An overview on human serum lectins.

An extensive literature survey done on the various naturally occurring lectins in human serum upon its salient features such as methods of detection, level and sites of synthesis, binding specificity, cation dependency, modes of isolation, molecular and functional characterization way back from 1930s to till date was presented in a tabulated section. In addition, the generation of lectin and other immune molecules in vertebrates upon treatment with exogenous elicitors has also been framed in a tabular form. Furthermore, ANEW lectin induced in human serum for the very first time by an exogenous elicitor was detected, isolated and characterized by us whose features are also tabulated explicitly.

Isolation, purification and characterization of beta-1,3-glucan binding protein from the plasma of marine mussel Perna viridis.

A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.

Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis.

A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B. The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds. The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin. The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D. affinis. In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation. Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of D. affinis agglutinin 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.

Activation of prophenoloxidase in the plasma and haemocytes of the marine mussel Perna viridis Linnaeus.

Phenoloxidase activity was detected in plasma and haemocytes of the marine mussel Perna viridis. This enzyme exists as a proenzyme, prophenoloxidase (proPO), in both these haemolymph fractions and could be activated in vitro by exogenous proteases (trypsin and alpha-chymotrypsin) and a detergent (sodium dodecyl sulphate). In addition, laminarin (a polymer of beta-1,3 glucan) and bacterial lipopolysaccharides (LPSa) effectively triggered proPO activation in these haemolymph fractions. The activation of proPO by non-self molecules was dependent upon calcium ions at a low concentration. This activation process appeared to involve a limited proteolysis, since serine protease inhibitors (soybean trypsin inhibitor, benzamidine or p-nitrophenyl-p'-guanidinobenzoate) suppressed conversion of proPO to the active enzyme. This study demonstrates the selective response of plasma and haemocytic proPO to activation by different types of bacterial LPS tested and suggests that proPO system in both plasma and haemocytes of P. viridis serves an important function in non-self recognition and host immune reactions.

A lipopolysaccharide-binding hemagglutinin with specificity for acetylated aminosugars in the serum of the hermit crab Diogenes affinis (Henderson).

A naturally occurring hemagglutinin was detected in the serum of the hermit crab Diogenes affinis, and its erythrocyte (RBC) binding activities, physicochemical properties, and carbohydrate binding specificity were characterized. Both the hemagglutination profile and the pattern of cross-reactivity of the serum with different RBC types in cross-adsorption tests suggested a strong affinity of the serum agglutinin for rat RBC. Further analysis revealed that the agglutinin was specifically dependent on Ca2+ for its hemagglutinating activity and reversibly sensitive to EDTA. The activity was found to be stable between pH 6.0 and 7.5, heat-labile, and completely precipitable by ammonium sulphate or TCA, suggesting the proteinaceous nature of the serum agglutinin. In hemagglutination-inhibition assays, the serum agglutinin of D. affinis showed a distinct and unique specificity for acetyl group-containing carbohydrates and glycoprotein. Furthermore, the hemagglutinating activity of the serum agglutinin was also inhibited by lipopolysaccharide from Salmonella abortus equi, which might indicate a significant role of humoral agglutinin in the immune response of crustaceans against bacterial infection.

Characterization of a natural hemagglutinin in the serum of a freshwater crab Parathelphusa hydrodromus (Herbst).

A naturally occurring hemagglutinin (HA) was detected in the serum of the freshwater crab Parathelphusa hydrodromus using mammalian erythrocytes (RBC) as indicator cells. The serum gave the highest HA titer with rabbit RBC. In cross adsorption tests, this RBC type completely adsorbed all HA activities from serum. An analysis of the physico-chemical properties of HA showed it to be specifically dependent on the presence of Ca2+ for its activity, irreversibly sensitive to EDTA, stable between pH 7.5 and 10.0, and heat-labile. Further studies demonstrated that the HA is proteinaceous as it was precipitable by conventional deproteinizing agents, and susceptible to the action of proteases and 2-mercaptoethanol. HA-inhibition assays performed with 44 carbohydrates revealed that the serum HA was specific for non-reducing terminal glucose with alpha 1-2 glycosidic linkage. Thus this agglutinin appears to be unique among all the known crustacean agglutinins.

Comparative analysis of agglutinins from hemolymph and albumin gland of Helix pomatia.

