Isolation and characterization of an exopolymer produced by Bacillus licheniformis: In vitro antiviral activity against enveloped viruses.

The exopolymer (EPSp) produced by the strain B. licheniformis IDN-EC was isolated and characterized using different techniques (MALDI-TOF, NMR, ATR-FTIR, TGA, DSC, SEM). The results showed that the low molecular weight EPSp contained a long polyglutamic acid and an extracellular teichoic acid polysaccharide. The latter was composed of poly(glycerol phosphate) and was substituted at the 2-position of the glycerol residues with a αGal and αGlcNH2. The αGal O-6 position was also found to be substituted by a phosphate group. The antiviral capability of this EPSp was also tested on both enveloped (herpesviruses HSV, PRV and vesicular stomatitis VSV) and non-enveloped (MVM) viruses. The EPSp was efficient at inhibiting viral entry for the herpesviruses and VSV but was not effective against non-enveloped viruses. The in vivo assay of the EPSp in mice showed no signs of toxicity which could allow for its application in the healthcare sector.

Anaemia in advanced chronic fasciolosis.

The association between fasciolosis-induced anaemia and related factors has been quantified in a rodent model. Haematological parameters were analysed in Wistar rats at 20 and 60 weeks post-infection (p.i.). Pigment stones and bile specimens were collected. Serum IgG1, IgG2a and IgE were determined in rat serum samples. Cytokine levels have been correlated with haematological parameters. The screening for gastrointestinal bleeding was carried out. Bacteriological bile cultures revealed viable bacteria in 53.8% of specimens at 60 weeks p.i. The results show that the type of anaemia in fasciolosis might be considered a biomarker of the chronicity period of the disease, changing from normocytic to macrocytic in the early chronic period (20 weeks p.i.) and to microcytic in the advanced chronic period (60 weeks p.i.). Likewise, changing from normochromic in the early chronic period to hypochromic in the advanced chronic period. Multivariate analysis suggested an association between anaemia and the following factors: fluke burden, eggs per gram of faeces, body area of parasite, presence of blood in faeces, IgG1 and eosinophil levels, and % of splenic weight. Of all variables analysed, the fluke burden is the one which presents the highest anaemia risk, even exceeding the variable presence of blood in faeces. The development of anaemia appears to be complex and may involve multiple mechanisms. However, to the mechanisms that until now explain Fascioliosis-related anaemia (compensatory increase in erythrocyte production and a continuous drain on iron stores resulting from the parasites' blood-sucking activities) the following causes ought to be added: haemolysis of red blood cells, the general effects of inflammation on erythropoiesis, concomitant parasitic and bacterial infections and pre-morbid nutritional abnormalities. Extrapolation to human fasciolosis is discussed. The results of the rodent model lead to the assumption that a high risk of anaemia in subjects with a heavy parasitic burden in human hyperendemic areas of fasciolosis is to be expected.

The efficacy of photochemical treatment with methylene blue and light for the reduction of Trypanosoma cruzi in infected plasma.

BACKGROUND AND OBJECTIVES: Chagas disease is a transfusion-transmitted infection. This study evaluates the efficacy of a methylene blue (MB) and light system for reducing the viability of Trypanosoma cruzi in plasma. MATERIALS AND METHODS Trypanosoma cruzi strains were spiked in plasma pools. Treatment arms included combined filtration, MB, light and freezing. Post-treatment parasite viability was assayed through in vitro cultures and in vivo inoculation in inducible nitric oxide synthase- and interferon-gamma-receptor-deficient mice. RESULTS: The filtration, MB and light combined treatment showed a log reduction of > 3.4 in in vitro cultures, and log reductions that ranged from > 4.9 to > 5.8 in deficient mice inoculated with different T. cruzi strains. CONCLUSION: The treatment of plasma units with the MB and light system reduces the T. cruzi burden and could be useful in preventing transfusion-transmitted Chagas disease.

Trypanosoma cruzi-induced molecular mimicry and Chagas' disease.

