Sialic Acid-specific Lectin Research Articles

Revisiting legume lectins: Structural organization and carbohydrate-binding properties

Legume lectins are a diverse family of carbohydrate-binding proteins that share significant similarities in their primary, secondary, and tertiary structures, yet exhibit remarkable variability in their quaternary structures and carbohydrate-binding specificities. The tertiary structure of legume lectins, characterized by a conserved β-sandwich fold, provides the scaffold for the formation of a carbohydrate-recognition domain (CRD) responsible for ligand binding. The structural basis for the binding is similar between members of the family, with key residues interacting with the sugar through hydrogen bonds, hydrophobic interactions, and van der Waals forces. Variability in substructures and residues within the CRD are responsible for the large array of specificities and enable legume lectins to recognize diverse sugar structures, while maintaining a consistent structural fold. Therefore, legume lectins can be classified into several specificity groups based on their preferred ligands, including mannose/glucose-specific, N-acetyl-d-galactosamine/galactose-specific, N-acetyl-d-glucosamine-specific, l-fucose-specific, and α-2,3 sialic acid-specific lectins. In this context, this review examined the structural aspects and carbohydrate-binding properties of representative legume lectins and their specific ligands in detail. Understanding the structure/binding relationships of lectins continues to provide valuable insights into their biological roles, while also assisting in the potential applications of these proteins in glycobiology, diagnostics, and therapeutics.

Antimicrobial, anti-inflammatory and anti-arthritic activity of hemolymph lectin (NagLec) isolated from the freshwater crab, Oziotelphusanaga

Lectins are non-immune glycoproteins or proteins having a unique capacity to interact with carbohydrate ligands found on the surface of their host cells. In the present investigation, the lectin was purified from the hemolymph of freshwater crab, Oziotelphusa naga and its antimicrobial, anti-inflammatory and anti-arthritic activity was analysed. The preliminary characterization of the hemagglutinin was carried out to identify the erythrocyte and sugar specificity, optimum pH and temperature and cation dependency. The agglutinin was found to be highly specific to rabbit erythrocyte and inhibited by fetuin and α-lactose. Maximum hemagglutination activity was noted at pH 7.5–8 and temperature 20–40 °C. An O-acetyl sialic acid specific 75 kDa hemolymph lectin, designated as NagLec was isolated from the freshwater crab, Oziotelphusa naga by affinity chromatography on fetuin coupled Sepharose 4 B, with a purification fold of 185. The bacteria Staphylococcus aureus, Proteus mirabilis and fungus Candida albicans had the greatest zone of inhibition when treated with NagLec. The results of the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assays showed that the purified lectin inhibited the growth of Staphylococcus aureus at 0.031 and 0.065 μg/ml, which proved the bactericidal property of NagLec. NagLec generated alterations on the bacterial cells and led to protein leakage, which was dosage (24 and 48 μg/ml) and time dependent (10–40 min). COX and LOX enzyme was inhibited to 49.43% and 61.81% with 100 μg/ml concentration of NagLec respectively, demonstrating NagLec's ability to reduce inflammation. Furthermore, NagLec (500 μg) suppressed protein denaturation up to 77.12% whereas diclofenac sodium (a standard drug) was inhibited by 89.36%. The results indicate that NagLec, a sialic acid specific lectin isolated from the freshwater crab O. naga could be formulated as a nano drug in future owing to its antimicrobial, anti-inflammatory and anti-arthritic potential that could be targeted to specific pathogenic microbes and treat arthritis.

Identification and characterization of non-small cell lung cancer associated sialoglycoproteins

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-β were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-β may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SignificanceOur contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

NEU1 is more abundant in uveitic retina with concomitant desialylation of retinal cells.

Desialylation of cell surface glycoproteins carried out by sialidases affects various immunological processes. However, the role of neuraminidase 1 (NEU1), one of the four mammalian sialidases, in inflammation and autoimmune disease is not completely unraveled to date. In this study, we analyzed the retinal expression of NEU1 in equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis. Mass spectrometry revealed significantly higher abundance of NEU1 in retinal Müller glial cells (RMG) of ERU-diseased horses compared to healthy controls. Immunohistochemistry uncovered NEU1 expression along the whole Müller cell body in healthy and uveitic states and confirmed higher abundance in inflamed retina. Müller glial cells are the principal macroglial cells of the retina and play a crucial role in uveitis pathogenesis. To determine whether higher expression levels of NEU1 in uveitic RMG correlate with the desialylation of retinal cells, we performed lectin-binding assays with sialic acid-specific lectins. Through these experiments, we could demonstrate a profound loss of both α2-3- and α2-6-linked terminal sialic acids in uveitis. Hence, we hypothesize that the higher abundance of NEU1 in uveitic RMG plays an important role in the pathogenesis of uveitis by desialylation of retinal cells. As RMG become activated in the course of uveitis and actively promote inflammation, we propose that NEU1 might represent a novel activation marker for inflammatory RMG. Our data provide novel insights in the expression and implication of NEU1 in inflammation and autoimmune disease.

Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818)

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.

The Human Lung Glycome Reveals Novel Glycan Ligands for Influenza A Virus

Glycans within human lungs are recognized by many pathogens such as influenza A virus (IAV), yet little is known about their structures. Here we present the first analysis of the N- and O- and glycosphingolipid-glycans from total human lungs, along with histological analyses of IAV binding. The N-glycome of human lung contains extremely large complex-type N-glycans with linear poly-N-acetyllactosamine (PL) [-3Galβ1–4GlcNAcβ1-]n extensions, which are predominantly terminated in α2,3-linked sialic acid. By contrast, smaller N-glycans lack PL and are enriched in α2,6-linked sialic acids. In addition, we observed large glycosphingolipid (GSL)-glycans, which also consists of linear PL, terminating in mainly α2,3-linked sialic acid. Histological staining revealed that IAV binds to sialylated and non-sialylated glycans and binding is not concordant with respect to binding by sialic acid-specific lectins. These results extend our understanding of the types of glycans that may serve as binding sites for human lung pathogens.

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization.

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.

Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay.

Cerebrospinal fluid (CSF) leakage can lead to brain and spine pathologies and there is an urgent need for a rapid diagnostic method for determining CSF leakage. Beta-2 transferrin (β2TF), asialotransferrin, is a specific CSF glycoprotein biomarker used to determine CSF leakage when distinguished from serum sialotransferrin (sTF).Methods: We detected β2TF using an immunochromatographic assay (ICA), which can be potentially developed as a point-of-care (POC) testing platform. Sialic acid-specific lectin selectively captures sTF in multiple deletion lines within an ICA test strip, enabling the detection of β2TF. A sample pre-treatment process efficiently captures excess sTF increasing sensitivity for CSF leakage detection.Results: An optimal cut-off value for determining the presence of CSF in test samples was obtained from receiver operating characteristic (ROC) analysis of the ratio of the test signal intensity and the deletion lines. On 47 clinical samples, ICA test strips discriminated CSF positive from negative samples with statistically significant (positive versus negative t-test; P =0.00027). Additional artificial positive samples, prepared by mixing CSF positive and negative clinical samples, were used as a further challenge. These positive samples were clearly discriminated from the negative samples (mixture versus negative t-test; P =0.00103) and CSF leakage was determined with 97.1% specificity and 96.2% sensitivity.Conclusions: ICA represents a promising approach for POC diagnosis of CSF leakage. While requiring 70 min assay time inconvenient for POC testing, our method was significantly shorter than conventional electrophoresis-based detection methods for β2TF.

Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

Immune response of anti-lectin Pjlec antibody in freshwater crab Paratelphusa jacquemontii

Sialic acid specific lectin Pjlec isolated from serum of the freshwater crab Paratelphusa jacquemontii served as an antigen for the production of immunoglobulin (Ig) in Balb/c mice sera. Enzyme-linked immunosorbent assay (ELISA) of mice anti-sera with Pjlec lectin affirmed the induction and production of antibody. Anti-Pjlec antibody was isolated from the antisera of mice by Protein A Sepharose affinity chromatography and checked for purity by immunoblot with lectin. Mass spectrometry (MS/MS) of papain digethe peptide sequence of antigen binding fragment (Fab) and fragment crystallizable (Fc). Coatingsted anti-Pjlec revealed of anti-Pjlec to the target cell, rabbit erythrocyte failed to enhance in vitro phagocytosis in the crab. However, inoculation of anti-Pjlec in the hemolymph of the crab elicited in vitro phagocytosis. Proteins in hemocyte lysate supernatant (HLS) were separated by electrophoresis failed to immunoblot with Pjlec or anti-Pjlec. Peptide sequences of trypsin digested lectin protein appeared homologous to deuterostome chordate. The protostome crab that lack the ability to synthesize sialic acid however bind to sialic acid a deuterostome sugar to suggest the complexity in innate immune system of invertebrates. The application of lectin and its antibody require further study on application of pathological conditions associated with alterations in sialylated cell surface.

