TACHYCITIN, A SMALL GRANULAR COMPONENT IN HORSESHOE-CRAB HEMOCYTES, IS AN ANTIMICROBIAL PROTEIN WITH CHITIN-BINDING ACTIVITY

Small granules of horseshoe crab hemocytes contain two known major ant imicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions, In the pr esent study, we examined the biological properties and primary structu re of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein wit h an apparent M(r)=8,500 on Tricine-SDS-polyacrylamide gel electrophor esis, Ultracentrifugation analysis revealed tachycitin to be present i n monomer form in solution, Tachycitin inhibited the growth of both Gr am-negative and -positive bacteria, and fungi, with a bacterial agglut inating property. Moreover, tachycitin and big defensin acted synergis tically in antimicrobial activities, The amino acid sequence and intra chain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mat ure tachycitin consisted of 73 amino acid residues containing five dis ulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded fo r an NH2-terminal signal sequence followed by the mature peptide and a n extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding pe ptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, an d laminarin. Tachycitin may represent a new class of chitin-binding pr otein family in animals.