Mannose-containing Glycoproteins Research Articles

EDEM3 Modulates Plasma Triglyceride Level through Its Regulation of LRP1 Expression.

SummaryHuman genetics studies have uncovered genetic variants that can be used to guide biological research and prioritize molecular targets for therapeutic intervention for complex diseases. We have identified a missense variant (P746S) in EDEM3 associated with lower blood triglyceride (TG) levels in >300,000 individuals. Functional analyses in cell and mouse models show that EDEM3 deficiency strongly increased the uptake of very-low-density lipoprotein and thereby reduced the plasma TG level, as a result of up-regulated expression of LRP1 receptor. We demonstrate that EDEM3 deletion up-regulated the pathways for RNA and endoplasmic reticulum protein processing and transport, and consequently increased the cell surface mannose-containing glycoproteins, including LRP1. Metabolomics analyses reveal a cellular TG accumulation under EDEM3 deficiency, a profile consistent with individuals carrying EDEM3 P746S. Our study identifies EDEM3 as a regulator of blood TG, and targeted inhibition of EDEM3 may provide a complementary approach for lowering elevated blood TG concentrations.

Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains.

Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging.

EDEM3 Modulates Plasma Triglyceride Level Through Its Regulation of LRP1 Expression

Human genetics studies have uncovered genetic variants that can be used to guide biological research and prioritize molecular targets for therapeutic intervention for complex diseases and metabolic conditions. We have identified a missense variant (P746S) in EDEM3 associated with lower blood triglyceride (TG) levels in >300,000 individuals. Functional analyses in cell and mouse models show that EDEM3 deficiency strongly increased the uptake of very low-density lipoprotein and thereby reduced the plasma TG level, as a result of up-regulated expression of LRP1 receptor. We demonstrate that EDEM3 deletion up-regulated the pathways for RNA and ER protein processing and transport, and consequently increased the cell surface mannose-containing glycoproteins, including LRP1. Metabolomics analyses reveal a cellular TG accumulation under EDEM3 deficiency, a profile consistent with individuals with carrying EDEM3 P746S. Our study identifies EDEM3 as a regulator of blood TG, and targeted inhibition of EDEM3 may provide a complementary approach for lowering elevated blood TG concentrations.

Abstract 16650: EDEM3 Modulates the Plasma Triglyceride Level via Its Regulation of LRP1 Expression

Population genetic studies have provided abundant insight about the development of diseases in all kinds, which has been validated to be instrumental to prioritize biological research and molecular targets for therapeutic intervention. Here we show another example of endoplasmic reticulum (ER) - degradation alpha -mannosidase like protein 3 (EDEM3) in this aspect. We first used a population genetic approach to highlight a variant in the coding region of EDEM3 that is significantly associated with lower blood triglyceride level in a cohort with >30,000 individuals worldwide. Our functional analysis show that the deletion of EDEM3 gene induced a strong uptake of very low-density lipoprotein (VLDL), but not LDL, as a result of up-regulated expression of low-density lipoprotein receptor (LDLR) - related protein 1 (LRP1). We found that the deletion of EDEM3 gene increased more mannose-containing glycoproteins on cell surface, but without altering the sensitivity of cells to ER stress. Our pathway analyses demonstrate that the gene deletion up-regulated the pathways for RNA and ER protein processing and transport, while down-regulated the metabolic pathways, which may help to maintain a normal response to ER stress, and facilitate the processing and transport of the mannose-containing glycoproteins including LRP1 to cell surface. Lipid metabolite analyses reveal a cellular accumulation of TG under EDEM3 deficiency, in particular those with shorter carbon-chains, a profile largely opposite to the one of the plasma from individuals carrying the EDEM3 missense mutation, suggesting that the data from our functional analysis are consistent with the phenotype of genetic perturbation of EDEM3. Thus, our study identifies EDEM3 as a regulator of plasma TG, and targeted inhibition of EDEM3 expression may provide opportunity for plasma TG lowering.

Selective capture of glycoproteins using lectin-modified nanoporous gold monolith

The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of α-lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31×1018 and 1.85×1015moleculesm−2, respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989×1018 and 1.32×1015moleculesm−2, respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A=2.3 and BSA:Con A=0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.

ОЧИЩЕННЯ І ХАРАКТЕРИСТИКА НОВОГО МАНОЗОСПЕЦИФІЧНОГО ЛЕКТИНУ ІЗ ЦИБУЛИН ГІАЦИНТИКА ГОСТРОЛОПАТЕВОГО (Hyacinthella аcutiloba K. Perss.)

