Tunicate C-type Lectins as Acute Phase Reactants

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9406649 Vasta As knowledge of the biochemical properties and gene sequences of animal lectins has accumulated in recent years, we have begun to understand not only their complex molecular and gene organization, but also their evolution and multiple biological roles. The goal of this research is to characterize the molecular and gene structures and binding properties of lectins from selected invertebrates in order to understand their evolution as self/non- self recognition molecules, and to elucidate the molecular mechanisms of their biological functions. With prior NSF support considerable progress has been made in isolation, purification, and biochemical characterization of four distinct L-fucosyl binding lectins, CPL-I, -II, -III, and -IV, from plasma and hemocytes of the protochordate Clavelina picta. Serological and primary structure evidence suggest that C. picta lectins are related to acute phase reactants from vertebrates, namely, human pro-clotting factor VIIIc (F VIIIc), C-reactive protein (CRP), and serum amyloid P (SAP). The C.picta lectin binding properties, including the fine specificity for L-fucose-containing oligosaccharides, affinity constants, divalent cation dependence and effect of toxic metals, have been extensively characterized, and the amino acid residues of the polypeptide chain involved in the interactions with the ligand have been identified by specific chemical modification. C. picta lectins bind preferentially to oligosaccharides that exhibit non- reducing terminal fucose but surprisingly, also bind phosphocholine, galactans, sialic and muramic acids, and proteoglycans. Preliminary studies to identify the "natural" ligands, both exogenous (environmental and colony-associated bacterial isolates) and endogenous (tunic sulfated galactan) have begun. Based on experimental results thus far, it appears that C. picta lectins are multifunctional recognition/effector mosaic molecules, involved not only in recognition and opsonization o f potentially pathogenic bacteria from the environment but also in wound repair upon injury of the body wall. Additional lectins have now been isolated from C. picta that have been characterized as belonging to the S-type. Until recently, S-type lectins were believed to be restricted to vertebrate species. This finding resulted not only in the isolation of S-type lectins in a variety of invertebrate species but also in the crystallization and resolution of the three dimensional structure of an animal lectin for the first time. To obtain the complete nucleotide sequence that will allow alignment of the extensive amino acid sequence obtained so far, a hemocyte cDNA library was constructed in the expression vector lunizap and through PCR techniques, a nucleic acid probe for CPL-III was developed. The hemocyte cDNA library was screened with this probe and positive recombinant phage clones were isolated. All the clones selected yield a product of similar size when used as templates for PCR with the same primers. These are currently being sequenced. Goals for the next three years are to conduct studies in two areas: 1) Protein and cDNA structure of C. picta lectins: By the end of the current funding period it is expected that substantial nucleotide sequence of CPL-III will be in hand, and the extent of homology to the acute phase reactants CRP, SAP, and F VIIIc will be determined. By the end of the new funding period equivalent information should be available for the other three C.picta lectins. 2) Biological role of C.picta lectins: Endogenous and exogenous "natural" ligands, i.e. tunic sulfated galactans and polysaccharides from selected environmental bacterial strains, will be sought and characterized. in vitro studies of the interactions of the characterized "natural" ligands or their relevant determinants on the C.picta hemocytes will be studied, and the inducibility of C.picta lectins by the putative natural ligands will be examined. %%% The goal of this research is to characterize the molecular structure, properties and biological functions of certain sugar- binding molecules ("lectins") from selected invertebrate animals to understand their evolution as self/non-self recognition molecules, and to clarify the molecular mechanisms of their biological functions which may be important in the defense mechanisms of a diversity of animals. ***