Novel 3,4-di- and 3,4,5-trihydroxyphenylalanine-containing polypeptides from the blood cells of the ascidians Ascidia ceratodes and Molgula manhattensis

Abstract

Acetic acid urea extraction of the blood cells of two ascidian species yielded four novel families of polypeptides (3500-5300 Da) containing 3,4-dihydroxyphenylalanine (DOPA) and 3,4,5-trihydroxyphenylalanine (TOPA) from the Phlebobranch Ascidia ceratodes and two DOPA proteins (9-10 kDa) from the Stolidobranch Molgula manhattensis. 3,4,5-Trihydroxyphenylalanine residues have not been found previously in polypeptides in any biological system. The DOPA content of the M. manhattensis proteins is the highest yet reported for a naturally occurring DOPA protein. The successful isolation of proteinaceous components from A. ceratodes blood cells requires the incorporation of high concentrations of complexing agent in the extraction buffers to inactivate vanadium(III) which forms intractable organometallic polymers. The A. ceratodes blood cell polypeptides are all rich in alanine and TOPA residues, have neutral to basic pI values, and, while all give single bands on acid urea-polyacrylamide electrophoresis, they exhibit extensive microheterogeneity. This is reflected by their large molecular weight distribution as determined by matrix-assisted laser desorption ionization-mass spectrometry, the variation in the ratio of their 280-nm absorption to chromophores of unknown structure in the polypeptide's electronic absorption spectra, and the N-terminal sequence analysis. The two M. manhattensis proteins consist largely of four amino acids (alanine, valine, phenylalanine, and DOPA) with DOPA accounting for upward of 40 mol%. Both give an N-terminus of Ala-Phe-Tyr before resisting further progress by Edman degradation. Proteins and polypeptides from both ascidians are extremely resistant to proteases, a property which, while hampering characterization by sequence analysis, appears ideally suited to their proposed function of forming an impervious hemostat at the site of vascular injury. The yield of proteins and polypeptides relative to tunichromes appears to be seasonally dependent. The presence of DOPA and TOPA moieties in the proteins and polypeptides implicates them, as well as the tunichromes, as potential metal-sequestering agents.