A new lectin with 6.5 mg/kg yield was purified from fresh bulb Hyacinthella аcutiloba K. Perss. by combination of affinity chromathography on yeast mannan and by ion exchange chromatography on DEAE-toyopearl. The best lectin inhibitor among tested monoand disaccharides was D-turanose (Glcp α1-3 Fruf). Very powerful inhibitor of lectin activity was yeast mannan. The lectin revealed weak affinity to α-methyl-Dmannoside, D-fructose and 2-acetamido-D-galactopyranoside. The lectin interacted also with pig liver glycogen, starch and mannose-containing glycoproteins (ovoalbumin, ovomucoid and calf thyroglobulin), but don’t interacted with horsera dish peroxidase and calf intestine alkaline phosphatase. According to electrophoresis, in 20% SDS-PAAG containing SDS-Na the lectin consists with subunits of molecular weight 12 kDa. Molecular weight of the lectin is 48 kDa according to gel-chromatography on Toyopearl HW-55. The lectin agglutinated best of all rabbit erythrocytes and worse agglutinated guinea-pig, very weak — rat erythrocytes and did not agglutinate human and goat erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin did not lose hemaglutinating activity and endured heating to +65 °C during one hour.

Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins

A procedure for determination of apparent affinity constants KDapp between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10−7 to 9×10−6M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.

Biochemical Characterization and Functional Studies of Acanthamoeba Mannose-Binding Protein

Acanthamoebae produce a painful, sight-threatening corneal infection. The adhesion of parasites to the host cells is a critical first step in the pathogenesis of infection. Subsequent to adhesion, the parasites produce a potent cytopathic effect (CPE) leading to target cell death. Recent studies showing that acanthamoebae express a mannose-binding protein (MBP) and that free alpha-mannose (alpha-Man) specifically inhibits the adhesion of parasites to host cells suggest that the MBP plays a key role in the pathogenesis of Acanthamoeba infection by mediating host-parasite interactions. However, direct evidence showing that Acanthamoeba MBP is a virulence protein has been lacking. In this study, we demonstrate that the polyclonal immunoglobulin Y (IgY) antibodies prepared against affinity-purified Acanthamoeba MBP markedly inhibit the adhesion of parasites to host cells. The antibody also inhibited the Acanthamoeba-induced CPE on host cells. In contrast, preimmune IgY did not influence either the adhesion of the parasites to host cells or the amoeba-induced CPE. Using a variety of approaches, including affinity chromatography on an alpha-Man gel, electrophoresis under native and denaturing conditions, biotinylation of cell surface proteins, and immunostaining, it was conclusively established that Acanthamoeba MBP is located on the surface membranes of the parasites. Neutral-sugar analysis and lectin binding experiments using succinylated concanavalin A, a plant lectin with high affinity for mannose, revealed that Acanthamoeba MBP is itself a mannose-containing glycoprotein. N-Glycanase treatment to remove N-linked oligosaccharides shifted the subunit molecular mass of MBP from 130 kDa to 110 kDa. Hexosamine analysis revealed that Acanthamoeba MBP lacks detectable levels of GalNAc, suggesting the absence of O-linked oligosaccharides. In summary, we have characterized Acanthamoeba MBP and have shown that it is a major virulence protein responsible for host-parasite interactions and the parasite-induced target cell destruction.

Recognition of Entamoeba histolytica 115-kDa surface protein by human secretory immunoglobulin A antibodies from asymptomatic carriers.

Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.

Binding of free immunoglobulin light chains to VpreB3 inhibits their maturation and secretion in chicken B cells.

The VpreB3 gene product was first characterized as an immunoglobulin (Ig) mu heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM(+) immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.

Purification, Characterization, Molecular Cloning, and Expression of Novel Members of Jacalin-related Lectins from Rhizomes of the True Fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.

The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum.

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.

Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDPMP) is essential for the formation of GDP-mannose, the central activated mannose donor in glycosylation reactions. Deletion of its gene is lethal in fungi, most likely as a consequence of disrupted glycoconjugate biosynthesis. Furthermore, absence of GDPMP enzyme activity and the expected loss of all mannose-containing glycoconjugates have so far not been observed in any eukaryotic organism. In this study we have cloned and characterized the gene encoding GDPMP from the eukaryotic protozoan parasite Leishmania mexicana. We report the generation of GDPMP gene deletion mutants of this human pathogen that are devoid of detectable GDPMP activity and completely lack mannose-containing glycoproteins and glycolipids, such as lipophosphoglycan, proteophosphoglycans, glycosylphosphatidylinositol protein membrane anchors, glycoinositolphospholipids and N-glycans. The loss of GDPMP renders the parasites unable to infect macrophages or mice, while gene addback restores virulence. Our study demonstrates that GDP-mannose biosynthesis is not essential for Leishmania viability in culture, but constitutes a virulence pathway in these human pathogens.

Calreticulin and Calsequestrin are Differentially Distributed in Canine Heart

Cardiac sarcoplasmic reticulum (SR) consists of three continuous yet distinct regions: longitudinal, corbular, and junctional. Ca2+uptake, catalyzed by the Ca2+-dependent ATPase, is thought to occur throughout the SR whereas Ca2+release occurs in the region of the junctional SR. In the SR, Ca2+is stored in a complex with Ca2+-binding proteins such as calsequestrin. In the present study, the distribution of the high-affinity calcium-binding protein, calreticulin, in canine cardiac SR was determined and compared with the distribution of other SR proteins. Three types of distribution were observed. Many proteins, including the Ca2+-ATPase and two mannose-containing glycoproteins (52 and 131 kDa), were present in all subfractions. These proteins were absent or depleted from the sarcolemma-enriched fraction. The ryanodine receptor/Ca2+release channel and calsequestrin were localized to the junctional SR. Calreticulin immunoreactivity was detected in fractions containing longitudinal SR but not in fractions enriched in sarcolemma or junctional SR. Hence calreticulin is present in a unique vesicular fraction and the Ca2+-binding proteins calreticulin and calsequestrin display different patterns of distribution in canine heart.

