Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes. Results: Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt. Conclusion: Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.

The modes of binding methyl-alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A--a theoretical approach

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A: synthesis, thermodynamic and NMR spectroscopy studies

α-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl α-d-mannopyranoside (Ka values in the range of 104 M−1). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1–3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.

Chemical modification studies on Abrus agglutinin Involvement of tryptophan residues in sugar binding

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydratebinding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2- hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

Unusual structural stability and ligand induced alterations in oligomerization of a galectin

Abstract L-14, a 14-kDa S-type lectin shows the jelly roll tertiary structural fold akin to legume lectins yet, unlike them, it does not dissociate on thermal unfolding. In the absence of ligand L-14 displays denaturation transitions corresponding to tetrameric and octameric entities. The presence of complementary ligand reduces the association of L-14, which is in stark contrast with legume lectins where no alterations in quaternary structures are brought about by saccharides. From the magnitude of the increase in denaturation temperature induced by disaccharides the binding constants calculated from differential scanning calorimetry are comparable with those extrapolated from titration calorimetry indicating that L-14 interacts with ligands essentially in the folded state.

Studies on the variants of the protein toxins ricin and abrin

his study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

Further characterization of the saccharide specificity of peanut (Arachis hypogaea) agglutinin

2-Dansylamino-2-deoxy-D-galactose (GalNDns) has been shown to bind to peanut (Arachis hypogaea) agglutinin (PNA) in a saccharide-specific manner. This binding was accompanied by a five-fold increase in the fluorescence of GalNDns. The interaction was characterized by an association constant of 0.15 mM at 15° and ΔH and ΔS values of -57.04 kJ·mol-1 and -118.1 J·mol-1.K-1, respectively. Binding of a variety of other mono-, di- and oligo-saccharides to PNA, studied by monitoring their ability to dissociate the PNA-GalNDns complex, revealed that PNA interacts with several T-antigen-related structures, such as β-d-Galp-(1→3)-D-GalNAc, β-D-Galp-(1→3)-α-D-GalpNAcOMe, and β-D-Galp-(1→3)-α-D-GalpNAc(1→3)-Ser, as well as the asialo-G(M1) tetrasaccharide, with comparable affinity, thus showing that this lectin does not discriminate between saccharides in which the penultimate sugar of the β-D-Galp-(1→3)-D-GalNAc unit is the α or β anomer, in contrast to jacalin (Artocarpus integrifolia agglutinin), another anti T-lectin which preferentially binds to β-D-Galp-(1→3)-α-D-GalNAc and does not recognize β-D-Galp-(1→3)-β-D-GalNAc or the related asialo-G(M1) oligosaccharide. These studies also indicated that, in the extended combining region of PNA which accommodates a disaccharide, the primary subsite (subsite A) is highly specific for D-galactose, whereas the secondary subsite (subsite B) is less specific and can accommodate various structures, such as D-galactose, 2-acetamido-2-deoxy-D-galactose, D-glucose, and 2-acetamido-2-deoxy-D-glucose.

Cloning and sequencing of winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBA I): presence of second glycosylation site and its implications in quaternary structure

We report cloning of the DNA encoding winged bean basic agglutinin (WBA I). Using oligonucleotide primers corresponding to N- and C-termini of the mature lectin, the complete coding sequence for WBA I could be amplified from genomic DNA. DNA sequence determination by the chain termination method revealed the absence of any intervening sequences in the gene. The DNA deduced amino acid sequence of WBA I displayed some differences with its primary structure established previously by chemical means. Comparison of the sequence of WBA I with that of other legume lectins highlighted several interesting features, including the existence of the largest specificity determining loop which might account for its oligosaccharide-binding specificity and the presence of an additional N-glycosylation site. These data also throw some light on the relationship between the primary structure of the protein and its probable mode of dimerization.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

Structure, dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X-ray studies

Molecular dynamics simulations have been carried out on all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations, and the scatter of the locations of carbohydrate and carbohydrate-binding residues are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands, and the complexes indicate a combination of conformational selection and induced fit. Proteins 2009; 77:760-777.

Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.

