Lectin Site Research Articles

Purification, some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 °C; however, at 55 °C EiLL lost more than 90% of its activity and at 60 °C it was totally inactivated. The p I of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.

Localization of the Lectin, ERp57 Binding, and Polypeptide Binding Sites of Calnexin and Calreticulin

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.

Dietary lectins can stimulate pancreatic growth in the rat.

Lectins are proteins or glycoproteins of nonimmune origin, which bind specifically to carbohydrate structures. They are widespread in the human diet, and many are resistant to digestion. High doses of lectins have been shown to stimulate intestinal and pancreatic growth. The aim of the present study was to investigate the long-term actions of low doses of lectins on the rat intestine and pancreas. A long-term carcinogenesis study was performed using low levels (40 micro g/rat/day) of peanut (PNA) or mushroom lectin (ABA) which bind to O-linked (mucin-type) oligosaccharides in the gut. While this was primarily designed as a colon carcinogenesis study, the pancreas was also investigated. No significant changes in colon carcinogenesis were seen, however, the colons were slightly heavier in the lectin treated groups. The weight of the pancreas was significantly greater (by 18 and 23%) in both lectin treated groups (P < 0.03/0.001). The weights of the acini and septal tissue were also increased by 39-46% in PNA and ABA fed animals, respectively (P < 0.002); there was no significant change in the endocrine pancreas. In conclusion, long-term feeding of low doses of lectin can influence pancreatic growth, and this trophic action may have potential adverse implications for the development of pancreatic cancer in humans.

Lectin Receptor Kinases in Plants

Referee: Dr. Philip Becraft, Zoology and Genetics/Agronomy Depts., 2116 Molecular Building, lowa State University, Ames, IA 50011Forty-two lectin receptor kinase (lecRK)-related sequences and nine related soluble legume lectin sequences were identified in the Arabidopsis thaliana genome. The genes are scattered as a single or gathered copies at different loci throughout the five chromosomes, and four predicted lecRK probably correspond to pseudogenes. Both structural alignments and molecular modeling revealed striking similarities between the lectinlike domain of lecRK, and related A. thaliana soluble lectins and legume lectins. The hydrophobic cavity is extremely conserved, whereas most of the residues forming the monosaccharide-binding site and the bivalent cation-binding site of legume lectins are poorly conserved. LecRK should be unable to bind the simple sugars usually recognized by genuine legume lectins. Molecular modeling of the kinase domain suggests that, except for two apparently inactive receptors, all other lecRK contain a putative functional Ser/Thr kinase catalytic domain. Both the juxtamembrane and C-terminal domains, which are considered important regions for regulating the kinase activity, exhibit a few specific stretches of amino acid residues. Some phylogenetic relationships are inferred from the phylogenetic trees built up from the different lecRK domain sequences. LecRK cluster in three distinct classes (A,B,C), one of them (B) being more closely related to soluble lectins of A. thaliana and legume lectins.

HIV-1 gp41 Envelope Residues 650–685 Exposed on Native Virus Act as a Lectin to Bind Epithelial Cell Galactosyl Ceramide

The initial step in the interaction between human immunodeficiency virus (HIV-1) and epithelial cells is the binding of HIV-1 envelope glycoproteins to the epithelial cell galactosyl ceramide (GalCer). Here we show that HIV-1 envelope gp41 residues 650-685 bind GalCer in a galactose-specific manner. The gp41 residues that display this lectin activity are highly conserved among HIV-1 isolates and constitute three regions: residues 650-661, which encompass a charged helix; residues 662-667, referred to as the conserved epitope ELDKWA, the epitope recognized by antibodies that neutralize HIV-1 entry in epithelial and CD4(+)-mononucleated cells; and residues 668-685, a hydrophobic Trp-rich sequence that stabilizes the structure of the galactose binding site. Similar to other galactose-specific lectins, the gp41 lectin site is active only as an oligomer. Finally the orientation of the galactose toward the gp41 lectin site appears to be controlled by the lipid microenvironment of the epithelial membrane. From the experimental data we construct a theoretical model of the interaction between gp41 and GalCer based on thermodynamic considerations. This model integrates the dynamics and the spatial organization of the viral envelope glycoproteins, GalCer organized in raft microdomains in the apical region of the epithelial cell membrane and the interfacial water. Characterization of the minimal sequence and structure of gp41 in direct interaction with GalCer may help unravel the still unknown immunogenic determinant able to elicit antibodies against ELDKWA and target of one of the rare neutralizing antibodies against gp41.

