Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland

Summary A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.Abbreviations BSA bovine serum albumin - F BSA Fluoresceinylated BSA - GlcNAc N-Acetylglucosamine - Lac lactose - Man mannose - Man 6-P mannose-6-phosphate - MES morpholino ethanesulfonic acid - PBS phosphate buffered saline - Tg thyroglobulin     

Interactions of a mammalian beta-galactoside-binding lectin with hamster fibroblasts

A beta-galactoside-binding endogenous lectin extracted from bovine heart binds to the surface of baby hamster kidney (BHK) cells. The binding to and agglutination of cells is reduced in certain ricin-resistant mutants (Ric cells) in parallel with the decreased number of binding sites for the selective agent, ricin, a galactose-specific plant lectin. However, clear differences in the binding specificities of bovine lectin and ricin are shown by the effect of neuraminidase. BHK cells and Ric mutant cells treated with neuraminidase bind similar amounts of the bovine lectin compared with untreated cells, and ricin binding is greatly increased. The mammalian lectin immobilised on inert glass mediates the attachment and spreading of normal BHK cells and agglutinates these cells in solution. Ricin-resistant mutant cells respond poorly. These results are consistent with a role of endogenous lectins in cellular adhesiveness and show that cell adhesion may be regulated by the density of specific surface receptors for lectins.     

Chicken tissue binding sites for a purified chicken lectin

A lactose-binding lectin previously purified from embryonic chicken muscle and adult chicken liver, and here referred to as chicken-lactose-lectin-I (CLL-I), was added to sections of various adult chicken tissues to detect available binding sites. Both the sites of binding of added CLL-I as well as the tissue distribution of endogenous CLL-I were determined by indirect immunofluorescence using a rabbit antibody to CLL-I followed by fluorescent goat anti-rabbit IgG. Some tissues such as intestine and kidney showed abundant extracellular binding sites for the lectin, primarily between cells, in basement membrane, and in material on the luminal surface. In contrast, adult heart showed no significant binding sites for CLL-I. Adult pancreas showed considerable endogenous CLL-I in an extracellular site surrounding exocrine lobules, but added CLL-I did not bind substantially. The distribution of CLL-I binding sites in intestine were mimicked by those of purpurin, another lactose-binding lectin. CLL-I binding sites were also detected on the surface of cultured chick embryo skin fibroblasts. The factors controlling the specific distribution of occupied and unoccupied CLL-I binding sites are not known.     

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.     

Development of screening methods for detection of carbohydrate-binding proteins by use of soluble glycosylated polyacrylamide-based copolymers.

Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Detection of sarcolectin-specific receptors like the cytokine macrophage migration inhibitory factor in rheumatoid nodules.

The objective of this study was the evaluation of the relation between the N-acetyl-neuraminic acid-binding endogenous lectin sarcolectin and the cytokine macrophage migration inhibitory factor (MIF) during development of rheumatoid nodules (RN) in seropositive rheumatoid arthritis (RA). Sarcolectin was purified and biotinylated. The binding patterns of this probe were analyzed in RN from patients with RA (n = 23) and compared with the distribution of antibodies with specificity for MIF, fibrin, fibronectin. In early RN, all areas of the inflammatory tissue displayed presence of receptors for sarcolectin. Macrophages were especially positive. In mature rheumatoid nodules binding of sarcolectin was restricted to the periphery of necrotic areas, to endothelial cells and perivascular connective tissue of marginal zones. Distribution patterns of MIF were similar but not identical. The histological staining characteristics demonstrate sarcolectin-binding receptors in RN that are altered upon disease progression. The finding suggests that specific interactions between this endogenous lectin and MIF may be involved in the course of RA.     

Anomer specificity of the 14 kDa galactose-binding lectin: A reappraisal

A β-anomer preference among galactosides has been attributed to the S-type 14 kDa galactose binding lectin. Here the anomeric preference of this lectin from bovine brain (BBL) is reexamined using inhibition of lectin-mediated haemagglutination, binding of the lectin to dot-blotted glycoproteins and affinity electrophoresis of the lectin through polysaccharide-containing gels. 1.0-methyl α-D-galactoside was 8 times better inhibitor of BBL than the corresponding ß-anomer. The terminal galactose in bovine thyroglobulin (exclusively. α-linked) were also nearly 8 times more inhibitory than those in asialofetuin (exclusively ß-linked). The terminal α-galactose-containing endogenous glycoproteins of bovine brain were nearly 4 times better inhibitors of BBL than laminin. Removal of terminal α-galactose units by α-galactosidase fully abolished the BBL binding of thyroglobulin and endogenous glycoproteins. BBL was also sugar-specifically retarded by polyacrylamide gel containing guar galactommannan which bears only α-linked galactose. Data indicated that α-galactosides were sometimes better than their β-anomers in binding to BBL. The significance of this observation to the physiological role of galactose-binding lectins is discussed.     