Rational Design and Development of Polysialic Acid-Binding Peptides

Polysialic acid (PSA) is a unique post-translational modification found on the neural cell adhesion molecule (NCAM) and plays an important physiological role in nervous system development through modulation of neuronal interactions. While the size and hydrophilicity of PSA result in a large excluded volume that serves its non-adhesive properties well, a number of proteins act as specific ligands to PSA, including Siglec-11 – a sialic-acid specific lectin that recognizes alpha-2,8-linked N-acetylneuraminic acid. Understanding of biomolecular interactions involving PSA may be applied in the targeting of PSA in biotechnological applications, as for the development of ligands, such as peptides, to serve as biosensors, modulators in infections caused by PSA-expressing microbes, or novel affinity agents for neural stem cell targeting. In addition to being small, relatively stable molecules, peptides offer the possibility to study and exploit natural interactions as the basis for rational design. In this work, we demonstrate the development of PSA-binding peptides through rational design, beginning with epitope mapping of Siglec-11 for understanding of PSA-lectin interaction regions and identification of lead candidates. High-throughput synthesis and screening of peptide libraries, with screening in a spatially addressable manner on peptide microarrays, enabled study of peptide-PSA binding interactions. Lead peptides isolated show overlap with the binding pocket of Siglec-11, as evident from molecular dynamics (MD) simulations of PSA with a homology model of the Siglec-11 N-terminal domain. Peptides were further studied through mutational analyses for examination of critical binding residues and for understanding the affinity and selectivity of PSA-peptide interactions. Binding data from microarray studies were corroborated with surface plasmon resonance (SPR) spectroscopy and fluorescence anisotropy assays. Library level data, combined with MD simulations of peptide-PSA interactions, suggest the importance of spatial positioning along with identity of peptide residues. That is, while the electrostatic component of binding is high, as expected, the geometry of residues plays an important role and hence offers potential for development of selectivity. For purposes of comparison to our natural-partners based rational approach, phage display screening of a random peptide library was also conducted and lead peptides were studied through mutational analyses as above. The approach used in development and study of PSA-binding peptides thus contributes to the understanding of carbohydrate-peptide interactions and has implications for small-molecule targeting of specific glycosylation patterns in disease-related applications.

Sialic acid-specific lectin participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus merguiensis.

A sialic acid-specific lectin was purified from the hemolymph of Fenneropenaeus merguiensis by repetitive affinity fetuin-agarose column chromatography. The purified F. merguiensis lectin (called FmL) consisted of two distinct 30.9 and 32kDa subunits with identical N-terminal amino acid sequences of ten residues. FmL was also composed of sugar moieties; glucosamine, glucose, mannose and N-acetyl neuraminic acid but not N-glycolyl neuraminic acid. It was postulated to be a glycoprotein as it was positively stained by glycoprotein staining kit and detected by some bionylated plant lectins. Deglycosylation by either peptide N-glycosidase F or trifluoromethanesulfonic acid turned both types of FmL subunits to 28kDa peptides. The internal peptide sequence of FmL was similar to a fibrinogen-related domain of human ficolin and the horseshoe crab lectin. Determination of the lectin concentrations in the hemolymph was performed by ELISA while its hemaglutinating activity (HA) was tested by hemagglutination. Both specific lectin concentrations and HA increased as shrimp developed ovarian maturation stages 2 to 4. Their constitutive levels were found in pre-vitellogenic females and higher than those of males. Both specific lectin concentrations and HA of FmL were inducible to the highest levels at 12h after F. merguiensis was challenged by pathogenic Vibrio harveyi. The FmL-induced agglutination of V. harveyi was specifically abolished by sialic acid, fetuin and bacterial cell wall components. These findings might indicate the implication in an immune response of FmL to protect the shrimp themselves or their spawning eggs towards pathogenic bacteria in surrounding environment.

Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues.

Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. Animal experiment. Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA) strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues) specific Griffonia simplicifolia-1 (GSL-1) was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc) specific wheat germ agglutinin (WGA) was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells.

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid.

In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; β-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (P<0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.

