Trichosanthin interacts with and enters cells via LDL receptor family members

The type-I ribosome-inactivating protein trichosanthin displays selective cytotoxicity, suggesting specific mechanisms for entry into cells. Here we show that trichosanthin binds specifically to the endocytic receptors LRP and megalin, and that binding as well as uptake into cells is inhibited by the receptor-associated protein (RAP). The results suggest that the known abortifacient and renotoxic actions of trichosanthin are caused by LRP-mediated uptake in trophoblasts and megalin-mediated uptake in proximal tubule epithelial cells, respectively.     

Lectin-like molecules on the murine macrophage cell surface

Lectin-like molecules on the surface of murine peritoneal exudate macrophages induced by thioglycolate or an anti-tumor streptococcal preparation, OK-432, were investigated and isolated. Furthermore, their sugar-binding specificities and their role in macrophage-mediated tumor cytotoxicity were examined. A neoglycoprotein, D-galactose (Gal)-bovine serum albumin, bound to these murine peritoneal macrophages. This binding of Gal-bovine serum albumin was inhibited by D-galactose, and by complex-type oligosaccharides (unit B) and high mannose-type oligosaccharides (unit A) prepared from porcine thyroglobulin. When thioglycolate-elicited macrophages were activated by lipopolysaccharide and/or the culture supernatant of concanavalin A-activated mouse spleen cells, they became tumoricidal and the number of the lectin-like molecules on the macrophage surface was found to increase. Since the binding and cytotoxic activities of these tumoricidal macrophages toward tumor cells were partially inhibited by D-galactose, the D-galactose-binding lectin-like molecules on the surface of tumoricidal macrophages might play an important role in macrophage-mediated cytotoxicity. These lectin-like molecules were then isolated from solubilized murine peritoneal exudate cells labeled with pyridoxal 5'-phosphate and sodium [3H]borohydride by affinity chromatography on columns of asialo unit B oligosaccharide-Sepharose 4B and/or beta-D-galactose-Bio-Gel P-100. The proteins bound to the asialo unit B oligosaccharide-Sepharose 4B column and eluted specifically were found to have approximate molecular weights of 79 000 and 18 000, and the protein bound to and eluted from the beta-D-galactose-Bio-Gel P-100 column had an approximate molecular weight of 77 000. These isolated proteins bound to the surface of glutaraldehyde-fixed tumor cells, and their binding was inhibited by D-galactose and also by D-mannose. Since most of the 77 kDa protein bound to the asialo unit B oligosaccharide-Sepharose 4B, this protein was assumed to be identical with the 79 kDa protein. These results suggest that the lectin-like molecules on murine macrophages have wide specificity and that one lectin-like molecule can bind both D-galactose and D-mannose.     

Molecular basis of host epithelial cell recognition by Trichomonas vaginalis

Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65,000 daltons (65 kDa; AP65), 51 kDa (AP51), 33 kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long-term-grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti-adhesin antibodies reacted by immunoblot only with the respective protein and detected, by indirect immunofluorescence, each adhesion on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact-dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesions also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.     

Heterogeneity of the ribosome-inactivating protein trichosanthin in Trichosanthes kirilowii tubers

The ribosome-inactivating protein trichosanthin isolated from the tubers of Trichosanthes kirilowii, the Chinese drug Tianhuafen, has a molecular mass of approximately 26 kDa. We show here that T. kirilowii tubers also contain ribosome-inactivating proteins with a small extent of structural variation from and a larger molecular mass than trichosanthin.     

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.     

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP-ribose) synthetase. It was classified as IgG1/kappa and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA-binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross-reactivity was examined using DNA-binding proteins from 6 different sources, 1F4 reacted with the 120- and 32-kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures of poly(ADP-ribose) synthetase and its degradation products are highly conserved in various animal cells.     

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.     

Identification and characterization of nuclear location signal-binding proteins in nuclear envelopes