Garlic (Allium sativum) Lectins Bind to High Mannose Oligosaccharide Chains

Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 +/- 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manalpha1-3Man) < mannotriose (Manalpha1-6Manalpha1-3Man) approximately mannopentaose << Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both alpha1-3- and alpha1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several alpha1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.

Binding of Acanthamoeba to 23 mannose-glycoproteins of corneal epithelium: effect of injury

Purpose. Acanthamoeba keratitis is a sight-threatening corneal infection. It is known that: (i) more amoebae bind to the surface of injured corneas than to the normal corneal surface and (ii) mannose-containing glycoproteins (GPs) possess binding sites for Acanthamoeba. The present study was undertaken to determine whether subtle corneal surface injury exposes mannose-GPs and whether more amoebae bind to the mannose-GPs of injured corneas than to those of normal corneas.Methods. Corneal cup assays were developed to determine whether corneal surface injury exposes binding sites for a mannose/glucose-specific lectin, succinylated-concanavalin A (s-ConA). To determine whether injury exposes mannose-GPs, corneal surface proteins were biotinylated, biotin-labeled mannose-GPs were allowed to bind to s-ConA-agarose beads and were analyzed by SDS-polyaerylamide gel electrophoresis (PAGE). Amoeba binding to mannose-GPs of corneal epithelia was analyzed by PAGE-blot overlay assays.Results. S-ConA binding site density was 2.4 times greater on the injured corneal surface than on the surface of normal corneas. Based on the analysis of the s-ConA-bound, biotin-labeled corneal surface proteins, approximately 5.2 times greater amounts of mannose-GPs were present on the surface of injured corneas than on the normal corneal surface. PAGE-blot overlay assays of s-ConA bound GPs of unlabeled corneal epithelia revealed that, on a per mg total cell protein basis, injured corneal epithelium contained 1.8 times greater amounts of Acanthamoeba-reactive mannose-GPs than normal corneal epithelium.Conclusions. Subtle corneal injury exposes mannose-GPs on the surface of injured corneas. The newly exposed GPs may serve to provide additional attachment sites for the amoebae. This, in turn, could render the cornea susceptible to the infection. Curr. Eye Res. 17:770–776, 1998.

Pathogenesis of Acanthamoeba keratitis: carbohydrate-mediated host-parasite interactions.

Acanthamoeba keratitis is a sight-threatening corneal infection. In a recent study, the saccharide mannose has been shown to inhibit the binding of Acanthamoeba organisms to the epithelium of the cornea (L. D. Morton, G. L. McLaughlin, and H. E. Whiteley, Infect. Immun. 59:3819-3822, 1991). In an attempt to determine the molecular mechanism by which acanthamoebae adhere to the surface of the cornea, the present study was designed to determine whether Acanthamoeba castellanii derived from an infected human cornea (i) binds to mannose-containing glycoproteins (mannose-GPs) of corneal epithelium and (ii) expresses one or more mannose-binding proteins. Mannose-GPs of primary cell cultures of rabbit corneal epithelium were isolated by using three different agarose-conjugated, mannose-specific lectins. By electrophoresis blot-overlay assays, 35S-labeled acanthamoebae were shown to bind to mannose-GPs of corneal epithelium and to a neoglycoprotein, mannose-bovine serum albumin (mannose-BSA). 35S-labeled acanthamoebae also bound to microtiter wells coated with mannose-BSA in a concentration-dependent manner. The binding of amoebae to mannose-GPs was blocked by free methyl-alpha-D-mannopyranoside. The parasites did not bind to galactose-BSA or to many other proteins lacking mannose residues. A membrane-associated mannose-binding protein (136 kDa) of A. castellanii was isolated by affinity chromatography of detergent extracts of unlabeled parasites and of cell surface biotin-labeled parasites on a p-aminophenyl alpha-D-mannopyranoside-agarose column. The affinity-purified protein of the amoeba was shown to bind specifically to mannose-BSA. In summary, a mannose-binding protein is present on the surface membranes of Acanthamoeba, and corneal epithelial cells express Acanthamoeba-reactive GPs. One of the mechanisms of Acanthamoeba adhesion to the corneal surface may involve interactions between the mannose-binding protein of Acanthamoeba and mannose-GPs on the surface of corneal epithelium.

Characterization of rod outer segment plasma membrane proteins which bind to the mannose receptor.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Epithelial glucosphingolipid expression as a determinant of bacterial adherence and cytokine production.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo-GSLs) (derived from the precursor Gal alpha 1-4Gal beta 1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P-fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.