Genetic variation as a tool for histochemical localization of a nonspecific esterase

Summary A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Genetic variation as a tool for histochemical localization of a nonspecific esterase

A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.     

The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin

The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle.     

Human galectin-2: expression profiling by RT-PCR/immunohistochemistry and its introduction as a histochemical tool for ligand localization

Sugar-encoded information of glyco-conjugates is translated into cellular responses by endogenous lectins. Galectins stand out against other lectin families due to their wide range of functions including cell adhesion, tissue invasion or growth regulation exerted at extracellular, membrane, cytoplasmic and nuclear sites. This remarkable versatility warrants close scrutiny of their emerging network, in this study with focus on homodimeric human galectin-2. We first detected presence of specific mRNA in various tissue types by processing post mortem and surgical specimens by RT-PCR protocols. Overlap of gene expression was noted with proto-type galectins-1 and -7 and also family members from the other two subgroups. To monitor expression on the level of protein a polyclonal anti-galectin-2 antibody was raised. Immunopositivity was semi-quantitatively assessed in sections of 209 human samples establishing an array both of normal tissues and samples with inflammation or benign/malignant growth. In general, positivity was predominantly epithelial without restriction of staining to certain tissue types, as fittingly indicated by our RT-PCR analysis. Staining was not limited to the cytoplasm but also included nuclear sites. To examine the suitability of the labeled lectin as a histochemical probe we biotinylated galectin-2 under activity-preserving conditions and introduced it to tissue profiling. Specific cytoplasmic staining proved the validity of the concept. Our results encourage systematic histopathologic studies by immuno- and lectin histochemistry, especially by adding galectin-2 as study object to galectin fingerprinting which has already yielded prognostic information on galectins-1, -3, -4 and -8 and hereby contributed to define functional overlap/divergence in this lectin family.     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.     

Heparin-binding lectin from human placenta: further characterization of ligand binding and structural properties and its relationship to histones and heparin-binding growth factors

We have previously demonstrated that the heparin-binding lectin of human placenta dissociates into up to four distinct polypeptides with molecular weights of 14,400, 15,000, 16,200, and 16,700 (Kohnke-Godt, B., & Gabius, H.-J. (1989) Biochemistry 28, 6531-6538). Stable complexes to ligands can shift the molecular weight appearance of the lectin to higher values. They can be dissociated in the additional presence of 9 M urea or by enzymatic degradation of heparin in model studies. The binding of heparin is rather stable over a range of salt concentrations from 1 to 3 M NaCl. Chemical modification with group-specific reagents to arginine, lysine, histidine, tyrosine, and tryptophan results in substantial inactivation of binding activity. Further amino-terminal sequence analyses point to a high-scoring relationship in this region to histone sequences, namely, histone H2B, but to no published sequences for any heparin-binding growth factor. Calculation of relatedness on the basis of differences in amino acid composition corroborates the conclusion of molecular distinction between the lectin, histones H2A and H2B, and the fibroblast growth factor as well as angiogenin. Histones only weakly agglutinate type II erythrocytes in contrast to the lectin. The immobilized lectin exhibits two classes of binding sites with KD values of 3 and 110 nM in contrast to one estimated KD value of 250 nM with a commercially available histone fraction. Both fractions retain binding activity to biotinylated heparin in transblots and are immunologically cross-reactive to antibodies, raised against the lectin as antigen. Subcellular fractionation clearly demonstrates that heparin-inhibitable hemagglutination activity and immunologically cross-reactive protein bands, characteristic for the lectin, but not unequivocally distinguishable from certain histone fractions in blots, are not confined to the nuclear fraction in the human placenta.     

Alkanethiol containing glycopolymers: a tool for the detection of lectin binding

Glycopolymers are useful macromolecules with a non-carbohydrate backbone for presenting saccharides in multivalent form. Here, glycopolymers containing mannose and alkanethiol linker were synthesized through substituting preactivated poly [N-(acryloyloxy) succinimide] (pNAS) with amine-containing monomer. With the obtained glycopolymers, a glycosurface was generated on the gold surface of quartz crystal microbalance (QCM) through self-assembled strategy by the use of alkanethiol functional group. Furthermore, the resulting glycosurface was used to detect the binding of mannose specific lectin concanavalin A (Con A).     

Differential localization of lectin binding sites and neuropeptides in human dorsal root ganglia

The subpopulations were compared of neurons in human dorsal root ganglia (DRG), as substance P, identified by somatostatin, Glycine max lectin (SBA) specific to terminal N-acetylgalactosamine, and Ulex europaeus I agglutinin (UEA-I) specific to l-fucose. The lectins and neuropeptides all bound to neurons of small diameter. Furthermore, the majority of the SBA binding neurons or somatostatin positive neurons were also UEA-I binding neurons. However, SBA binding neurons were not colocalized with somatostatin or substance P. Less than 20% of substance P positive neurons showed colocalization with l-fucosyl residues, and approximately 10% of l-fucosyl residues showed colocalization with substance P. Our results suggest that both l-fucose and terminal N-acetylgalactosamine containing neurons in the human DRG are subjected to different subpopulations from substance P or somatostatin positive neurons.     

Albumin immobilized on agarose as a tool for measuring ligand binding of proteins or peptides.

