Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.     

Sequence studies of peanut agglutinin

Sequence studies have been performed on affinity purified peanut agglutinin, a galactose binding lectin. 161 residues have been compared to homologous residues in soybean agglutinin and favin. Extensive similarities have been uncovered, confirming the conservation of lectin sequences among all legume lectins. Evidence is presented for the existence of internal duplications and/or isolectins.     

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

Abstract To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli . Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli , respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.     

The macromolecular properties of peanut agglutinin

The dependence upon solution conditions of the quaternary structure and gross conformation of peanut agglutinin was examined by sedimentation equilibrium, sedimentation velocity, gel chromatography, and circular dichroism. At pH 8, the protein exists as a compactly folded tetramer of molecular weight 98,000. Between pH 4.75 and pH 3.0, the molecular reversibly dissociates to a (still globular) dimer. In the presence of denaturants such as SDS or guanidinium chloride, the protein dissociates to its four equal-sized constituents polypeptide chains. The circular dichroic spectrum of peanut lectin exhibits changes in the near ultraviolet upon binding of lactose, whereas the far ultraviolet spectrum remains unchanged. Dissociation to the dimeric state produces subtle changes in both the near and far ultraviolet circular dichroic spectrum.     

In vitro effect of thymosin alpha 1 on the expression of peanut agglutinin binding by murine thymocytes

Thymosin alpha 1 induces the loss of PNA binding ability by subpopulation of thymic cells. This loss is probably due to an endocytic process. Nevertheless this disappearance is not a permanent one, suggesting a recycling of the PNA binding molecule. The cells that modulate their PNA binding sites after exposure to Thymosin alpha 1 are a small proportion of the total PNA+ thymocytes, indicating that not all thymocytes are susceptible to the thymic hormone Thymosin alpha 1. Conversely the exposure of thymocytes to Thymosin alpha 1 induces the disappearance of the binding sites for this ligand without further recycling, behavior expected for the receptor of a regulatory ligand. These results also indicate that the Thymosin alpha 1 and the PNA binding sites are on different molecules on the surface of the PNA+ thymocytes.     

Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick

 Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons. Accepted: 23 February 1998     

Influence of sulphate groups in the binding of peanut agglutinin. Histochemical demonstration with ligh- and electron-microscopy

Summary The influence of sulphation of mucus glycoproteins in the binding of peanut agglutinin (PNA) to tissue sections has been investigated by means of histochemical techniques at the light- and electron-microscopic level. A sequential methylation-saponification procedure was applied for the desulphation of tissue samples. Labelling by peroxidase- and colloidal gold-conjugated PNA was compared in control and desulphated samples of rat intestinal mucosa. The high-iron-diamine (HID) technique was used as a control for the effectiveness of the desulphation technique, and the Alcian Blue, pH 2.5 (AB 2.5), PAS and phosphotungstic acid-HCl (acid-PTA) techniques served as controls for the integrity of the oligosaccharide chains, respectively. In general, a marked increase of PNA reactivity was observed in desulphated samples when compared with control sections. These findings indicate that sulphation of galactose inhibits the binding of PNA to carbohydrate moieties in tissue sections. Staining patterns obtained with HID, PNA and the desulphation-PNA sequence in the goblet cells of the large intestine suggest a modification of the secretory product stored in these cells as the cell matures and moves from the lower crypt region toward the luminal surface. These modifications were not detected in the small intestine. Ultrastructural detection of PNA-binding sites suggests that galactose residues are incorporated into the oligosaccharide chains of O-liked glycoproteins at the medial cisternae of the Golgi apparatus. However, sulphation occurs at the trans side of the Golgi complex and the trans Golgi network. In conclusion, desulphation procedures are useful for revealing PNA-binding sites.     

The amino acid sequence of peanut agglutinin

The amino acid sequence of peanut (Arachis hypogaea) agglutinin was determined from three major fragments obtained by mild acid cleavage at Asp-Pro peptide bonds. The sequence of 236 amino acids has residues identical to those that form the metal-binding site and the hydrophobic pocket in concanavalin A and other lectins, although the overall similarity is only 42%. In the segments of peanut agglutinin that correspond to the four loops that form the carbohydrate-binding site in concanavalin A and favin, several central residues are homologous, while others show changes to smaller side chains, such as Tyr----Gly. The carbohydrate-binding site of peanut agglutinin may therefore have a similar peptide-backbone architecture, but form a considerably more open cleft.     