Immune mechanisms in atherosclerosis; Focus on the role of the complement system

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the inner layer of the arterial wall. During atherogenesis, various structures that are recognized as non-self by the immune system, such as modified lipoproteins, are deposited in the arterial wall. Accordingly, atherosclerotic lesions and blood of humans and animals with atherosclerotic lesions show signs of activation of both innate and adaptive immune responses. Although immune attack is initially a self-protective reaction, which is meant to destroy or remove harmful agents, a chronic inflammatory state in the arterial wall accelerates atherosclerosis. Indeed, various modulations of the immune system of atherosclerosis-prone animals have provided us with convincing evidence that immunological mechanisms play an important role in the pathogenesis of atherosclerosis. This thesis focuses on the role of complement system, a player of the innate immunity, in atherosclerosis. Complement activation via any of the three different pathways (classical, alternative, lectin) proceeds as a self-amplifying cascade, which leads to the generation of opsonins, anaphylatoxins C3a and C5a, and terminal membrane-attack complex (MAC, C5b-9), all of which regulate the inflammatory response and act in concert to destroy their target structures. To prevent uncontrolled complement activation or its attack against normal host cells, complement needs to be under strict control by regulatory proteins. The complement system has been shown to be activated in atherosclerotic lesions, modified lipoproteins and immune complexes containing oxLDL, for instance, being its activators. First, we investigated the presence and role of complement regulators in human atherosclerotic lesions. We found that inhibitors of the classical and alternative pathways, C4b-binding protein and factor H, respectively, were present in atherosclerotic lesions, where they localized in the superficial proteoglycan-rich layer. In addition, both inhibitors were found to bind to arterial proteoglycans in vitro. Immunohistochemical stainings revealed that, in the superficial layer of the intima, complement activation had been limited to the C3 level, whereas in the deeper intimal layers, complement activation had proceeded to the terminal C5b-9 level. We were also able to show that arterial proteoglycans inhibit complement activation in vitro. These findings suggested to us that the proteoglycan-rich layer of the arterial intima contains matrix-bound complement inhibitors and forms a protective zone, in which complement activation is restricted to the C3 level. Thus, complement activation is regulated in atherosclerotic lesions, and the extracellular matrix is involved in this process. Next, we studied whether the receptors for the two complement derived effectors, anaphylatoxins C3a and C5a, are expressed in human coronary atherosclerotic lesions. Our results of immunohistochemistry and RT-PCR analysis showed that, in contrast to normal intima, C3aR and C5aR were highly expressed in atherosclerotic lesions. In atherosclerotic plaques, the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR, and a small fraction of them also expressed C3aR, mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. These results suggested that intimal cells can respond to and become activated by complement-derived anaphylatoxins. Finally, we wanted to learn, whether oxLDL-IgG immune complexes, activators of the classical complement pathway, could have direct cellular effects in atherogenesis. Thus, we tested whether oxLDL-IgG immune complexes affect the survival of human monocytes, the precursors of macrophages, which are the most abundant inflammatory cell type in atherosclerotic lesions. We found that OxLDL-IgG immune complexes, in addition to transforming monocytes into foam cells, promoted their survival by decreasing their spontaneous apoptosis. This effect was mediated by cross-linking Fc receptors with ensuing activation of Akt-dependent survival signaling. Our finding revealed a novel mechanism by which oxLDL-IgG immune complexes can directly affect the accumulation of monocyte-macrophages in human atherosclerotic lesions and thus play a role in atherogenesis.

A kinetic analysis of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside antigen—lectin interaction

A kinetic study of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (T-antigen) with lectin peanut agglutinin is described. The disaccharide antigen was synthesized by chemical methods and was functionalized suitably for immobilization onto a carboxy-methylated sensor chip. The ligand immobilized surface was allowed interaction with the lectin peanut agglutinin, which acted as the analyte and the interaction was studied by the surface plasmon resonance method. The ligand—lectin interaction was characterized by the kinetic on-off rates and a bivalent analyte binding model was found to describe the observed kinetic constants. It was identified that the antigen-lectin interaction had a faster association rate constant (k a1) and a slower dissociation rate constant (k d1) in the initial binding step. The subsequent binding step showed much reduced kinetic rates. The antigen-lectin interaction was compared with the kinetic rates of the interaction of a galactopyranosyl-(1→4)-β-d-galactopyranoside derivative and a mannopyranoside derivative with the lectin.