Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor.

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.

Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation.

Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.

The effect of high and low Dk/L soft contact lenses on the glycocalyx layer of the corneal epithelium and on the membrane associated receptors for lectins.

PURPOSE: Bacterial adherence or binding to the target cell is a prerequisite for the initial stage of most infections and seems to be mediated by lectin-like ligands on the bacterial surface and specific receptors on the target cell membrane. The purpose of this study was to establish whether contact lens wear under closed eye conditions changes the glycocalyx layer, whether it exposes more lectin receptors than eye closure without a contact lens, and whether wear of low oxygen transmissibility (Dk/L) contact lenses exposes more receptors than high Dk/L contact lenses. METHODS: The eyes of six rabbits under general anesthesia were fit with either a high Dk/L soft contact lens (40 x 10(-9), boundary corrected) or a low Dk/L soft contact lens (2 x 10[-9]) or were left without a lens as controls. All eyes were kept closed by suturing for 24 hours. After removal of the contact lenses, all corneas were excised, put in glutaraldehydeforfixation, rinsed, incubated with plant-derived lectins (wheat-germ agglutinin [WGA]) conjugated with gold particles, and prepared for electron microscopy. Membrane associated gold particles were counted and the results were processed statistically. RESULTS: After 24 hours of lens wear under closed eye conditions, the glycocalyx layer showed physical changes in the form of thinning or compression and signs of biochemical changes reflected as an increase in number of WGA receptors. The average number of membrane associated gold particles per 750 micro length of corneal epithelium in control corneas was 1,287.5 +/- 92.5. Particles were significantly (P<0.001) more numerous after wear of high Dk/L contact lenses (3,230.0 +/- 294.5) and after wear of low Dk/L contact lenses (4,611.3 +/- 223.3). The figure after wear of low Dk/L contact lenses was significantly (P<0.01) higher than the figure after wear of high Dk/L contact lenses. CONCLUSION: Our results indicate that lens wear under closed eye conditions seems to change the corneal glycocalyx layer physically as well as biochemically. Significantly larger numbers of WGA receptors were exposed after contact lens wear than without a contact lens. Significantly more receptors were exposed after wear of low Dk/L contact lenses than after wear of high Dk/L contact lenses. These changes may be of importance in relation to the risk of bacterial keratitis.

Ultrastructural localization of lectin receptors in the preimplantation ovine embryo.

BACKGROUND: Preimplantation development of mammalia is characterized by cell surface changes functioning in intercellular communication and adhesion. The glycoconjugate role in cellular interactions has been analysed for several groups but not in sheep embryos. The binding patterns of eleven lectins during sheep preimplantation development were investigated and the role of glycoconjugates in early development was discussed. METHODS: Ultrathin sections from preimplantation ovine embryos (3-7 days) were incubated with different colloidal gold conjugated lectins and the frequency of gold particles on the cell membrane, some organelles, and the zona pellucida was evaluated. RESULTS AND CONCLUSIONS: We observed a higher staining of WGA, DBA, and SBA lectins in the intercellular contact zone with respect to the free cell surface of blastomeres during cleavage. This indicates that the N-acetyl galactosamine and N-acetyl glucosamine residues may be involved in sheep morula compaction. In contrast, the trophoblast cell displays an increase of staining of some lectins previously identified during cleavage (LcH, WGA, SBA, MPA, and PNA) on the free membrane, and a lack of sugar residues in the intercellular surface. This polarization of the trophoblast cell surface is not observed in the inner cell mass and could provide a mechanism for differentiation within the blastocyst. Intracytoplasmic vesicles show a cytochemical identity with lysosomes in the blastocyst (abundant GlcNAc and Man/Glc residues) that may reflect a functional relationship between both organelles in an intracellular cycle. The zona pellucida presents abundant GalNAc, GlcNAc, and Gal residues during preimplantation ovine development.

Ultrastructural localization of wheat germ agglutinin binding sites on the sperm surface of water buffalo (Bubalus bubalis). A fracture label study.

In the present study we have examined the plasma membrane surface organization employing fluorescein isothiocyanate linked wheat germ agglutinin (WGA) of the cauda epididymal and ejaculated spermatozoa of water buffalo. Intramembrane particle distribution pattern in the various segments of the spermatozoa has also been observed. WGA-ovomucoid gold has been used to study the distribution of sialoproteins on the sperm surface. With fracture label, WGA receptor sites have been identified on the fractured membrane halves of the sperm plasma membrane overlying the acrosome as well as the middle piece and the principle piece.

