Concepción Agundis

Review: Immunity mechanisms in crustaceans

Crustacean aquaculture represents a major industry in tropical developing countries. As a result of high culture densities and increasing extension of aquaculture farms, the presence of diseases has also increased, inducing economic losses. Invertebrates, which lack adaptive immune systems, have developed defense systems that respond against antigens on the surface of potential pathogens. The defense mechanisms of crustaceans depend completely on the innate immune system that is activated when pathogen-associated molecular patterns are recognized by soluble or by cell surface host proteins, such as lectins, antimicrobial, clotting, and pattern recognition proteins, which, in turn, activate cellular or humoral effector mechanisms to destroy invading pathogens. This work is aimed at presenting the main characteristics of the crustacean proteins that participate in immune defense by specific recognition of carbohydrate containing molecules, i.e. glycans, glycolipids, glycoproteins, peptidoglycans, or lipopolysaccharides from Gram-negative and Gram-positive bacteria, viruses, or fungi. We review some basic aspects of crustacean effector defense processes, like agglutination, encapsulation, phagocytosis, clottable proteins, and bactericidal activity, induced by these carbohydrate-driven recognition patterns.

Participation of a sialic acid-specific lectin from freshwater prawn Macrobrachium rosenbergii hemocytes in the recognition of non-self cells

Phagocytic activity of circulating hemocytes from freshwater prawns is mediated by a small group of granulocytes. Recognition of nonself cells by these cells seems to be mediated by two independent mechanisms: specific, via O-acetylsialic acid, as well as N-acetylated sugars on recognized cells and a nonspecific one. Both mechanisms show differences in their optimal temperature and time differences for activity. Hemocytes with phagocytic activity interact positively with rabbit IgG raised against the serum lectin. Attempts to elucidate the nature of the membrane-lectin on hemocytes performed by electroimmunotransfer blot assays on cell lysates indicates the presence of two major proteins with a molecular weight of 68-72 kDa. Electron microscopy revealed a regular distribution of lectin on hemocyte plasma membranes. Our results suggest the active participation of a membrane lectin in the recognition of nonself.

MORPHOLOGY OF HEMOCYTES FROM THE FRESHWATER PRAWN MACROBRACHIUM ROSENBERGII

Using morphological criteria, we identified three types of blood cells in the freshwater prawn Macrobrachium rosenbergii. Hyaline hemocytes, the most abundant type, have few large cytoplasmic granules, a large nucleocytoplasmic ratio, and lyse spontaneously in the absence of anticoagulant. Granular hemocytes are heterogeneous in size and in density of their granules. They are phagocytic and readily spread on substrates. The third type of hemocytes, identified as undifferentiated hemocytes, are the least abundant. The hemocytes of this economically relevant crustacean are compared with blood cells of other decapods. J. Morphol. 234:147–153, 1997.

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii.

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.

Quantification of lectin in freshwater prawn (Macrobrachium rosenbergii ) hemolymph by ELISA.

An enzyme-linked immunoabsorbent assay was developed to quantify the lectin present in the hemolymph of the freshwater prawn Macrobrachium rosenbergii. This method involves the use of murine monoclonal IgG1 with kappa light chain (designated as 3G1) antibodies raised against the purified lectin, the assay that we developed recognized as little as 30 ng/ml of lectin, and was used to measure the lectin concentration in animals at different maturation stages. The highest concentration of lectin was identified in the hemolymph from post-larval prawns and the lowest in molt stage adult animals. The hemagglutination activity of the lectin was four-fold higher in adult than in juvenile specimens, although in all cases N-acetylated sugar residues, such as N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetyl-D-neuraminic acid were inhibitors of the lectin activity, suggesting that lectin plays a role in the transport of N-acetylated sugar in juvenile prawns. Our results indicate that lectin concentration and hemagglutinating activity could be influenced by developmental conditions of the freshwater prawn.

Analysis of B-cell epitopes from the allergen Hev b 6.02 revealed by using blocking antibodies

Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.

Inhibition of phagocytic activity by the N-acetyl-D-galactosamine-specific lectin from Amaranthus leucocarpus.

Amaranthus leucocarpus lectin (ALL), specific for N-acetyl-D-galactosamine, induces inhibition of the erythrophagocytic activity of resident murine peritoneal macrophages and of the macrophage-like cell line J-774. This effect was observed only in macrophages that were Mac-2 (CD11c/CD18 or CR4) negative, indicating that macrophage activation induces important modification to the glycosylation (mainly O-glycosylation) of the membrane. Receptors for IgM and C3b remain unaltered after lectin treatment. Ultrastructural analysis revealed (a) that ALL induced the formation of pinocytic vacuoles, and (b) a regular distribution over the macrophage membrane as well as endosomal vesicles of the gold labeled ALL. Our results suggest that macrophage membrane glycoproteins with constitutive N-acetyl-D-galactosamine residues participate in the regulation of pinocytic-phagocytic vacuole formation.

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.

Overexpression of glycosylated proteins in cervical cancer recognized by the Machaerocereus eruca agglutinin

In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fuca1,2 (GalNAca1,3) Galb1,4) showed increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity; healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA’s recognition; moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs’ recognized peptides revealed that the latter matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that play a relevant role in cervical cancer’s invasive capacity. O-glycosylation participates in the disassembly of intercellular junctions favoring cancer progression.