Bovine serum albumin immobilized on agarose has been tested in competitive binding studies as a means of measuring the binding of cortisol, tryptophan, fatty acids, and bilirubin to a number of albumins and albumin fragments. Chemical coupling of albumin to agarose does not appear to alter the primary binding sites for most ligands and the degree of ligand binding by immobilized albumin is comparable to that by soluble albumin. Evaluation of ligand binding by a protein based on its competition with immobilized protein is suggested as a convenient procedure particularly well suited to proteins and ligands whose size precludes investigation by dialysis or whose instability demands a rapid procedure.     

Glycoconjugates of the normal human colorectum: a lectin histochemical study

Summary Previous studies of the normal human colorectum by lectin histochemistry have used a mixture of tissues, including those derived from colons harbouring neoplasia and inflammatory bowel diseases. In the current investigation, tissues from patients without either of these conditions have been examined with a wide panel of lectins, encompassing specificities directed against both N- and O-linked sequences, using an avidin peroxidase revealing system and evaluated with a semiquantitative scoring method. The results of binding of these lectins have been compared with those seen in the resection margins of (at least 5 cm away from) colorectal carcinomas. Consistent regional variations were noted between right- and left-sided colonie tissues, with more diverse glycan structures and a greater sialyl content in the distal colon. There was evidence of graduation of formation of oligosaccharide chains in developing crypts, possibly related to the maturation and expression of glycosyl transferases responsible for the incorporation of mannose residues of N-linked oligosaccharides and of N-acetylgalactosamine and N-acetylglucosamine. Comparison with previous reports has revealed some variations, possibly related to tissue fixation and processing and to lectin concentrations employed, which raises the question of standardization of methodologies in lectin histochemical investigations.     

Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Glycoconjugates of the human trabecular meshwork: a lectin histochemical study

Summary Twelve specimens of resin-embedded human trabecular meshwork were probed with a panel of 21 biotinylated lectins, using an avidin-biotin peroxidase revealing system, in order to determine the normal pattern of saccharide expression in this tissue. High-mannose, intermediate and hybrid N-linked glycans, and complex N-linked bisected and non-bisected bi/tri-antennate glycans, as shown by the binding ofCanavalia ensiformis (ConA),Pisum sativum (PSA),Lens culinaris (LCA) agglutinins andPhaseolus vulgaris erythroagglutinin (ePHA), were strongly expressed by the canal of Schlemm endothelium and juxtacanalicular tissue, but less so by the corneoscleral meshwork. Highly branched complex glycans were not found, as there was no binding byPhaseolus vulgaris leukoagglutinin (1PHA). Sialyl residues, especially thoseα2,6-linked as demonstrated by strongSambucus nigra (SNA) lectin staining, were also abundant in this area.N-acetyllactosamine sequences and some O-linked glycans were present in the trabecular meshwork, as shown bySolanum tuberosum (STA),Datura stramonium (DSA), andJacalin (Jac) lectin binding, while fucose residues were not detected byTetragonolobus purpureas (LTA) orUlex europaeus-1 (UEA-1) agglutinins. These results indicate similarities with renal glomerular and vascular endothelium, although the lack of binding with UEA-1 agglutinin suggests differences which may relate to the specialized function of the trabecular meshwork. This study provides a baseline for comparative analysis of the glycans of human trabecular meshwork in pathological conditions such as primary open-angle glaucoma.     

Identification of endogenous sugar-binding proteins (lectins) in human placenta by histochemical localization and biochemical characterization

Human placentas of different stages of development were histochemically analyzed for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificity for beta-galactosides, alpha-galactosides, alpha-mannosides, alpha-fucosides and alpha-glucosides. Temporal differences in the expression of sugar-binding proteins and different patterns of staining of the component cell types of human placenta were discerned, especially pronounced for alpha-fucoside-specific binding in the trophoblast and alpha-glucoside-specific binding in fetal and maternal macrophages. Fractionation of salt and detergent extracts from human placentas by affinity chromatography on columns with immobilized carbohydrates or glycoproteins substantiated the histochemically detectable temporal changes on the basis of alterations in the pattern of individual sugar-binding proteins, as determined by gel electrophoresis under denaturing conditions. Analysis of the trophoblastic layer primarily disclosed the presence of several additional sugar-binding proteins (lectins) in comparison to full-term placenta. The presence and developmental changes of such endogenous sugar receptors may lead to specific carbohydrate-protein interactions of physiological significance with similarly developmentally regulated carbohydrated portions of glyco-conjugates, already detected in human placenta by plant lectins.     

Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand

We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP-sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino-efaroxan blocks ATP-sensitive potassium channels in human pancreatic beta-cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Further characterization and structural studies on human placenta lectin

The properties of a previously purified beta-galactoside-binding lectin of human placenta were studied in detail. Isoelectric focusing gave multiple bands around pH 4.9, although the lectin preparation was homogenous in SDS-polyacrylamide gel electrophoresis. High-performance gel chromatography suggested that the lectin exists mainly as the monomer and that a small fraction forms a dimer. From all the criteria examined, human placenta lectin resembles one of the chick lectins obtained from embryonic skin or adult intestine (subunit molecular weight: 14,000). The lectin was inactivated by thiol-modifying reagents, p-chloromercuribenzoic acid and N-ethylmaleimide. Reduced and carboxymethylated lectin contained five carboxymethylated cysteines per subunit, and five free thiol groups were titrated by using 5,5'-dithio-bis(2-nitrobenzoic acid). Preliminary sequence analysis showed the presence of a region highly homologous to the corresponding region of the chick lectin (13 identical residues out of 18 from number 70 to 87 of the chick lectin), suggesting a close evolutionary relation between these lectins and the importance of this conserved region in the function of the lectins.