Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

A lectin from the Asian horseshoe crabTachypleus tridentatus: purification,specificity and interaction with tumour cells

A lectin from the haemolymph of the Asian horseshoe crabTachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-boundN-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues,N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin theN-acetyl group and the glyceryl side chain ofN-acetylneuraminic acid are important, while presence of an aglycon, specially an -glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin andCollocalia mucin, all beingO-chain glycoproteins but carrying completely different carbohydrate chains. The majority ofN-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the Tachypleus tridentatus agglutinin-receptor which is present on red cells andO-chain glycoproteins.Abbreviations TTA Tachypleus tridentatus agglutinin - SDS sodium dodecyl sulfate - BSM bovine sub-maxillary mucin - VCS Vibrio cholerae sialidase - OSM ovine submaxillary mucin - WGA Wheat germ agglutinin - NeuAc N-acetylneuraminic acid.     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.     

A New Metabolite from Aspergillus versicolor

Neurospora sitophila produced extracellular and cell wall-associated lectins. The addition of l-sorbose to a culture resulted in a decrease in the production of the former lectin and complete abolition of the latter. The lectin in the culture filtrate was purified by bovine submaxillary mucin-conjugated Sepharose chromatography. The molecular weight of the lectin was calculated to be approx. 40,000 by Sephacryl S-200 gel filtration, and that of the subunit to be approx. 22,000 by SDS/polyacrylamide- gel electrophoresis. The lectin was not inhibited by simple sugars or their homopolymers. It was inhibited strongly by glycoproteins from human erythrocyte membrane and bovine submaxillary mucin, and moderately by α1-acid glycoprotein from human plasma, human IgA and IgM, and fetal calf fetuin. The lectin agglutinated human type A, B and O erythrocytes to the same degree. Erythrocytes from chick, horse, rabbit and sheep were more efficiently agglutinated.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.     

Binding properties of Clostridium botulinum type C progenitor toxin to mucins

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.     

Specificity of submaxillary gland sialyltransferases

A method has been developed to determine the activities of specific sialyltransferases by analysis of the products of the reaction. This method, which utilizes high performance liquid chromatography, distinguishes addition of sialic acid to the N-acetylgalactosamine vs. galactose residues of the mucin disaccharide Galβ(1→3)GalNac, and can be used to distinguish formation of the 3′- and 6′-isomers of sialyllactose. For the bovine, ovine, and porcine submaxillary extracts, more than 95% of the activity with asialo ovine submaxillary mucin is due to formation of NeuAc α(2→6)GalNAc. With lactose as the acceptor, more than 95% of the α(2→3) isomer is produced. Activity with asialofetuin is due solely to the O-linked chain, with relative activity toward the galactose vs. GalNAc residues of 0.32, 1.5, and 0.10 for bovine, ovine, and porcine, respectively. The rat submaxillary gland extract showed equal formation of 3′- and 6′-sialyllactose, and very low activity with asialo ovine submaxillary mucin. However, at least 40% of the activity toward the Galβ(1→3)GalNAc disaccharide of asialofetuin was directed toward the GalNAc residue. The relative preference of the N-acetylgalactosaminide α(2→6) sialyltransferase for a monosaccharide vs. a substituted GalNAc may play a role in regulation of chain length during mucin synthesis.     

A d-galactose-binding lectin purified from coronate moon turban,Turbo (Lunella) coreensis,with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids

A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by d-galactose, N-acetyl d-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5–11 and temperature until 50 °C for 1 h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

BackgroundSialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.MethodsNeuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.ResultsClostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.ConclusionsImmobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.General significanceImmobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.     

Characterization of an endo-alpha-N-acetyl galactosaminidase from Diplococcus pneumoniae.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.     

Neuraminidase in Bacteroides fragilis.

A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin.     

Gel filtration of sialoglycoproteins.

The role of sialic acid in the gel-filtration behaviour of sialoglycoproteins was investigated by using the separated isoenzymes of purified human liver alpha-L-fucosidase and several other well-known sialic acid-containing glycoproteins (fetuin, alpha1-acid glycoprotein, thyroglobulin and bovine submaxillary mucin). For each glycoprotein studied, gel filtration of its desialylated derivative gave an apparent molecular weights much less than that expected just from removal of sialic acid. For the lower-molecular-weight glycoproteins (fetuin and alpha1-acid glyocprotein), gel filtration of the sialylated molecules led to apparent molecular weights much larger than the known values. The data indicate that gel filtration cannot be used for accurately determining the molecular weights of at least some sialoglycoproteins.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

Engineering a versatile tandem repeat-type α2-6sialic acid-binding lectin

Previously, we developed an α2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for α2,6-sialylated N-glycans (in the order of 10⁻⁶ M in K_d), almost comparable to a natural α2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.     

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.     

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.