Lectin histochemical study of bovine lingual glands.

Bovine lingual glands consist of mucous acini capped by demilunes. Information on the chemical structure of their secretory glycoconjugates was obtained by means of a battery of peroxidase-conjugated lectins with affinity for specific terminal sugars. Sialidase procedures followed by lectin staining were also used to visualize the sugar sequences. Stored secretions in mucous acinar cells contained fucose, N-acetylglucosamine, alpha and beta-N-acetylgalactosamine as terminal sugar residues and beta-galactose as penultimate sugar in a heterogeneous distribution. Demilunar cells failed to react with any of the lectins examined except that of Dolichos biflorus.

Growth of olfactory epithelial tissue in vitro: lectin staining of axons.

In vitro methods have been used to study several aspects of development of olfactory epithelium. In this paper, the value of growing olfactory tissue in explant cultures is reviewed and some experiments are reported on the identification of lectin receptors on olfactory axons by the use of lectin-gold complexes. Both concanavalin A-gold (con A-gold) and wheat germ agglutinin-gold consistently decorated olfactory axons in explant cultures. Con A-gold also bound to the tips of growth cone filopodia, suggesting the glycoconjugate molecules containing alpha-methyl-pyranoside are important in adherence of growth cones to their substrate. The wide range in density of lectin-gold particles suggested that axons, and the sensory cells from which they arise, are not a uniform population, i.e., they have different molecular fingerprints. This was supported by the observation that soybean agglutinin-gold stained some axons very well, but others remained unstained. Peanut agglutinin did not bind to any axons.

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.

Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

Ultrastructural detection in vitro of WGA-, RCA I-, and Con A-binding sites involved in the invasion of heart muscle cells by Trypanosoma cruzi.

The presence of carbohydrate residues in the plasma membrane of normal and Trypanosoma cruzi-infected heart muscle cells was investigated cytochemically using ruthenium red, lanthanum nitrate, periodic acid-Schiff/thiocarbohydrazide/silver, and gold- and ferritin-lectin complexes. The study combined conventional electron microscopy with the new analytical technique of electron spectroscopic imaging (ESI). Galactosyl, mannosyl, and sialyl residues were detected in regions of host-cell plasma membrane that undergo interiorization together with the parasite. Lectin-binding sites were sometimes found to show a punctate or patchy distribution in the endocytic vacuole membrane. These findings suggest the that glycoconjugates cytochemically detected in the host-cell plasma membrane participate in the invasion of heart muscle cells by T. cruzi.

Ultrastructural localization of lectin-binding sites on the surface of the guinea pig conjunctival epithelium.

The cell-surface binding sites of two lectins, concanavalin A (Con A) and wheat-germ agglutinin (WGA) in the guinea pig conjunctiva were investigated at the ultrastructural level by means of pre-embedding staining of the tissue with lectin-colloidal gold complexes. Wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, showed prominent and fairly uniform binding to the microvilli. The binding was markedly increased in the vicinity of the goblet cells, indicating that the same carbohydrate ligands were also present in the mucus. In contrast, no binding of concanavalin A, which recognizes mannose and N-glucose, was observed. The results suggest the presence of sialic acid and galactose as the constituent carbohydrates of glycoconjugates in the surface membrane of conjunctival epithelial cells as well as in the mucus produced by the goblet cells.

Light and electron microscopic demonstration of wheat germ agglutinin binding in the pericellular matrix of rat mandibular condylar cartilage.

Binding of wheat germ agglutinin to rat mandibular condylar cartilage was investigated with the avidin-biotin peroxidase complex. Binding sites were observed in the pericellular matrix of the hypertrophic cell zone. Two distinct patterns were identified: one showed binding to the pericellular matrix without apparent contour; in the other binding was confined to the matrix but with conspicuous condensation forming a pericellular rim. The first binding pattern was seen particularly in the upper part of the hypertrophic cell zone adjoining the mature cell zone; the second was localized in the lower part of this zone adjacent to the site of endochondral calcification. The binding sites in the pericellular matrix are assumed to be NANA and/or GlcNAc in view of the sugar specificity of this lectin. The presence of these binding sites in this region may be due to a structural alteration or modification of proteoglycans in the course of preparation for endochondral calcification.