Application and efficacy test of siRNA vaccine against nervous necrosis virus infection

Sialic acid specific lectin Pjlec isolated from serum of the freshwater crab Paratelphusa jacquemontii served as an antigen for the production of immunoglobulin (Ig) in Balb/c mice sera. Enzyme-linked immunosorbent assay (ELISA) of mice anti-sera with Pjlec lectin affirmed the induction and production of antibody. Anti-Pjlec antibody was isolated from the antisera of mice by Protein A Sepharose affinity chromatography and checked for purity by immunoblot with lectin. Mass spectrometry (MS/MS) of papain digethe peptide sequence of antigen binding fragment (Fab) and fragment crystallizable (Fc). Coatingsted anti-Pjlec revealed of anti-Pjlec to the target cell, rabbit erythrocyte failed to enhance in vitro phagocytosis in the crab. However, inoculation of anti-Pjlec in the hemolymph of the crab elicited in vitro phagocytosis. Proteins in hemocyte lysate supernatant (HLS) were separated by electrophoresis failed to immunoblot with Pjlec or anti-Pjlec. Peptide sequences of trypsin digested lectin protein appeared homologous to deuterostome chordate. The protostome crab that lack the ability to synthesize sialic acid however bind to sialic acid a deuterostome sugar to suggest the complexity in innate immune system of invertebrates. The application of lectin and its antibody require further study on application of pathological conditions associated with alterations in sialylated cell surface.

Use of Spectroscopic Techniques to Reveal the Nature of the Interactions of Two Sialic Acid Specific Lectins with Gold Nanoparticles.

From UV-vis absorption, steady state and time resolved fluorescence measurements coupled with circular dichroism (CD) spectral studies, it was revealed that among the two lectins: Sambucus nigra agglutinin (SNA) and Saraca indica (saracin II), SNA forms stronger binding complex in the ground state with gold nanoparticles (GNPs). From the measurements of Stern-Volmer (SV) constants Ksv, and binding constants K(A) and number of binding sites two important inferences could be drawn. Firstly, the fluorescence quenching is primarily due to static quenching and secondly SNA forms stronger binding with GNPs relative to the other lectin saracin II. Synchronous fluorescence spectral measurements further substantiate this proposition of exhibiting the fully exposed tryptophan residue in case of SNA. It appears that the lectin SNA adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with GNPs. CD measurements demonstrate that gold nanoparticles when interact with the lectins (glycoproteins), no significant distortion in the structural pattern of the later occurs. The unaltered identity in the secondary structural pattern of both SNA and saracin II in presence of gold nanoparticles hints that GNPs may be used as useful drug or drug delivery systems.

Opsonic function of sialic acid specific lectin in freshwater crab Paratelphusa jacquemontii.

The sialic acid specific humoral lectin, Pjlec of the freshwater crab Paratelphusa jacquemontii was investigated for its opsonin function with rabbit erythrocyte as target cell for phagocytosis by the crab’s hemocyte. The untreated or trypsin treated erythrocyte induced lectin response after challenge however failed when treated with neuraminidase evidently indicating glycan dependency for elicited immune response. Our observation of in vitro phagocytosis of the erythrocyte untreated or coated with serum, clarified serum appeared to be recognized and engulfed by hemocytes but when coated with isolated lectin Pjlec, the response was elicited. Moreover, with trypsin treated erythrocyte the response remained unchanged but neuraminidase or O-glycosidase treatment eliminated the response reaction. This suggested the sialic acid specific reaction of lectin with the erythrocyte and was essential for recognition to allow the lectin Pjlec to act as an opsonin. The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte. The efficiency of in vitro hemolysis of Pjlec coated erythrocyte with hemocyte when compared to untreated erythrocyte with or without hemocyte also established the opsonic function of the lectin. The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface. The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316.

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

Comparison of the nature of interactions of two sialic acid specific lectins Saraca indica and Sambucus nigra with N-acetylneuraminic acid by spectroscopic techniques

The present paper deals with the isolation and purification of a new sialic acid binding lectin from the seed integument of Saraca indica (Ashok) and the purified lectin was designated Saracin II. Comparative studies on the interactions of saracin II and another sialic acid specific lectin Sambucus nigra agglutinin (SNA) with N-acetylneuraminic acid (NANA) were made using UV–vis absorption, steady state and time resolved fluorescence along with circular dichroism (CD) spectroscopy to reveal the nature and mechanisms of binding of these two lectins with NANA. The experimental observations obtained from UV–vis, steady state and time resolved fluorescence measurements demonstrated that SNA–NANA system formed relatively stronger ground state complex than saracin II–NANA pair. CD measurements further substantiated the propositions made from steady state and time resolved spectroscopic investigations. It was inferred that during interaction of SNA with NANA, the lectin adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with NANA.

Occurrence of novel Cu(2+)-dependent sialic acid-specific lectin, on the outer surface of mature caprine spermatozoa.

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu(2+)) ion (300 μM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 μM concentration. However, asialofetuin is not capable of inhibiting this Cu(2+)-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu(2+) ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu(2+) dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu(2+)-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.