Role of protein kinase C in the phosphorylation of CD33 (Siglec-3) and its effect on lectin activity.

CD33 (Siglec-3) is a marker of myeloid progenitor cells, mature myeloid cells, and most myeloid leukemias. Although its biologic role remains unknown, it has been demonstrated to function as a sialic acid-specific lectin and a cell adhesion molecule. Many of the Siglecs (including CD33) have been reported to be tyrosine phosphorylated in the cytosolic tails under specific stimulation conditions. Here we report that CD33 is also a serine/threonine phosphoprotein, containing at least 2 sites of serine phosphorylation in its cytoplasmic domain, catalyzed by protein kinase C (PKC). Phosphorylation could be augmented by exposure to the protein kinase-activating cytokines interleukin 3, erythropoietin, or granulocyte-macrophage colony-stimulating factor, in a cytokine-dependent cell line, TF-1. The CD33 cytoplasmic tail was phosphorylated by PKC in vitro, in a Ca(++)/lipid-dependent manner. CHOK1 cells stably expressing CD33 with cytoplasmic tails of various length also showed phorbol myristate acetate (PMA)-dependent phosphorylation of CD33. Inhibition of CD33 phosphorylation with pharmacologic agents resulted in an increase of sialic acid-dependent rosette formation. Furthermore, the occupancy of the lectin site affected its basal level of phosphorylation. Rosette formation by COS cells expressing a form of CD33 lacking its cytoplasmic domain was not affected by these same agents. These data indicate that CD33 is a phosphoprotein, that its phosphorylation may be controlled by PKC downstream of cytokine stimulation, and that its phosphorylation is cross-regulated with its lectin activity. Notably, although this is the first example of serine/threonine phosphorylation in the subfamily of CD33-like Siglecs, some of the other members also have putative target sites in their cytoplasmic tails.

Crystal structures of artocarpin, a Moraceae lectin with mannose specificity, and its complex with methyl-α- d-mannose: implications to the generation of carbohydrate specificity

The seeds of jack fruit ( Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the β-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 Å resolution, respectively. The structure of the lectin complexed with methyl-α-mannose, has also been determined at 2.9 Å resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the β-prism I fold.

Role of the lectin domain of Mac-1/CR3 (CD11b/CD18) in regulating intercellular adhesion.

Leukocyte diapedesis requires that Mac-1/CR3-dependent adhesion be regulated so that cells can move from one attachment site to another. The high affinity adhesion state of Mac-1/CR3 is generated when it forms a lectin-dependent complex with the receptor for urokinase plasminogen activator (uPAR; CD87). The extensively glycosylated uPAR binds to the same C-terminal lectin domain of CD11b that had previously been shown to prime Mac-1/CR3 for cytotoxic degranulation in response to beta-glucan. uPAR and beta-glucan compete for a lectin site that is near to the CBRM1/23 epitope (residues 943-1047) at the C-terminus of CD11b, and thus the lectin domain is critical to both the adhesion and cytotoxic functions of Mac-1/CR3. Adhesion is reversed when the uPA enzyme is captured by its receptor (uPAR), causing uPAR to bind to CD11b at a second site (residues 424-440) that is in between the N-terminal I-domain and the divalent cation binding region.

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules.

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble beta-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein-Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble beta-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I+ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcgammaRIIIA (CD16).