Binding of 4-methylumbelliferyl-galactosides to peanut agglutinin

The binding of 4-methylumbelliferyl-α-D-galactopyranoside, -β-D-galactopyranoside and -D-Galβ(1→3)DGalNac to peanut agglutinin was studied by fluorescence. Peanut agglutinin quenched the fluorescence intensity of 4-methylumbelliferyl-α-D-galactopyranoside but enhanced that of the two 4-methylumbelliferyl-β-galactosides. For α-D-galactopyranoside, the association constants measured at 4 and 25°C were 3.4 × 10³ and 1.7 × 10³ M^?1 respectively, and for D-Galβ(1→3)DGalNac, 1.5 × 10⁵ and 3.3 × 10⁴ M^?1. The binding enthalpies estimated from these values are consistent with the existence of extended sugar binding sites in the peanut agglutinin molecule.     

Effects of pH on K depolarization and contracture in frog muscles.

Membrane potential changes and mechanical tension provoked by 20 or 80 mM K at pH 8.0 or 4.5 were measured in muscle fiber bundles from Rana temporaria and Rana esculenta. The bathing solution contained Cl as the main anion. Low pH increased the depolarizing efficiency of high K to about the same degree in muscles of both species of frogs. The tension-[K] relationship was shifted toward lower [K] in muscles from R. temporaria and toward higher [K] in muscles from R. esculenta. It was concluded that the depolarization-contraction coupling was insensitive to pH changes in the former but not in the latter frog species.     

Identification of peanut agglutinin receptors on mouse testicular germ cells

Peanut agglutinin receptors expressed specifically in mouse testicular germ cells have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lectin blotting techniques. Two major components were estimated to have molecular weights of 86,000 and isoelectric points of 6.1 +/- 0.3 and 6.2 +/- 0.3. Minor components with molecular weights of 71,000-74,000 and isoelectric points of 6.1 +/- 0.3 were also detected. Specific expression of these receptors on testicular germ cells was confirmed using the testes of mutant mice, Sld/Sld, devoid of germ cells.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.     

Effects of caffeine on the intracellular distribution of calcium in frog sartorius muscle

The influence of caffeine on the intracellular distribution of calcium in the frog sartorius muscle was studied by differential centrifugation in an attempt to identify the locus of action of this alkaloid. The problem was approached in two ways. In the first, the locus of action was sought by relating the kinetic functions of ⁴⁵Ca washout curves of muscles to changes in the distribution of ⁴⁵Ca in the isolated fractions from the same muscles. It was not possible to make any correlation of the ⁴⁵Ca-washout curves to the activity in the fractions; the relative distribution of this nuclide remained essentially unchanged at 1-, 2-, and 3-hour intervals along the curve. The washout curves appear to be the net effect of a complex interaction of the calcium in pools containing both readily exchangeable calcium and calcium which has a slow exchange or turnover rate. The second approach centered upon the examination of the effect of caffeine on the intracellular distribution of ⁴⁵Ca and of calcium among the cellular fractions. Caffeine treatment resulted in a distinct increase in the calcium content of the mitochondrial fraction and a decrease in the calcium of the microsomal fraction. Electron micrographic studies revealed significant morphological changes in the whole muscle and in the isolated mitochondrial fraction after the muscle had been exposed to caffeine in a concentration producing irreversible contracture or rigor (10 mM). The increase in calcium content of the mitochondrial fraction after caffeine treatment may be due to an actual accumulation of calcium by the mitochondria or may be the consequence of the appearance of granular vesicles in the fraction.     

Effect of cytostatics on the development of denervation disorders in skeletal muscles

Experiments on rats were made to study the effect of cytostatics on the rest membrane potentials (RMP) of muscle fibres and chemosensitivity of the botulinum toxin (BT) poisoned m. soleus. Intramuscular injection of the sublethal dose of BT on the 5th day evoked the blockade of the synaptic neuromuscular transmission, depolarization of the muscle cells and the decreased sensitivity to acetylcholine. Daily intraperitoneal injections of vincristine (25 micrograms/100 g) and fluorouracil (5 mg/100 g) to rats did not affect the development of the neuromuscular transmission blockade induced by BT. The cytostatics did not change the RMP of the myocytes or chemosensitivity of the normal muscles. However, both the drugs prevented the depolarization of myocytes and the decreased chemosensitivity of the muscles paralyzed with BT. It is assumed that the delayed appearance of the cytostatic-induced denervation is a consequence of the suppressed division of the satellite cells.     

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

Mutational analysis at Asn-41 in peanut agglutinin. A residue critical for the binding of the tumor-associated Thomsen-Friedenreich antigen.

Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor-associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the water-mediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. Of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity.