SPINALIN, A NEW GLYCINE-RICH AND HISTIDINE-RICH PROTEIN IN SPINES OF HYDRA NEMATOCYSTS

Here we present the cloning, expression and immunocytochemical localiz ation of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clone s were identified by in situ hybridization to differentiating nematocy tes. Sequencing of a full-length clone revealed the presence of an N-t erminal signal peptide, suggesting that the mature protein is sorted v ia the endoplasmic reticulum to the post-Golgi vacuole in which the ne matocyst is formed. The N-terminal region of spinalin (154 residues) i s very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly a s pairs. For both regions a polyglycine-like structure is likely and t his may be stabilized by hydrogen bond-mediated chain association. Sim ilar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues ) and an acidic region (9 glutamates and 9 aspartates out of 32 residu es). Western blot analysis with a polyclonal antibody generated agains t a recombinant 19 kDa fragment of spinalin showed that spinalin is lo calized in nematocysts. Following dissociation of the nematocyst's cap sule wall with DTT, spinalin was found in the insoluble fraction conta ining spines and the operculum, Immunocytochemical analysis of develop ing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogene sis to form spines on the external surface of the inverted tubule and the operculum.