Molecular analysis of the Gal/GalNAc adhesin of Entamoeba histolytica

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactose/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E.coli, retained at least some of its native conformation.     

Infection and immunity mediated by the carbohydrate recognition domain of the Entamoeba histolytica Gal/GalNAc lectin

Entamoeba histolytica causes invasive amebiasis, a major parasitic disease of the developing world, whose primary symptoms are liver abscess and colitis. All strains of E. histolytica express a 260-kDa surface Gal/GalNAc lectin that is antigenically conserved and immunogenic. The lectin is required for adherence to human intestinal epithelial cells and contact-dependent killing of immune effector cells. By expression cloning, the carbohydrate recognition domain (CRD) was identified within the lectin heavy-subunit cysteine-rich region. Of interest for a hepatic parasite, the CRD had sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF) and competed with HGF for binding to the c-Met HGF receptor. In an animal model of invasive disease, immunization with the CRD inhibited liver-abscess formation, yet in humans, a naturally acquired immune response against the CRD did not persist.     

The virtual hospital: the digital library moves from dream to reality

We examined the effects of passive immunization with a monoclonal antibody (EH3015) that recognizes a 150-kDa surface lectin of Entamoeba histolytica on amebic liver-abscess formation in hamsters. The hamsters were inoculated i.p with 0.1, 1.0, or 10 mg of EH3015 at 24 h prior to an intrahepatic challenge with 10(5) trophozoites of E. histolytica. In hamsters treated with 1.0 and 10 mg of EH3015 the incidence of liver abscesses was significantly reduced. These results demonstrate that monoclonal antibody EH3015 can prevent the development of amebic liver abscesses and that the 150-kDa lectin may be a protective antigen on the surface of E. histolytica.     

Amaranthus leucocarpus lectin recognizes human naive T cell subpopulations

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc residue found in the inner core of Gal beta 1,3GalNAc alpha 1,O-Ser/Thr disaccharide (T-antigen) or GalNAc alpha 1,O-Ser/Thr (Tn-antigen). Flow cytometric analysis using fluorescein-labeled lectin and monoclonal antibodies against human cell surface markers indicated that 5.7% of mononuclear cells from human healthy donors are recognized by ALL. These cells have the phenotype CD2+CD4+CD19- and most of the lymphocytes recognized are also CD27+, CD45RA+ and CD43+. ALL possesses mitogenic activity on lymphocytes after neuraminidase treatment. Our results indicate that the receptors recognized by ALL could be considered surface markers for naive human T lymphocyte subsets.     

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.     

Elderberry (Sambucus nigra) bark contains two structurally different Neu5Ac(alpha2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins

A second NeuAc(alpha2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sambucus nigra) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(alpha2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate-binding specificity and ribosome-inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.     

Cardiovascular effects of microinjections of opioid agonists into the ''Depressor Region'' of the ventrolateral periaqueductal gray region

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.     

GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.     

Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells

The biosynthesis of the prohormone convertase PC2 was studied in Chinese hamster ovary cells stably transfected with PC2 cDNA (CHO/PC2) and in rat insulinoma cells (Rin5f). The major form of PC2 synthesized by CHO/PC2 cells was a 75-kDa protein corresponding to proPC2; this protein was retained intracellularly for 2-4 h following synthesis, suggesting prolonged intracellular residence. In contrast, the major form of PC2 within Rin cells initially exhibited a molecular mass of 72 kDa and was then progressively converted to a 64-kDa species. This 64-kDa species, which required 1-2 h to be released, was the major PC2 form detectable in Rin cell medium. Calcium-dependent benzyloxycarbonyl-Arg-Ser-Lys-Arg-aminomethylcoumarin cleaving activity was found in spent Rin cell medium; this activity could be immunoprecipitated with a carboxyl-terminal PC2 antibody, but not with preimmune serum. In neither cell line did intracellular PC2 become endoglycosidase H-resistant over time. PC2 released from Rin cells was also endoglycosidase H-sensitive. Microsequencing and endoglycosidase H results indicate that 75-kDa CHO cell PC2 and 72-kDa Rin cell PC2 both represent proPC2. We speculate that (a) PC2 undergoes unusual glycosylation, which may be related to its slow release from cells, and (b) the 64-kDa molecule detectable in spent Rin cell medium represents the enzymatically active form of PC2.     