Chagas' disease, caused by Trypanosoma cruzi, has been considered a paradigm of infection-induced autoimmune disease. Thus, the scarcity of parasites in the chronic phase of the disease contrasts with the severe cardiac pathology observed in approximately 30% of chronic patients and suggested a role for autoimmunity as the origin of the pathology. Antigen-specific and antigen-non-specific mechanisms have been described by which T. cruzi infection might activate T and B cells, leading to autoimmunity. Among the first mechanisms, molecular mimicry has been claimed as the most important mechanism leading to autoimmunity and pathology in the chronic phase of this disease. In this regard, various T. cruzi antigens, such as B13, cruzipain and Cha, cross-react with host antigens at the B or T cell level and their role in pathogenesis has been widely studied. Immunization with those antigens and/or passive transfer of autoreactive T lymphocytes in mice lead to clinical disturbances similar to those found in Chagas' disease patients. On the other hand, the parasite is becoming increasingly detected in chronically infected hosts and may also be the cause of pathology either directly or through parasite-specific mediated inflammatory responses. Thus, the issue of autoimmunity versus parasite persistence as the cause of Chagas' disease pathology is hotly debated among many researchers in the field. We critically review here the evidence in favor of and against autoimmunity through molecular mimicry as responsible for Chagas' disease pathology from clinical, pathological and immunological perspectives.

¿Existe algún papel patogénico para la autoimmunidad en la enfermedad de Chagas? / Is there a pathogenic role of autoimmune responses in Chagas' disease?

Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, un paradigma de enfermedad autoimmune inducida por infección. La escasez de parásitos en la fase crónica de la enfermedad contrasta con la severa patología cardíaca observada en aproximadamente un 30 por ciento de los pacientes crónicos, y sugiere un papel para la autoinmunidad en el orígen de la patología. Dependiendo de la respuesta inmunitaria contra el parásito, se han descrito mecanismos antígeno específicos y antígeno no-específicos que pueden activar células T y B y causar autoinmunidad. Entre los primeros, el mimetismo molecular (similitud de secuencias entre antígenos del agente infeccioso y del hospedador) se considera el mecanismo más importante que puede conducir a autoinmunidad y patología en la fase crónica de esta enfermedad. Con el uso de técnicas más sensibles, el parásito se detecta cada vez más facilmente en el hospedador infectado, el cual puede padecer patología bien directamente o a través de la respuesta inflamatoria específica contra el parásito. Por ello, el tema de la autoinmunidad versus persistencia del parásito como causa de la patología en la enfermedad de Chagas es extensamente debatido entre los investigadores del área. Nuevos argumentos a favor y en contra de cada hipótesis han sido publicados recientemente. En el presente trabajo pasamos revista a ambas hipótesis de forma crítica y desde perspectivas clínicas, patológicas e inmunológicas (AU)

Diagnóstico de la enfermedad de Chagas en niños / Diagnosis of Chagas disease in children

La enfermedad de Chagas, endémica en América Central .y del Sur, se debe a la infección por T. cruzi; sin embargo, se ha propuesto que el origen de la enfermedad podría ser autoinmune. Gracias a las medidas preventivas de control del insecto vector, en la actualidad el mayor riesgo de transmisión de la enfermedad es a través de transfusiones sanguíneas y por transmisión materna; por ello, el estudio de la incidencia de la entidad en la población infantil es un indicador de la eficiencia de la prevención. No obstante, el diagnóstico sigue siendo complejo, y se requiere la combinación de al menos tres tests para determinar la enfermedad. En el presente estudio hemos utilizado un péptido sintético, denominado R3, correspondiente a un autoantígeno humano que es reconocido en ELISA por los sueros chagásicos. Dicho test mostró un 92,4 por ciento de sensibilidad y un 100 por ciento de especificidad para sueros chagásicos. Esta sensibilidad y especificidad fueron mayores que para otros autoantígenos descritos hasta la fecha. No se detectó reactividad cruzada con sueros de pacientes infectados con Leishmania, en sueros de pacientes con cardiomiopatía dilatada idiopática, ni en sueros de individuos sanos de la zona endémica. Además, la inmunorreactividad del ELISA R3 correlacionó con otros tests para la enfermedad, como inmunofluorescencia indirecta y ensayos de ELISA con extractos de T. cruzi, así como hemaglutinación indirecta. Los niveles de anticuerpos anti-R3 aumentaron con la progresión y la sintomatología de la enfermedad de Chagas. Cabe destacar que se observó una disminución en el título de anticuerpos anti-R3 en pacientes tratados con drogas antiparas tarias. Estos resultados sugieren que la presencia de anticuerpos anti-Cha es un marcador altamente específico de la enfermedad de Chagas y que el ELISA R3 podría ser de ayuda para el diagnóstico y la monitorización de la enfermedad de Chagas (AU)

Antibodies to an epitope from the Cha human autoantigen are markers of Chagas' disease.