The hemolymph of Helix pomatia contains a weak agglutinating activity. This lectin concentration was calculated to be about 1.8 micrograms.ml-1. Among the different red blood cells tested, pronase-treated sheep erythrocytes were found to be the most suitable indicator cells. Their agglutination could be inhibited by GalNAc and GlcNAc. The serum agglutinin was isolated by affinity chromatography using Sephadex G-200 as the matrix. It exhibited a single band in discontinuous PAGE. In the presence of SDS, subunits of 27,000 daltons were obtained which, after addition of 2-mercaptoethanol, partly dissociated into 13,000-dalton subunits. The biochemical properties observed were compared with those of the well-known blood group A-specific lectin from the albumin gland of H. pomatia.

Lectin-dependent recognition of foreign cells by hemocytes of the mussel, Mytilus edulis.

Phagocytosis of human erythrocytes (rbc) by hemocytes of the mussel Mytilus edulis was found to be influenced by four heterologous lectins. The effects were examined in the absence of Ca++ ions under three experimental conditions: when the lectins were bound to 1) both hemocytes and rbc, 2) only hemocytes, but not to rbc, and 3) only rbc, but not to hemocytes. The lectins used included: albumen gland agglutinin from Helix pomatia (HPA), wheat germ agglutinin (WGA), Ricinus-120 (Ric-120) and Concanavalin-A (Con A). HPA, WGA and Ric-120, for which both hemocytes and A-rbc possess receptors, strongly enhanced uptake of A-rbc. This lectin-mediated phagocytosis was abolished by addition of specific sugars either to lectin-pretreated rbc (HPA, WGA) or to a pretreated hemocyte monolayer (Ric-120); this indicated the stimulation of phagocytosis by the binding of lectin to carbohydrate determinants at the surface of hemocytes and target cells. On the other hand, HPA which binds to hemocytes, but not to O-rbc, did not influence phagocytosis of these rbc; and Con A which binds to A-rbc, but not to hemocytes, also failed to stimulate phagocytosis. These findings reveal the importance of carbohydrate determinants on the surface of hemocytes as well as on target cells in recognition and in lectin-mediated phagocytosis of foreign cells by Mytilus hemocytes.

Crustacean defense strategies II. Recognition, clearance, accumulation and externalization of soluble foreign proteins by the mud-crab Scylla serrata.

The Defense reactions of Scylla serrata have been investigated by challenging crabs with two metalloproteins, horse radish peroxidase (HRP:M.W. 40,000) and hemoglobin (Hb:64,000). The defense response analyzed include 1) recognition of foreign proteins; 2) clearance from the haemocoel; 3) accumulation in tissues and 4) externalization. Protein clearance was similar to dye clearance in several respects: 1) clearance rate increased with higher a) molecular weight and concentration and b) at night and 2) decreased with crab size. HRP clearance rate was significantly increased after repeated injections of HRP but not saline. Clearance was not enhanced after repeated injections of Hb. About 70% of HRP accumulates in gills within 2 hours after injection, when there were no signs of HRP accumulation in the hepatopancreas. But about the same percent of injected Hb accumulated in both gills and hepatopancreas after 2 hours of injection. When 300 ug HRP declined in gills after 8 hour, similar amounts of HRP accumulated in the hepatopancreas. Moreover when 325 ug of Hb was lost from gills after 4 hours, the hepatopancreas accumulated about 300 ug, suggesting that foreign proteins may undergo antigenic alteration in the gills prior to mobilization to the hepatopancreas. There is no evidence of degradation of proteins in gills. When HRP and Hb level declined in the hepatopancreas, the levels of these proteins increased in gut contents indicating externalization of injected proteins via gut.

Crustacean defense strategies. I. Molecular weight dependent clearance of dyes in the mud crab Scylla serrata (Forskal) (Portunidae: Brachyura).

Clearance rates of dyes injected into the hemocoel of the mud crab, Scylla serrata, revealed several patterns of clearance and retention associated with physico-chemical and biological variables. Clearance rates remained constant: 1) when the molar concentrations of the injected dyes were equal, 2) after repeated injections of dyes and 3) after serum opsonization. Rates increased: 1) with higher molecular weight irrespective of charge, 2) at higher dye concentration and 3) at night concomitant with an increase in the hemocyte population. In contrast, clearance rates decreased with increasing crab size. Dye cleared from the hemolymph accumulated in the gills but not in the hepatopancreas, antennary glands nor gut. The retention of dyes in the gills increased with higher molecular weights. Granular hemocytes accumulated in the gill rachis and lamellae of dye treated but not in gills of normal and saline injected crabs. A role for hemocytes in molecular weight and concentration dependent clearance of dyes is suggested. Our findings are important for understanding the molecular basis of recognition in crustaceans.