Stage-specific isozymes of ascaris suum complex II: the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae have the common iron-sulfur subunit

Abstract

The cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b5. It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b5, but possesses an NH2-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165–172]. The nematode cytochrome b5 is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b5 with a complete presequence (b5wt) and its NH2 terminus-truncated derivatives, b5Δ18 and b5Δ28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Δ18, and tb5Δ28, produced recombinant proteins with a histidine-tagged NH2-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.