Identification and characterization of homologues of vertebrate β-thymosin in the marine mollusk Aplysia californica

The β-thymosins have been known as actin-sequestering proteins, but now are recognized as molecules with multiple and diverse intracellular and extracellular functions. Two closely related proteins, β-thymosin ^His and β-thymosin^Gln, have been de novo sequenced by top-down mass spectrometry in the common neurobiology model, Aplysia californica. As determined by nanoelectrospray quadrupole-enhanced Fourier-Transform mass spectrometry with collisionally activated and electron-capture dissociations, both of these Aplysia β-thymosins are acetylated and differ by a single residue in the central actin-binding domain. Profiling of individual cells and tissue by matrix-assisted laser desorption/ionization mass spectrometry reveals that these proteins are widely expressed in the Aplysia central nervous system, including in individual identified neurons, neuronal clusters, nerves and connective tissues. Newly identified β-thymosin^His and β-thymosin^Gln are also detected by mass spectrometry in hemolymph, and in releasates collected from whole ganglia. When applied exogenously, β-thymosin proteins, purified from nerve cell extract, support the anchoring of neurons, and increase neurite sprouting and total neurite outgrowth in culture. These positive effects on neurite regeneration in cell culture suggest that the β-thymosin proteins have an extracellular function in the central nervous system of Aplysia californica.