Chagas' disease is a prevalent disease in South America that is thought to have an autoimmune etiology. We previously identified human Cha as a new autoantigen recognized by chagasic sera. Those sera recognized an epitope spanning amino acids 120 to 129 of Cha, named R3. In the present study we have used the synthetic R3 peptide for the detection of serum immunoglobulin G antibodies from patients at different stages of Chagas' disease, including a therapeutically treated group. The immunoreactivity with R3 by enzyme-linked immunosorbent assay (ELISA) showed 92.4% sensitivity and 100% specificity for Chagas' disease sera. This sensitivity and specificity were higher than for any other autoantigen described to date. No anti-R3 antibodies were detected in sera from Leishmania-infected or idiopathic dilated cardiomyopathy patients or healthy controls from the same areas. Moreover, anti-R3 antibody reactivity detected by ELISA correlated with conventional serological tests as indirect immunofluorescence and ELISA assays with Trypanosoma cruzi extracts and other diagnostic tests as indirect hemagglutination. The levels of anti-R3 antibodies increased with progression and symptomatology of Chagas' disease. More interestingly, a statistically significant fall in anti-R3 antibody titer was observed in patients treated with antiparasitic drugs. Those results suggest that the presence of anti-R3 antibodies is a highly specific marker of Chagas' disease and that R3 ELISA could be helpful in the diagnosis and monitoring of this disease.

Dominant T- and B-cell epitopes in an autoantigen linked to Chagas' disease.

In Chagas' disease caused by Trypanosoma cruzi, a paradigm of autoimmune disease, both autoantibodies and autoreactive T cells have been described. We have identified a novel dominant autoantigen, named Cha, recognized by the majority of sera from T. cruzi-infected humans and mice. We noted significant homologies between amino acids 120-129 of Cha, where the B-cell epitope maps, and an expressed sequence tag from T. cruzi, and also between amino acids 254-273 of Cha and a repeated amino acid sequence from the shed acute-phase antigen (SAPA) of T. cruzi. Moreover, T. cruzi-infected mice contain autoreactive T cells that can cross-react with Cha and the SAPA homologous peptides. Transfer of T cells from infected mice triggered anti-Cha (120-129) Ab production in naive recipients. Interestingly, heart tissue sections from those adoptive transferred mice showed cardiac pathology similar to T. cruzi-infected mice. Our results demonstrate the presence of both T- and B-cell cross-reactive epitopes in the Cha antigen. This dual mimicry may lead to T/B cell cooperation and give rise to a pathological immunodominant response against Cha in T. cruzi infected animals.

Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring.

Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

GP 50/55, a membrane antigen of Trypanosoma cruzi involved in autoimmunity and immunosuppression.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and the induction of autoantibodies crossreacting with host cells and tissues. We described here a GPI-linked 50/55 kDa antigen (GP50/55) present on the T. cruzi membrane, but not in the membrane of other parasites of the family Trypanosomatidae. We have obtained several monoclonal antibodies which specifically recognize this molecule. One of these GP50/55-specific mAbs (C10) crossreacts with a 28 kDa antigen expressed on the membrane of activated mouse and human T and B lymphocytes, after "in vitro" activation with mitogens, phorbol esters, or antigen, and on several murine T and B lymphocyte cell lines. Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from T. cruzi-infected mice or Chagasic patients but not from uninfected mice or control patients contain antibodies which recognize a similar p28 antigen and also suppress the proliferation of human T lymphocytes. These results suggest a possible role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis.

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.

Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid.

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.

A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis.