Biotinylated derivative of a human brain lectin: synthesis and use in affinoblotting for endogenous ligand studies

Coupling of biotin to an endogenous lectin yields a probe which can be used for selective nonradioactive detection of complementary endogenous ligands. To exemplify practical applications of this type of compounds, we have synthesized and characterized a biotinylated derivative of a beta-galactoside-specific human brain lectin. Proteins which bind this lectin can be located on nitrocellulose sheets after electrophoretic transfer from gradient polyacrylamide gels, by sequential incubation with biotinylated probes and streptavidin-peroxidase, with visualization by an insoluble reaction product (affinoblotting). Biotinylated galactoside-binding plant lectins were used in the same way to visualize human brain glycoproteins, and their binding specificity was compared with that of human brain lectin. The results obtained by means of these different probes showed the usefulness of the endogenous lectin derivative to actually identify its endogenous partners. Thus this approach may find extended applications in the study of biological activities of vertebrate lectins in homologous systems, i.e., with lectins and ligands coming from the same tissue origin.     

Bifunctional properties of lectins: lectins redefined

Both ‘classical’ lectins, originally identified as cell agglutinins, and other carbohydrate-binding proteins that were identified by different means, may contain a second type of binding site that is specific for a non-carbohydrate ligand. This, among other findings, is changing our view of endogenous lectin functions and of the proper definition of this group of proteins.     

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.     

Endogenous cell surface lectin in Dictyostelium: quantitation, elution by sugar, and elicitation by divalent immunoglobulin

The amount of total endogenous cellular and cell surface lectin in aggregating Dictyostelium purpureum was determined by a number of immunochemical techniques. The results show that of the 5 x 10(6) molecules of the lectin (called purpurin) per aggregating cell only about 2% (1 x 10(5) molecules) is present on the cell surface. Cell surface purpurin can be specially eluted by lactose, which indicates that it is held to the surface by its carbohydrate-binding site. The eluted purpurin is replaced on the cell surface within 45 min. Estimates of cell surface purpurin made by binding of specific immunoglobulin to the cells at 4 degrees C indicate that a much larger amount, about 1 x 10(6) molecules, becomes associated with the cell surface in the presence of this divalent ligand. In contrast, univalent antibody fragments do not have this effect.     

Monoclonal antibodies against a beta-galactoside-binding lectin of chick embryo

Monoclonal antibodies against an endogenous beta-galactoside-binding lectin (monomer molecular weight 14,000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34-66). However, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of beta-galactoside-binding lectin, namely 14K lectin (monomer molecular weight 16,000) and a third species which is assumed to be a hybrid molecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.     

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.     

Endocytosis of thyroglobulin is not mediated by mannose-6-phosphate receptors in thyrocytes. Evidence for low-affinity-binding sites operating in the uptake of thyroglobulin.

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate-(Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concentrations of thyroglobulin (20 microM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].     

Prostaglandin E binding by plasmatic membranes of thyroid gland cells]

The binding of prostaglandins E (PGE) with human and bovine thyrocyte plasmatic membranes was investigated by the new method with material from the kit for radioimmunoassay of PGE. There was a high affinity and a low affinity site for the specific PGE binding on human and bovine thyrocytes plasmic membranes. The effect of thyrotropin and cyclic nucleotides on the PGE binding by the membranes was revealed.     

Glycoproteins modulate adhesion in terminally differentiated keratinocytes

Summary The stratum corneum can be dissociated into single squames by homogenization in ether. We have reaggregated the free corneocytes into a multilayered lamellar structure resembling an intact stratum corneum. The reconstituted stratum corneum reacts with fluorescein-conjugated lectins, unlike the intact tissue. We infer that the lack of binding in the intact tissue is due to masking of saccharide sites by lipids (which are extracted by the ether). In an extension of the procedure, the ether is removed and replaced by acetone. This system permits us to modulate corneocyte reaggregation by the addition of appropriate agents. We have used this system to corroborate our hypothesis that a 40 kD cell-surface glycoprotein (an endogenous lectin specific for amino sugars), which we have isolated from the stratum corneum, is instrumental in adhesion of corneocytes by cross-linking with amino sugar sites on adjacent cells. The reaggregation is inhibited by the antibody to the 40 kD glycoprotein. It is also inhibited by either the addition of amino sugars which bind to the endogenous lectin, or the addition of exogenous lectins specific for amino sugars which bind to the ligand.     

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

Externalization of the endogenous intracellular lectin of a cellular slime mold

Intracellular purpurin, the endogenous lectin of Dictyostelium purpureum, has previously been shown to be externalized upon exposure of the cells to anti-purpurin IgG. Externalization of additional purpurin was presumably the consequence of cross-linking of the purpurin molecules already on the cell surface by the IgG, since binding univalent anti-purpurin Fab to the cell surface did not have this effect. In the present report we show that multivalent glycoconjugates that interact with purpurin—including asialo-bovine submaxillary mucin, a bacterial galactan, and bovine albumin derivatized with multiple chains of either lactose or lacto-N-neotetraose—all elicit the externalization of intracellular purpurin. Some of the externalized lectin is bound to the cell surface and some is found in the medium, presumably bound to the glycoconjugates. A similar effect is produced by exposure of the cells to high concentrations of purified purpurin. Several plant lectins, including conA, also have some effect, whereas others are inactive. Since cells in late stages of aggregation have about three times as much cell surface purpurin as those in early stages, this externalization reaction may have significance late in development. The results suggest that intracellular purpurin may be released in response to cross-linking of the endogenous cell surface glycoconjugates that are: (1) already bound to endogenous purpurin; (2) capable of binding purpurin; (3) capable of binding certain other lectins. The intracellular lectin may be a reserve pool, that functions only upon externalization.