[Fine structure studies and identification of lectin receptors on the viral envelope of two HIV-isolates]. / Feinstrukturuntersuchungen und Identifizierung von Lektinrezeptoren auf der Virushülle zweier HIV-Isolate.

The electron microscopic particle findings were compared with the levels of revertase in corresponding samples over a longer period of time, and a good correlation was found. Comparative investigations of the fine-structure of two HIV isolates did not reveal any morphological differences. It can be assumed, on the basis of the comparative studies on lectin receptors using Helix pomatia lectin, that the viral envelopes of the two isolates are equipped similarly with N-acetyl-d-galactosamine. The differences are not significant with mature particles.

[Partial characterization of the epithelial lectin binding sites of human gingiva and skin]. / Partielle Charakterisierung der epithelialen Lektinbindung humaner Gingiva und Haut.

Frozen sections of human gingiva and skin, fixed in acetone, were subjected to limited enzyme digestion (neuraminidase, proteinase K, trypsin) or, respectively, the application of solvents (chloroform/methanol, triton X-100) to allow a partial characterization of epithelial lectin binding sites. Gingiva differs from normal skin in that more conA-binding glycolipids are present in the lower cell layers. In the upper layers conA-fixing glycoproteins are prevailing. Psoriatic foci regularly exhibit an increased presence of conA-binding glycolipids. Gingiva and normal skin have some common features in the behavior of the lectin binding sites of HPA, WGA and UEA I. Analogies in the binding pattern of conA and UEA I in gingival tissue and in psoriatic foci are thus due to different lectin receptors.

[Membrane changes in Duchenne/Becker muscular dystrophy: lectin binding and localization of dystrophin]. / Membranveränderungen bei Duchenne/Becker-Muskeldystrophie: Lektinbindung und Dystrophinlokalisation.

An RCA I-lectin binding glycoprotein of Mr = 370 kD is missing from or altered in the plasma membrane of Duchenne muscular dystrophy (DMD) skeletal muscle. In the present study the carbohydrate chain of this glycoprotein was localized to the external face of the plasma membrane in human skeletal muscle, and dystrophin, the protein product of the DMD gene, was localized to the inner (cytoplasmic) face. On double labelled Western blots the two proteins appeared as closely apposed but distinctly separate bands. Comparison of the plasma membrane binding of five lectins with overlapping sugar specificities in skeletal muscle from patients with DMD and the allelic milder disease form, Becker muscular dystrophy (BMD) showed that the RCA I-binding glycoprotein also strongly binds to phytohaemagglutinin, thereby largely characterising the carbohydrate binding site. This glycoprotein was absent or altered in DMD and markedly reduced in clinically manifest BMD but present in preclinical clinical BMD. There was no general depletion of plasma membrane glycoproteins in DMD because consistent plasma membrane binding could be demonstrated by peanut and maclura pomifera lectin. The possible implications of these findings for the pathogenesis of DMD/BMD are discussed.

Disulfide-linked receptors for mitogenic lectins on piglet lymphocytes.

The interaction between leucoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), soybean agglutinin (SBA) and lentil lectin (LcH) with disulfide-linked cell surface receptors on lymphocytes from mesenteric lymph nodes of 3-day piglets (PMLN) was investigated. Surface radioiodinated PMLN lymphocytes were lysed with buffer containing Nonidet-P40. The lysates were adsorbed on lectin-agarose derivatives (or bovine serum albumin-agarose). Eluates from the lectin-agarose derivatives were analyzed by one-dimensional or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions. Among the various-lectin-binding polypeptides, L-PHA recognizes a single 92-kDa disulfide-linked moiety in piglet lymphocyte lysate, comprised of polydisperse 52-kDa subunits. In addition to this apparent homodimer, SBA, Con A and LcH bind a much less prominent 82-kDa heterodimer comprised of 47-kDa and 37-kDa polypeptides; these molecules are not observed in eluates of L-PHA. Binding of the 92- and 82-kDa molecules by LcH is inhibited by methyl-alpha-D-mannoside. These results indicate that there are two lectin-binding disulfide-linked glycoproteins on lymphocytes from 3-day piglets which bind preferentially to potent mitogens. The electrophoretic properties of these molecules, under both reducing and nonreducing conditions, as well as their lectin-binding properties are very similar to those observed for antigen receptor molecules on lymphocytes from other species.

The distribution of lectin receptor sites in human breast lesions.

Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.