A radioiodinated, photoactivable synthetic nonapeptide corresponding to the nuclear location signal (NLS) of SV40 large T antigen has been used in photolabelling reactions with purified mouse liver nuclei, nuclear envelopes and other cellular fractions, to identify specific NLS-binding proteins which may be involved in selective transport of karyophilic proteins. SDS-polyacrylamide gel analysis of photolabelled products demonstrates that a 60 kDa nuclear protein and four nuclear envelope proteins (67, 60, 53 and 47 kDa) bind specifically to the native NLS and not to a mutant NLS or unrelated sequences. This binding shows saturation kinetics, with highest affinity of the NLS for the 60 and 67 kDa proteins. The nuclear 60 kDa NLS-binding protein is identical to the nuclear envelope 60 kDa NLS-binding protein by two-dimensional gel analysis of labelled proteins. Biochemical fractionation of labelled nuclear envelopes suggests that the 53 and 47 kDa proteins are peripheral membrane proteins whereas the 67 and 60 kDa proteins can be localized to the pore complex. The NLS also binds to solubilized 67, 60, 53 and 47 kDa proteins but with decreased affinity. Our results suggest that one of the early steps in selective nuclear transport of proteins may be the recognition of the NLS by the 60 kDa and/or 67 kDa binding proteins present in the nuclear pore complex.     

In vitro cytotoxicity of non-cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells

A soil isolate designated 90-F-45-14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells. When activated with proteolytic processing, inclusion proteins of the isolate 90-F-45-14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC(50) value of 60.8 microg ml(-1), while showing extremely high activities on the human leukaemic T cells (MOLT-4) and the normal T cells with EC(50) values of 0.27 and 0.20 microg ml(-1), respectively. Anti-leukaemic cell activity of the 90-F-45-14 proteins was eight to nine times greater than that of the B. thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad-spectrum cytolysin. The cytopathy by the 90-F-45-14 proteins was characterized by marked cell-ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis. Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa. A 100% homology was observed in the sequence of 15 N-terminal amino acids between the proteins of 170 and 103 kDa. There was no N-terminal sequence homology between 90-F-45-14 proteins and the existing Cry/Cyt proteins of B. thuringiensis. Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.     

登革2型病毒结合血管内皮细胞分子机制的初步研究

以ECV304细胞为对象分析登革病毒感染血管内皮细胞的机制。2型登革病毒(DEN2)吸附后微量蚀斑法测定ECV304细胞上清释放的病毒滴度,证实该细胞对DEN2感染有一定的敏感性。机械刮取或胰蛋白酶消化法收集ECV304细胞分离膜蛋白,SDS—PAGE见胰酶处理样品缺失一43 kDa的膜蛋白。将ECV304细胞膜蛋白与^35S—Met标记的DEN2进行病毒重叠蛋白结合试验(VOPBA),有29、34和43kDa的3种膜蛋白可与DV结合,其中29 kDa的蛋白对胰酶耐受。培养的ECV304细胞中加入重组E蛋白(rEgp)对DEN2吸附进行阻断试验,微量蚀斑法与间接免疫荧光表明rEgp抑制DEN2感染该细胞。VOPBA中rEgp可阻断病毒与细胞膜蛋白的结合。结果表明ECV304细胞表面可能存在29、34、43 kDa的3种与DEN2结合的相关蛋白,DEN2E蛋白可直接介导DV感染血管内皮细胞。     

Identification of phosphatidylserine-binding proteins in human white blood cells.

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.     

A Novel and Neurotoxic Tetrahydroisoquinoline Derivative In Vivo: Formation of 1,3-Dimethyl-1,2,3,4-Tetrahydroisoquinoline, a Condensation Product of Amphetamines, in Brains of Rats Under Chronic Ethanol Treatment

Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm-derived cells that store 5-hydroxytryptamine (5-HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5-HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56-kDa form of SBP may be the precursor of the 45-kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine (NAP-5-HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV-visible absorption spectra. A 1 M excess of NAP-5-HT inhibited the binding of [3H]5-HT to SBP by 50%. NAP[3H]5-HT was also synthesized and attached to both high- and low-affinity binding sites on both forms of SBP. The high-affinity constants for 45-kDa and 56-kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low-affinity constants were 0.3 microM and 0.15 microM. When the high-affinity site of partially purified SBP was photoaffinity-labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15-fold molar excess of 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosolic copper-binding proteins in rat and mouse hepatocytes incubated continuously with Cu(II).

The proteins that bind copper when it first enters cells are likely to play roles in its intracellular distribution and utilization. When hepatocytes were incubated with 64Cu(II), the time-dependence of the subcellular distribution of 64Cu was consistent with one or more cytosolic proteins distributing copper to the mitochondrial and nuclear fractions. Cytosolic copper was reproducibly distributed among four protein fractions from Sephadex G-150 columns at the earliest time (1 min) and at the lowest concentration used [2 microM-64Cu(II)] with both rat and mouse hepatocytes. Copper binding to proteins in these functions was sensitive to copper metabolic status. Hepatocytes from nutritionally copper-deficient rats or neonatal (9-30 days old) developing rats showed an inverse correlation between copper binding to metallothionein and copper binding to proteins in fraction I (approximately 88 kDa apparent) and fraction II (approximately 38 kDa apparent). The distribution of cytosolic 64Cu from the brindled-mouse model of Menkes disease indicated decreased binding by a protein in fraction I. Brindled-mouse hepatocytes also contain decreased levels of a approximately 55 kDa protein or subunit, which most likely represents a liver-specific secondary response to the primary defect. The results are consistent with one or more copper-binding proteins in fractions I and II having significant functions in intracellular copper metabolism.     