Hepatic clearance of 99mTc-GSA in cases of postoperative biliary atresia--a retrospective comparison with hepatic clearance of 99mTc-PMT]

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology, and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin - mannose-binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 ( Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.     

Pharmacokinetics of L-arginine during chronic administration to patients with hypercholesterolaemia

The existence of at least two distinct alpha 1,2fucosyltransferases has been postulated for many years, and recently confirmed in humans with the cloning of the human and rabbit secretor type alpha 1,2fucosyltransferase. We now describe the cloning and analysis of PFUT2, the pig secretor type alpha 1,2fucosyltransferase, which shows a high level of amino acid identity with previously cloned alpha 1,2fucosyltransferases, but more so with human and rabbit FUT2. Expression of PFUT2 in COS cells showed cell surface staining for H substance with UEAI lectin and anti-H monoclonal antibody, but not for A blood group substance. Kinetic studies were consistent with PFUT2 having a preference for type 1 and type 3 acceptors, as do the human and rabbit homologues, in contrast to PFUT1 which shows a preference for type 2 substrates. Like HuFUT1 and PFUT1, PFUT2 was able to dominate over the pig alpha 1,3galactosyltransferase in co-expression studies in COS cells and give preferential expression of H substance and reduced expression of Gal alpha (1,3)Gal. Cotransfection studies demonstrate that a combination of FUT1 and FUT2 cDNAs has an additive effect in suppressing expression of Gal alpha (1,3)Gal.     

Growth hormone-promoted tyrosyl phosphorylation of a 121-kDa growth hormone receptor-associated protein

Previous work in multiple cell types has shown that endogenous GH receptors, as well as the cloned liver GH receptor, associate with a tyrosine kinase. However, in SDS-PAGE gels of highly purified, kinase-active GH receptor preparations from 35S-labeled 3T3-F442A cells, only one broad band was detected corresponding to the molecular weight of the GH receptor rather than two bands which might be expected to result from a kinase-receptor heterocomplex. In the present study, a transfected Chinese hamster ovary (CHO) cell line (CHO4) that expresses an 84-kDa GH receptor rather than a 121-kDa GH receptor was used to examine whether the GH receptor might form a complex with a protein (e.g. tyrosine kinase) that comigrates on SDS-polyacrylamide gel electrophoresis gels with the endogenous GH receptor (M(r) 121,000) in 3T3-F442A cells. GH-GH receptor complexes were immunoprecipitated with anti-GH antibody from GH-treated CHO4 cells and incubated with [gamma-32P]ATP. 32P was incorporated into a 121-kDa protein as well as the 84-kDa GH receptor. Phosphorylation of both the 84-kDa GH receptor and the 121-kDa protein was on tyrosyl residues as determined by Western blotting with anti-phosphotyrosine antibody. The 121-kDa protein does not appear to bind GH. It was also not detected in the immunoprecipitate when cells had not been incubated with GH or when untransfected CHO cells were used. These findings suggest that in CHO4 cells, the 121-kDa protein is precipitated by the GH antibody because of its ability to form a complex with the GH receptor (p84). Western blot analysis of whole cell lysates using anti-phosphotyrosine antibody revealed that GH promotes the tyrosyl phosphorylation of a 121-kDa protein and several other proteins (p97, p42, p39) in a dose- and time-dependent fashion. Taken together, these findings are consistent with either p121 being the tyrosine kinase that complexes with the GH receptor and is activated in response to GH binding or with p121 forming a ternary complex with both the GH receptor and a tyrosine kinase and serving as a substrate of the GH receptor-associated tyrosine kinase.     

Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.     