The rate of receptor-mediated endocytosis of diferric 125I-transferrin by Chinese-hamster ovary cells expressing human transferrin receptors was compared with the rate measured for cells expressing hamster transferrin receptors. It was observed that the rate of endocytosis of the human transferrin receptor was significantly higher than that for the hamster receptor. In order to examine the molecular basis for the difference between the observed rates of endocytosis, a cDNA clone corresponding to the cytoplasmic domain of the hamster receptor was isolated. The predicted primary sequence of the cytoplasmic domain of the hamster transferrin receptor is identical with that of the human receptor, except at position 20, where a tyrosine residue in the human sequence is replaced with a cysteine residue. To test the hypothesis that this structural change in the receptor is related to the difference in the rate of internalization, we used site-directed mutagenesis to examine the effect of the replacement of tyrosine-20 with a cysteine residue in the human transferrin receptor. It was observed that the substitution of tyrosine-20 with cysteine caused a 60% inhibition of the rate of iron accumulation by cells incubated with [59Fe]diferric transferrin. No significant difference between the rate of internalization of the mutant (cysteine-20) human receptor and the hamster receptor was observed. Thus the substitution of tyrosine-20 with a cysteine residue can account for the difference between the rate of endocytosis of the human and hamster transferrin receptors.

Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation.

The major sites of serine and threonine phosphorylation of the human epidermal growth factor (EGF) receptor observed in intact cells are Thr654, Thr669, Ser1046, and Ser1047. Phosphorylation of the EGF receptor is increased at these sites in cells treated with platelet-derived growth factor or phorbol ester. This increase in EGF receptor phosphorylation is associated with an inhibition of the high affinity binding of EGF to cell surface receptors and an inhibition of the receptor tyrosine protein kinase activity. In order to test the hypothesis that the phosphorylation of the EGF receptor is mechanistically related to the modulation of EGF receptor function, we replaced the major sites of serine and threonine phosphorylation with alanine residues. EGF receptors containing single point mutations or multiple mutations were expressed in Chinese hamster ovary cells. Analysis of the regulation of the EGF receptor tyrosine protein kinase activity demonstrated that phorbol ester caused an inhibition of the tyrosine phosphorylation of wild-type receptors and receptors lacking Thr669, Ser1046, or Ser1047. In contrast, the inhibition of EGF receptor tyrosine phosphorylation caused by phorbol ester was not observed for any of the mutated EGF receptors that lacked Thr654. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at Thr654 is required for the inhibition of the receptor tyrosine protein kinase activity caused by phorbol ester. Investigation of the apparent affinity of the EGF receptor demonstrated that treatment with phorbol ester caused an inhibition of the high affinity binding of 125I-EGF to cells expressing wild-type EGF receptors and each of the mutated EGF receptors examined. We conclude that the regulation of the apparent affinity of the EGF receptor is independent of the major sites of serine and threonine phosphorylation of the EGF receptor.

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.

Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells.

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.

Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor.

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells.

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.

Two alternative mechanisms control the interconversion of functional states of the epidermal growth factor receptor.

Epidermal growth factor (EGF) and transforming growth factor alpha bind to a common receptor at the cell surface. Both the affinity and the tyrosine protein kinase activity of the receptor are regulated by exogenous factors, such as platelet-derived growth factor. A protein kinase C-dependent (Ca2+/phospholipid-dependent enzyme) and independent regulatory mechanism have been described. The protein kinase C-dependent mechanism results in the inhibition of the affinity and tyrosine kinase activity of the EGF receptor. We describe in this report an alternative mechanism of regulation of the receptor that is mediated by sphingosine. Treatment of WI-38 human fetal lung fibroblasts with 5 microM sphingosine for 2 min at 37 degrees C caused a marked increase in the affinity of the EGF receptor. Similar results were obtained when isolated plasma membranes prepared from these cells were incubated with sphingosine. A stimulation of the EGF receptor tyrosine protein kinase activity was also observed after sphingosine-treatment of plasma membranes. Sphingosine caused a decrease in the Km for ATP and an increase in the Vmax for the tyrosine phosphorylation of a synthetic peptide substrate. Control experiments demonstrated that these actions of sphingosine were not secondary to the inhibition of protein kinase C. These data indicate that sphingosine causes the functional conversion of the EGF receptor into an activated state that expresses both a high affinity for EGF and an increased tyrosine kinase activity. We conclude that sphingosine is a bioactive molecule in human fibroblasts.