Ribosomal protein L10a, a bridge between trichosanthin and the ribosome

Trichosanthin is a type I ribosome-inactivating protein with many pharmacological activities. The trichosanthin-coupled Sepharose affinity purification revealed a protein, which was identified by mass spectrometry as the ribosomal protein L10a. The interaction between trichosanthin and recombinant L10a was further confirmed by in vitro binding assay. Kinetic analysis by surface plasmon resonance technology revealed that L10a had a high affinity to trichosanthin with a K(D) of 7.78nM. The study with mutated forms of trichosanthin demonstrated that this specific association correlates with the ribosome-inactivating activity of trichosanthin. This finding might provide insight into the mechanisms by which trichosanthin inactivates ribosome and that underlies its pharmacological effect.     

A 70 kDa microtubule-associated protein in NIL8 cells comigrates with the 70 kDa heat shock protein

When eukaryotic cells are exposed to environmental stress such as elevated temperature, the synthesis of heat shock proteins (HSP) is stimulated. We have raised a monoclonal antibody to a 70 kDa cytoskeleton-associated protein; this antibody also appears to recognize HSPs 68, 70 and 90, as well as an additional 40 kDa non-heat shock protein. We have used this monoclonal antibody to study the localization of the 70 kDa protein in the cytoskeletons of NIL8 hamster fibroblasts. By selective sequential solubilization of the components of NIL8 cells and analysis of the resulting cytoskeletal preparations by Western blot technique and indirect immunofluorescence, we have shown that the 70 kDa protein is associated with microtubules in mitotic and interphase cells and comigrates with HSP70 on 2-dimensional gel electrophoretigrams.     

The generation of active fragments of complement receptor type 2 by trypsin digestion

B lymphocytes and Raji cells express the complement receptor type 2 (CR2) of 145 kDa which recognises the C3d fragment of C3. When intact cells are treated with trypsin, CR2 is degraded. There is a parallel loss in C3d-mediated rosetting and in proteins which bind to C3d-Sepharose. Initially 97 and then 83 kDa fragments of CR2 are produced which retain C3d binding activity. These fragments are associated with the cell surface and mediate rosetting. Purified 125I-labelled CR2, solubilised in detergent, produces fragments of apparently identical size on treatment with trypsin. The 83 kDa fragment produced by trypsin treatment closely resembles the major C3d binding protein spontaneously released into Raji cell culture medium.     

Novel non-labile covalent binding of sulfamethoxazole reactive metabolites to cultured human lymphoid cells

Sulfamethoxazole (SMX) causes rare hypersensitivity syndrome reactions characterized by fever and multi-organ toxicity. Covalent binding of SMX reactive metabolites to cellular proteins has been demonstrated but the link between cytotoxicity and targets of covalent binding has not been explored. We therefore investigated the relationship between covalent binding of the reactive SMX-hydroxylamine (SMX-HA) metabolite, and its cytotoxicity to a hystiocytic lymphoma (U937) cell line. Incubation of U937 cells with 0-1 mM SMX-HA for 3 h resulted in dose-dependent cytotoxicity, as assessed by tetrazolium dye conversion at 24 h. SMX-HA caused dose-dependent covalent binding to cellular proteins as assessed by immunoblotting with SMX antisera at 3 and 24 h. Covalent binding was predominantly to proteins of approximately 45, 59 and 75 kDa, but other targets were also observed. The relative extent of binding to proteins was significantly different from the relative cytotoxicity at 24 h. Further, cells surviving at 24 h also had extensive covalent binding. Covalent binding was observed under reducing (beta-mercaptoethanol) and non-reducing conditions to plasma membrane and microsomal but not cytosolic proteins. This non-labile covalent binding has not been previously reported. These observations suggest that extensive covalent binding does not necessarily lead to cell death, allowing the accumulation of potentially immunogenic drug-protein conjugates. These observations in whole cells may be relevant to the immunopathogenesis of SMX hypersensitivity syndrome reactions.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.