima. Of the sera tested, activity was highest in fetal bovine serum, with fetal bovine greater than newborn bovine greater than horse greater than human; more than 95% of the activity is in the lipoprotein-free infranatant of serum (d greater than 1.21). This activity appears to be a hitherto undescribed sphingomyelinase. Its biological functions are not known but may subserve a special role in sphingomyelin catabolism in the circulation, in blood vessel walls, or in the tissue(s) of origin.     

Enhancement of DNA synthesis of human epithelial carcinoma cells by acidic fibroblast growth factor

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Ubiquinol inhibition of neutral sphingomyelinase in liver plasma membrane: specific inhibition of the Mg(2+)-dependent enzyme and role of isoprenoid chain

In this work, the specificity of ubiquinol as inhibitor of the neutral sphingomyelinases present at the plasma membrane (Mg(2+)-dependent and -independent) and structural requirements for such inhibition have been studied. Our results have shown that ubiquinol specifically inhibits Mg(2+)-dependent neutral sphingomyelinase activity in isolated liver plasma membranes, but no significant participation of the Mg(2+)-independent enzyme was observed. Both the reduction state of the (hydro)quinone ring and the length of the hydrophobic side chain were important determinants in neutral sphingomyelinase inhibition. Ubiquinols inhibited the nSMase more efficiently than ubiquinones, and hydrophobic homologs with six or nine isoprene units were the most effective inhibitors. Inhibition of nSMase by ubiquinols displayed similarities with inhibition by manumycin and the hydroquinones F11334's, suggesting that these compounds could act as structural analogs of ubiquinol. Beyond its participation in mitochondrial energy metabolism, and as antioxidant, this novel role for ubiquinol as a neutral sphingomyelinase inhibitor should be considered an important factor to regulate lipid signaling at the plasma membrane that could be related to its beneficial effects on cells, tissues, and organisms.     

Sphingomyelinase restricts the lateral diffusion of CD4 and inhibits human immunodeficiency virus fusion

Previously, we reported that treatment of cells with sphingomyelinase inhibits human immunodeficiency virus type 1 (HIV-1) entry. Here, we determined by measuring fluorescence recovery after photobleaching that the lateral diffusion of CD4 decreased 4-fold following sphingomyelinase treatment, while the effective diffusion rate of CCR5 remained unchanged. Notably, sphingomyelinase treatment of cells did not influence gp120 binding, HIV-1 attachment, or fluid-phase and receptor-mediated endocytosis. Furthermore, sphingomyelinase treatment did not affect the membrane disposition of the HIV receptor proteins CD4, CXCR4, and CCR5, as determined by Triton X-100 extraction. Restriction of CD4 diffusion by antibody cross-linking also inhibited HIV infection. We therefore interpret the decrease in CD4 lateral mobility following sphingomyelinase treatment in terms of clustering of CD4 molecules. Examination of fusion intermediates indicated that sphingomyelinase treatment inhibited HIV at a step in the fusion process after CD4 engagement. Maximal inhibition of fusion was observed following short coculture times and with target cells that express low levels of CD4. As HIV entry into cells requires the sequential engagement of viral envelope protein with CD4 and coreceptor, we propose that sphingomyelinase inhibits HIV infection by inducing CD4 clustering that prevents coreceptor engagement and HIV fusion.