Invertebrates, lower chordates and developing embryonic stages of higher vertebrates possess the capacity for precise discrimination between self and non-self even though they lack the classical immunoglobulin-mediated recognition system. Since lectins (specific saccharide-binding molecules) occur in the sera and on the cells of species and developmental stages which lack immunoglobulins, it is possible that these specific molecules might be directly involved in developmentally related recognition and in the earliest phases of defense, particularly against microorganisms. The chief aim of this proposal is to isolate, purify and determine the molecular structure of defined lectins from protochordates (tunicates) and a lower vertebrate, the sea lamprey (a cyclostome). On the basis of screening of 10 representative species of urocordates for the presence of lectins and the specificity of these molecules for defined sugars, we have selected a galactose-binding lectin of the tunicate Didemnum candidum and two lectins specific for sialoconjugates from the tunicates Halocynthia pyriformis and Styela plicata as suitable molecules for the serological and biochemical studies planned. Study of sialoconjugate-binding lectins from the serum and eggs of the sea lamprey (Petromyzon marinus) will serve as a direct comparison with non-immunoglobulin molecules of corresponding specificities. The lectins will be isolated by affinity chromatography using immobilized purified sugars and glycoproteins. The purity of the isolated lectins will be assessed serologically and by analytical techniques including polyacrylamide gel electrophoresis in sodium dodecylsulfate-containing buffers and by isoelectric focusing. The molecules will be characterized physicochemically in terms of molecular weight analyses, carbohydrate and amino acid composition analyses, determination of the number and affinity of binding sites and investigation of secondary structures using optical rotatory dispersion and circular dichroism. We have been able to silate mg quantitites of the Didemnum lectin and have initiated N-terminal amino acid sequence analysis. We plan to prepare peptides from the lectins using proteolytic enzymes of defined specificity and chemical agents such as CNBR and to resolve peptides by gel filtration chromatography using high performance liquid chromatography and by reverse phase peptide chromatography. The peptides will be sequenced using the Beckman automatic sequencer with derivatized amino acids determined using HPLC with the goal of obtaining complete amino acid sequences so that detailed comparisons with known recognition molecules can be made. These studies will provide a critical test of the hypothesis that animal lectins constitute a multigenic family of recognition molecules.
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