Relationship between Calnexin and BiP in Suppressing Aggregation and Promoting Refolding of Protein and Glycoprotein Substrates

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.

Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.

The syntheses of 6-C-alkyl derivatives of methyl α-isomaltoside for a study of the mechanism of hydrolysis by amyloglucosidase

The epimeric (6aR)- and (6aS)-C-alkyl (methyl, ethyl and isopropyl) derivatives of methyl α-isomaltoside (1) were synthesized in order to examine the effects of introducing alkyl groups of increasing bulk on the rate of catalysis for the hydrolysis of the interunit α-glycosidic bond by the enzyme amyloglucosidase, EC 3.2.1.3, commonly termed glucoamylase (AMG). It was previously established that methyl (6aR)-C-methyl α-isomaltoside is hydrolysed about 2 times faster than methyl α-isomaltoside and about 8 times faster than its S-isomer. The kinetics for the hydrolyses of the ethyl and isopropyl analogs were also recently published. As was expected from molecular model calculations, all the R-epimers are good substrates. A rationale is presented for the catalysis based on conventional mechanistic theories that includes the assistance for the decomposition of the activated complex to products by the presence of a hydrogen bond, which connects the 4a-hydroxyl group to the tryptophane and arginine units. It is proposed that activation of the initially formed complex to the transition state is assisted by the energy released as a result of both of the displacement of perturbed water molecules of hydration at the surfaces of both the polyamphiphilic substrate and the combining site and the establishment of intermolecular hydrogen bonds, i.e., micro-thermodynamics. The dissipation of the heat to the bulk solution is impeded by a shell of aromatic amino acids that surround the combining site. Such shields are known to be located around the combining sites of lectins and carbohydrate specific antibodies and are considered necessary to prevent the disruption of the intermolecular hydrogen bonds, which are of key importance for the stability of the complex. These features together with the exquisite stereoelectronic dispositions of the reacting molecules within the combining site offer a rationalization for the catalysis at ambient temperatures and near neutral pH. The syntheses involved the addition of alkyl Grignard reagents to methyl 6-aldehydo-α-D-glucopyranoside. The addition favoured formation of the S-epimers by over 90%. Useful amounts of the active R-isomers were obtained by epimerization of the chiral centers using conventional methods. Glycosylation of the resulting alcohols under conditions for bromide-ion catalysis, provided methyl (6aS)- and (6aR)-C-alkyl-hepta-O-benzyl-α-isomaltosides. Catalytic hydrogenolysis of the benzyl groups afforded the desired disaccharides. 1H NMR studies established the absolute configurations and provided evidence for conformational preferences.Key words: amyloglucosidase (AMG), exo-anomeric effect, 6-C-alkyl-α-D-glucopyranosides and isomaltosides, mechanism of enzyme catalysis.

Expression of binding properties of Gal/GalNAc reactive lectins by mammalian glycotopes (an updated report).

Expression of the binding properties of Gal/GalNAc specific lectins, based on the affinity of decreasing order of mammalian glycotopes (determinants) rather than monosaccharide inhibition pattern, is probably one of the best ways to express carbohydrate specifity and should facilitate the selection of lectins as structural probes for studying mammalian glycobiology. Eleven mammalian structural units have been selected to express the binding domain of applied lectins. They are: 1. F, GalNAcalpha1 --> 3GalNAc; 2. A, GalNAcalpha1 --> 3Gal; 3. T, Galbeta1 --> 3GalNAc; 4. I, Galbeta 1 --> 3GlcNAc; 5. II, Galbeta1 --> 4GlcNAc; 6. B, Galalpha1 --> 3Gal; 7. E, Galalpha1--> 4Gal; 8. L, Galbeta1 --> 4Glc; 9. P, GalNAcbeta1 --> 3Gal; 10. S, GalNAcbeta1 --> 4Gal and 11. Tn, GalNAcalpha1 --> 4Ser (Thr) of the peptide chain. Thus, the carbohydrate specificity of Gal/GalNAc reactive lectins can be divided into classes according to their highest affinity for the above disaccharides and/or Tn residue. Examples of the binding properties of these lectins can be demonstrated by Ricimus communis agglutinin (RCA1), grouped as II specific lectin and its binding property is II > I > B > T; Ahrus precatorius agglutinin (APA), classified as T and its carbohydrate specificity is T > I/II > E > B > Tn; Artocarpus integrifolia (jacalin, AIL), as T/Tn specific and its binding reactivity is T > Tn >> I (II) and Geodia cydonium (GCL), as F/A specific, and with affinity for F > Ah [GalNAcalpha1-->43(L(Fuc)alpha1-->2)Gal] >> I > L. Due to the multiple reactivity of lectins toward mammalian glycotopes, the possible existence of different combining sites or subsites in the same molecule has to be examined, and the differential binding properties of these combining sites (if any) have to be characterized. To establish the relationship among the amino acid sequences of the combining sites of plant lectins and mammalian glycotopes should be an important direction to be addressed in lectinology.

