Purification and Characterization of Vitellin from the Egg of the Suminoe Oyster Crassostrea ariakensis and Cross-Reactivity of Anti-vitellin Antibody with Other Marine Invertebrate Egg Proteins.

A polyclonal antibody specific to an egg protein of Suminoe oyster Crassostrea ariakensis was previously developed in our laboratory to assess the reproductive life cycle of the oyster. The present study was undertaken to investigate vitellin of C. ariakensis (CAVt). Vitellin is an essential component of egg proteins in marine invertebrates as it provides energy and nutrients to the embryo and larvae. CAVt was purified from eggs of the oyster using ammonium sulfate precipitation followed by affinity chromatography with Concanavalin A-agarose. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE showed that CAVt is a high molecular weight [532 kiloDaltons (kDa)] protein, with multiple subunits. Similar to other vitellin proteins, it is a phospholipoglycoprotein composed of phospholipids (12.06%), carbohydrates (mannose, 10.08% or glucose, 9.84%), and alkali-labile phosphates (4.16%). Affinity chromatography, enzyme-linked immunosorbent aasay (ELISA) and western blot analysis revealed that CAVt is only present in the ovary, and two subunits of CAVt (72 and 35 kDa) are believed to be incorporated from the hemolymph into the oocyte. The antibody specific to CAVt (anti-CAVt), raised in rabbit, strongly cross reacted with the egg proteins of oyster species and scallops, suggesting that the antigenic epitopes are highly conserved among species. Our results suggest that the anti-CAVt antibody can be used to develop a tool similar to ELISA or western blotting for investigation of the effect of microorganisms on reproduction as well as the effect of chemicals on the endocrine system in C. ariakensis.

Gal/GalNAc specific multiple lectins in marine bivalve Anadara granosa.

Complete lectin mapping of molluscs with their diversified recognition pattern and possible role in lectin-carbohydrate interaction based immune response triggering need much attention. In this communication, Gal/GalNAc specific three lectins AGL-IA (Anadara granosa lectin-IA), AGL-IB (A. granosa lectin-IB) and AGL-IV (A. granosa lectin-IV) and a lectin having hemolytic activity AGL-III (A. granosa lectin-III) were purified from the plasma of A. granosa bivalve by a combination of gel filtration and affinity chromatography. AGL-IA and IB were oligomeric lectins whereas, AGL-III and IV were monomeric. The molecular weight of AGL-IA, IB, III and IV were 375, 260, 45 and 33 kDa respectively. AGL-IA and IV agglutinated both rabbit and pronase treated human erythrocytes, whereas AGL-IB agglutinated only rabbit erythrocytes. AGL-III was found to agglutinate rabbit erythrocytes, however, it caused hemolysis of pronase treated human erythrocytes. The activity of all four lectins was calcium dependent and maximum at a pH range 7-8. Apart from Gal/GalNAc specific, the four lectins showed substantial differences in their carbohydrate recognition pattern. Moreover, there was a difference in the carbohydrate specificity between AGL-III and other three lectins (AGL-IA, AGL-IB and AGL-IV) towards polyvalent glycotope. On the one hand, 'cluster glycoside effect' i.e., an enhancement of the activity of a multivalent ligand, was observed for carbohydrate specificities of AGL-IA, AGL-IB, AGL-IV. On the other hand, the effect of multivalent ligands on the carbohydrate specificity of AGL-III was opposite of cluster glycoside effect. The affinity of AGL-IA, AGL-IB and AGL-IV for ligands can be ranked as follows: glycoproteins >> polysaccharide > oligosaccharides and monosaccharides. However, Gal related monosaccharides were the best inhibitors of AGL-III and the inhibitory activity decreased gradually in the following order: monosaccharide > disaccharide > polysaccharide. Thus, the diverse specificity of multiple lectins in A. granosa plasma possibly enables to recognize a wide range of microorganisms.

Detection of Hanganutziu-Deicher antigens in O-glycans from pig heart tissues by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: In the α1,3-galactosyltransferase knockout (α-GalT KO) pig era, identification of the non-Gal epitopes is necessary for successful pig-to-human xenotransplantation. Recently, we successfully detected α-Gal epitopes as well as Hanganutziu-Deicher (H-D) antigens from the N-glycans in the pig heart tissues, which have been considered as promising non-Gal antigens. However, the profiling of O-glycan from pig heart tissues had not been performed owing to the difficulty of O-glycan preparation. METHODS: In this study, we established the simple and sensitive method to profile O-glycans from pig heart aortic valve, aortic wall, pulmonary valve, pulmonary wall, and cardiac muscle tissues. To liberate O-glycans from the pig heart tissues, we used non-reductive ß-elimination reagent and subsequently purified the glycans. After permethylation, the glycans were qualitatively analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The comprehensive O-glycan analysis method was successfully validated using model glycoproteins such as bovine serum fetuin (BSF) and bovine submaxillary gland mucin (BSM) glycoproteins, and their O-glycan profiles were in accordance with the data of previous studies. Next, we applied the method for O-glycan release and characterization to analysis of various pig heart tissues. As a result, total 39, 33, 24, 36, and 25 of O-glycans were detected from aortic valve, aortic wall, pulmonary valve, pulmonary wall, and cardiac muscle, respectively. Furthermore, four in aortic valve, one in aortic wall, one in pulmonary valve, one in pulmonary wall, and one in cardiac muscle were particularly determined as terminally N-glycolylneuraminic acid-linked O-glycans, which is considered to be the H-D antigens. CONCLUSIONS: Here, we initially described the O-glycan structures of various pig heart tissues, and additionally, the existence of H-D antigen type O-glycans was firstly identified. These results will be fundamental information for overcoming the xenoantigenic carbohydrate-related immunological rejection in pig-to-human heart tissue xenotransplantation.