Differential glycosylation of auditory and vestibular hair bundle proteins revealed by peanut agglutinin

Up to four morphologically distinct types of cross-link are found between the stereocilia in the hair bundles of avian hair cells. These links are involved in mechanotransduction, force transmission across the bundle, and maintenance of hair bundle structure. They appear to be specialisations of the cell coat, but very little is known about their molecular composition. Chick inner ear tissues were therefore screened with a number of different lectins to find markers for specialisations of the hair bundle surface. One lectin, peanut agglutinin (PNA), which recognises the dissacharide Gal beta 1-3GalNAc, was found to be a fairly selective marker for vestibular hair bundles, but it does not stain the stereocilia of auditory hair cells. The staining patterns observed with PNA in the vestibular system closely resemble those seen with a monoclonal antibody (mab) directed against a 275 kD component of the hair cell's apical surface known as the hair-cell antigen (HCA). However, unlike PNA, the mab recognises both vestibular and auditory hair cells. A detailed comparison of the fluorescence staining patterns observed with PNA and the anti-HCA mab indicates that binding sites for both ligands spatially codistribute on the surface of vestibular hair cells. The lectin and the anti-HCA mab binding sites are both sensitive to trypsin treatment, and, with sections of the vestibular system, PNA pretreatment blocks subsequent anti-HCA mab staining. Immunoelectron microscopy of vestibular hair bundles shows that PNA and the anti-HCA mab both label a type of cross-link known as the shaft connector. This link type is present on both auditory and vestibular hair bundles but reacts with PNA only in the vestibular system. The lectin jacalin, which has greater specificity for Gal beta 1-3GalNAc than does PNA, also only labels vestibular and not auditory hair bundles. Although terminal sialic acid residues can block both PNA and jacalin binding, neuraminidase treatment does not unmask cryptic binding sites for these lectins on auditory hair cells but does reveal PNA and jacalin staining at a number of other locations in the inner ear. The results obtained with the lectins PNA and jacalin indicate that either the HCA or other components of the shaft links are differentially glycosylated in the vestibular and auditory epithelia of the bird. The functional significance for such a difference in glycosylation remains to be determined, but auditory and vestibular hair cells operate over different frequency ranges, and variations in glycosylation might confer different micromechanical properties on the hair bundles in these two systems.     

Concentrations of cytokines in plasma of patients with large burns: their relation to time after injury, burn size, inflammatory variables, infection, and outcome

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-alpha. In a previous study, we localized EGF/TGF-alpha receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF-alpha binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF-alpha receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF-alpha followed by cross-linking with bis(sulphosuccinimidyl)suberate showed that TGF-alpha binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF-alpha to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF-alpha-stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF/TGF-alpha receptor protein, which dimerizes on binding to TGF-alpha, suggesting that the receptor protein may be involved in the signal transduction of TGF-alpha actions in the chicken granulosa cells.     

Identification of a putative precursor to the major surface glycoprotein of Pneumocystis carinii

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5' end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.     

Immunochemical characterization of anti-H monoclonal antibodies obtained from a mouse immunized with human saliva

Three IgM class anti-H monoclonal antibodies (1E3, 1E5 and 3H1) were obtained from a BALB/c mouse immunized with human O type saliva. These antibodies were found to agglutinate red cells from O group and A and B subgroups but not from Bombay and para-Bombay individuals whose H antigen was barely detected by anti-H reagents. The agglutination reactions of these antibodies were inhibited by H antigens from human tissues. It was also demonstrated that both 1E3 and 3H1 reacted with H disaccharide (Fuc alpha 1-->2Gal beta), H type 1 (Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta), H type 2 (Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta), H type 3 (Fuc alpha 1-->2Gal beta 1-->3GalNAc alpha) and H type 4 (Fuc alpha 1-->2Gal beta 1-->3GalNAc beta) but not with Lea (Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), Leb (Fuc alpha 1-->2Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), X (Gal beta 1-->4[Fuc alpha-->3]GlcNAc beta) or Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta). On the other hand, 1E5 was found to react with H type 1, H type 2, Leb and Y. Because of the unique reactivities against various fucosyl linkages these monoclonal antibodies could be useful not only as anti-H reagents but also as reagents for the structural analysis of fucosylated glycoconjugates.     

Microalbuminuria, arterial hypertension and the cardiovascular risk

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.