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds.

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [beta-D-Gal-(1-->4)-D-Glc], N-acetyllactosamine [beta-D-Gal-(1-->4)-D-GlcNAc] and lacto-N-biose [beta-D-Gal-(1-->3)-D-GlcNAc].

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3

BackgroundAlthough MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.ResultsMP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction.ConclusionsMP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc(1)Man(7-9)GlcNAc(2)) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates.

Lectin site interaction with capsular polysaccharide mediates nonimmune phagocytosis of type III group B streptococci.

Group B Streptococcus (GBS) causes substantial morbidity but most individuals exposed to the organism remain healthy. These experiments tested the hypothesis that engagement of the complement receptor 3 (CR3) lectin site would effectively trigger neutrophil-mediated phagocytosis of complement-opsonized type III GBS by nonimmune human sera. Using an opsonophagocytosis assay, saccharides identified as interacting with the CR3 lectin site effectively inhibited neutrophil-mediated killing of type III, strain COH1. Fructose, which does not interact with the lectin site, promoted significantly less inhibition of opsonophagocytosis. Saccharide-mediated inhibition was reversed in a dose-related fashion by addition of type III, GBS capsular polysaccharide-specific immunoglobulin G. When capsule-deficient or asialo mutant type III strains were employed, the lectin site was not required. Structurally defined GBS serotypes with a side chain at least two sugars in length engaged the lectin site, and N-acetyl D-glucosamine was not a required component monosaccharide. Intact type III capsular polysaccharide interacted significantly more efficiently with the lectin site than did oligosaccharides representing approximately 5 or 20 repeating units, respectively. Taken together, these experiments indicate that interaction of type III GBS capsular polysaccharide with the lectin site of CR3 effects phagocytosis of these organisms by nonimmune serum. Use of this mechanism of innate immunity provides a potential explanation for the infrequency with which susceptible individuals exposed to type III GBS develop invasive infection.

Mapping of the lectin, ERp57-binding and chaperone sites of calnexin and calreticulin

Mapping of the lectin, ERp57-binding and chaperone sites of calnexin and calreticulin

Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation.

Complement receptor type 3 (CR3) was initially described as an opsonic receptor. Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among them Mycobacterium tuberculosis. Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms. In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasii and Mycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatis and Mycobacterium phlei). A combination of mannan and beta-glucan inhibited the phagocytosis of zymosan but had no effect on M. kansasii ingestion. Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibited M. kansasii phagocytosis. In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan. Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochrome c reduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killed M. kansasii did not. Furthermore, lack of superoxide anion production during phagocytosis of M. kansasii was not due to inhibition of NADPH oxidase per se or superoxide anion scavenging. Together, our results indicate that (i) nonopsonic phagocytosis of zymosan and M. kansasii by CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis.

Functional Relationship between Calreticulin, Calnexin, and the Endoplasmic Reticulum Luminal Domain of Calnexin

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.