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The heterodimeric vitronectin receptor (VNR) and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) are two members of the integrin family of cell adhesion receptors that share the same beta subunit (GPIIIa). These proteins are involved in binding to vitronectin, fibrinogen and fibronectin and in cytoskeleton-membrane interactions. The present study shows that the human placental syncytiotrophoblast brush border membrane contains a heterodimer of subunit Mr values of 140,000 and 90,000 (non-reduced) or 125,000 and 100,000 (reduced). This protein was recognized by a monoclonal antibody to GPIIIa, rabbit antisera to the VNR and a human alloantiserum to GPIIIa. Brush border VNR-related protein bound to an immobilized peptide containing the Arg-Gly-Asp sequence and, less avidly, to immobilized fibrinogen. Only a small fraction of brush border VNR was associated with a cytoskeleton fraction. Membrane-bound brush border GPIIIa was distinct from that of platelets in its resistance to digestion by trypsin and Staphylococcus aureus V8 protease, and had a slightly lower mobility on SDS/PAGE. In addition, lectin-binding studies indicate glycosylation differences between microvillar and platelet GPIIIa heterodimers. Thus, although placental syncytiotrophoblast expresses a beta 3 integrin in its apical brush border, differences in protease sensitivity and carbohydrate content suggest that it may lack or mask certain antigenic determinants. This may be beneficial in avoiding harmful maternal alloantibody responses during pregnancy. Immunohistology showed that the VNR was present in syncytiotrophoblast apical but not basal plasma membranes, and was absent from other forms of trophoblast. The brush border VNR could function in localizing Arg-Gly-Asp-sequence-containing plasma proteins to the materno-trophoblastic interface.
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PMID:A vitronectin-receptor-related molecule in human placental brush border membranes. 172 Jun 17

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.
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PMID:Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides. 172 59

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast-directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane-associated glycoprotein. Western blot analysis on disulfide bond-reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS-polyacrylamide gels. Silver staining of the purified antigen on SDS-polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N-linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O-linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N-terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type-specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast-mediated bone resorption.
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PMID:Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase. 172 67

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L-fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did.
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PMID:The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. 172 47

This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.
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PMID:Studies on the variants of the protein toxins ricin and abrin. 174 Jan 26

A new protein, called alboaggregin-B (AL-B), has been isolated from Trimeresurus albolabris venom by ion-exchange chromatography. It agglutinated platelets without the need for Ca2+ or any other cofactor. The purified protein showed an apparent molecular mass on SDS-PAGE and gel filtration of about 23 kDa under nonreducing conditions. Ristocetin did not alter the binding of AL-B to platelets or affect AL-B-induced platelet agglutination. Agglutinating activity was not dependent on either proteolytic or lectin-like activity in AL-B. Binding analysis showed that AL-B bound to platelets with high affinity (Kd = 13.6 +/- 9.3 nM) at approximately 30,800 +/- 14,300 binding sites per platelet. AL-B inhibited the binding of labeled bovine von Willebrand factor (vWF) to platelets. Monoclonal antibodies against the 45-kDa N-terminal domain of platelet glycoprotein Ib inhibited the binding both of AL-B and of bovine vWF to platelets, and also inhibited platelet agglutination induced by AL-B and bovine vWF. Specific removal of the N-terminal domain of GPIb by treatment of the platelets with elastase or Serratia marcescens protease reduced the binding of labeled AL-B and bovine vWF to platelets and blocked platelet agglutination caused by both agonists. Monoclonal antibodies to glycoprotein IIb/IIIa, to bovine vWF, and to bovine serum albumin did not show any effect on the binding of AL-B to platelets. Our results indicate that the binding domain for AL-B on platelet GPIb is close to or identical with the one for vWF. This new protein may be a very useful tool for studying the interaction between platelets and vWF.
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PMID:Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib. 174 71

The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.
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PMID:Identification of lectin binding proteins in human tears. 174 57

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.
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PMID:Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens. 175 26

This morphological and biochemical study examines the cytoplasmic synaptic determinant recognized by a monoclonal antibody (B16). This antibody was generated by using an immunosuppression protocol that generates antibodies to relatively rare antigens. The B16 antibody labels structures in the brain that are dot-shaped and in the retina that resemble synaptic ribbons in their location, size, developmental emergence, and biochemical composition. The antigen is apparently conserved across species as it is found in retinas from lizards, frogs, fish, birds, mice, rats, rabbits, cats, and monkeys. This paper focuses on observations in the murine retina. Labeling in the outer plexiform layer of the retina is confined to the margin between the outer plexiform layer (OPL) and the outer nuclear layer. The labeled structure resembles a semiellipse or an arc with the open end facing the OPL and the top facing the outer nuclear layer. Overall, the arc is approximately 1 micron in length and less than 0.5 micron thick. Approximately 10% of the labeled arcs occur in a proximal stratum of the OPL and form a planar cluster that resembles a flat plaque parallel to the OPL. Five to ten arcs are found in each plaque. The arcs found within the plaques are approximately 50% smaller than the larger isolated arcs. Counterstaining with peanut agglutinin (PNA), a lectin that recognizes cone photoreceptors and their associated processes, demonstrates that the plaques are associated with the cone pedicles. Animals that have a higher ratio of cones/rods than mice demonstrate a much higher ratio of plaques/isolated arcs in the OPL. The structure labeled in the inner plexiform layer resembles a short bar (0.8 micron long by less than 0.5 micron wide) that is confined to the inner half of the inner plexiform layer in mice. The relative mobility (Mr) of the B16 antigen obtained from mouse retinal and brain tissue is 88 kD, as determined by SDS-PAGE followed by Western blotting. The mouse 88 kD protein is relatively soluble (precipitates at 70% ammonium sulphate) and elutes at a pH of 7.3 from an isoelectric focusing column. It appears that the determinant recognized by the B16 antibody is a previously undescribed synaptic protein that is associated with the synaptic ribbons in photoreceptor and bipolar terminals of most vertebrate retinas.
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PMID:A synaptic antigen (B16) is localized in retinal synaptic ribbons. 176 43

Two lectin fractions (FI and FII) were obtained from seeds of Butea frondosa by affinity chromatography on a sorbent of macroporous glass coupled to the disaccharide alpha-D-GalNAc-(1----3)-beta-D-Gal. Both of these fractions, although different in their sugar specificity, were found on SDS-PAGE to consist of two polypeptide chains of 33 kDa and 35 kDa. In the native state the subunits associated to form a 250 kDa complex, possibly comprising four molecules of the 33 kDa polypeptide and four molecules of the 35 kDa polypeptide. The presence of a faint 70 kDa band when the 250 kDa complex was subjected to SDS-PAGE may indicate the existence of a sequential mechanism of aggregation. N-terminal amino acid sequence analysis revealed extensive homology between these lectins and those of other Leguminosae.
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PMID:Studies on the N-terminal sequences of lectins isolated from the seeds of Butea frondosa. 184 34


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