The sweets standing at the borderline between allo- and xenotransplantation.

Animal cells are densely covered with glycoconjugates, such as N-glycan, O-glycan, and glycosphingolipids, which are important for various biological and immunological events at the cell surface and in the extracellular matrix. Endothelial α-Gal carbohydrate epitopes (Galα3Gal-R) expressed on porcine tissue or cell surfaces are such glycoconjugates and directly mediate hyperacute immunological rejection in pig-to-human xenotransplantation. Although researchers have been able to develop α1,3-galactosyltransferase (GalT) gene knockout (KO) pigs, there remain unclarified non-Gal antigens that prevent xenotransplantation. Based on our expertise in the structural analysis of xenoantigenic carbohydrates, we describe the immunologically significant non-human carbohydrate antigens, including α-Gal antigens, analyzed as part of efforts to assess the antigens responsible for hyperacute immunological rejection in pig-to-human xenotransplantation. The importance of studying human, pig, and GalT-KO pig glycoprofiles, and of developing adequate pig-to-human glycan databases, is also discussed.

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum).

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.

Macoma birmanica agglutinin recognizes glycoside clusters of beta-GlcNAc/Glc and alpha-Man.

Macoma birmanica agglutinin (MBA) that seems to play crucial roles in the innate immunity of marine bivalve, M. birmanica has been earlier defined as GlcNAc/Man specific. However, most complementary carbohydrate structures to its binding domain and ligand clustering in its recognition profile have not been established. In this study, the complete recognition profile of MBA was examined by enzyme-linked lectin-sorbent assay and inhibition assay. Among the monosaccharides tested, GlcNAc was more reactive followed by Man and Glc, others were non-reactive; revealing the importance of equatorial -NAc group at C-2, -OH group at C-4 and C-6, and pyranose conformation of hexose. Moreover, beta-glycosides of GlcNAc and Glc were more potent whereas for Man it was alpha-glycoside. MBA recognized both exposed and internal alpha-Man and beta-GlcNAc/Glc residues well with most linkages except (beta1-4). This binding pattern was further extended and confirmed by polyvalent glycoside clusters of GlcNAc(beta1-2)Man(alpha1-, which was a better inhibitor than Man(alpha1-2/3/6)Man(alpha1- or Man(alpha1-3/6)Man(beta1- present in well-defined naturally occurring glycoproteins. This broad range specificity explains the importance of MBA as an important pattern recognition molecule that provides more realistic picture of carbohydrate-based immune response triggering.

Purification and characterization of an N-acetylglucosamine specific lectin from marine bivalve Macoma birmanica.

A calcium independent lectin of molecular mass 47kDa was isolated from the foot muscle of marine bivalve Macoma birmanica by ammonium sulphate precipitation followed by affinity chromatography on immobilized GlcNAc column and designated as M. birmanica agglutinin (MBA). The lectin agglutinated rabbit erythrocytes strongly compared to human erythrocytes over a wide pH range from 5 to 9 and up to 50 degrees C. MBA is a glycoprotein and consists of 7.63% sugar. Among the tested sugars for analysis of carbohydrate recognition properties, Me-betaGlcNAc was the most potent inhibitor followed by Me-alphaMan. Enzyme linked solid phase assay revealed that MBA interacted well with complex type N-linked glycans and moderately to high mannose type N-linked glycans. Fluorescence study of MBA indicated that tryptophan was present in a non-hydrophobic region and its binding to GlcNAc was neither quenched nor altered lambda(max) position. The denaturation of MBA induced by urea was a reversible process and urea could not significantly change the Trp environment. MBA interacted with both Gram-positive and Gram-negative bacteria by recognizing their surface exposed GlcNAc containing antigens.

Characterization, tissue expression, and immunohistochemical localization of MCL3, a C-type lectin produced by Perkinsus olseni-infected Manila clams (Ruditapes philippinarum).

A novel C-type lectin designated Manila clam lectin 3 (MCL3), with a molecular weight of 17.380kDa, was identified among haemocyte expressed sequence tags of Perkinsus olseni-infected Manila clams. MCL3 was expressed in Escherichia coli M15 cells and purified with a Ni-NTA His-binding resin matrix. MCL3 agglutinated rabbit erythrocytes in the presence of Ca(+2). MCL3-induced agglutination was partially inhibited by GalNAc, Man, lactose, and raffinose, whereas the polysaccharides bovine mucin type II and Candida mannan completely inhibited agglutination. MCL3 was expressed in the haemocytes of Manila clams 3 days after infection with P. olseni and 1 day after infection with Vibrio tapetis. Based on real-time PCR analysis, mRNA transcripts of MCL3 were present in the adductor, foot, gill, mantle, palp, and siphon of Perkinsus-infected Manila clams. Furthermore, MCL3 was detected in the foot, gill, mantle, gonad, digestive gland, gonad connective tissue, intestine, and stomach by immunohistochemical localization.

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes.

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>>>>monovalent Tn > or = Me-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits.

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.