Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation

A Single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extens ion (SP A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypepti de at a primary site (Cys(-1)-Asn(1)) and a secondary site (Gly(-4)-Ile(-3) ). In this study, we used site-directed mutagenesis and heterologous expres sion in vitro and in insect cells to the examine mechanisms that may lead t o alternative signal peptidase cleavage including alternative translation i nitiation at two in-frame AUGs (Met(-30) and Met(-20)), a suboptimal contex t for hydrolysis at the primary cleavage site, or cotranslational protein m odifications that expose an otherwise cryptic secondary cleavage site. In v itro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa prim ary translation products on SDS-PAGE analysis, while translation of cDNAs e ncoding Met(-30)Ala and Met(-20)Ala mutations resulted in only the single 2 8 and 29 kDa molecular mass species, respectively. These data are consisten t with translation initiation at both Met(-30) and Met(-20) during in vitro synthesis of SP-A. The Met(-30)Ala mutation reduced expression of the long er isoform in insect cells, indicating that the Met(-30) site also contribu tes to eucaryotic protein expression. Forcing translation initiation at Met (-30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-20) codon resulted in preferential expression of the l onger SP-A isoform but reduced overall expression of the protein almost 10- fold. Both isoforms were generated to some degree whether translation was i nitiated at the codon for Met(-30) or Met(-20) indicating that the site of translation initiation is not the sole determinant of isoform generation an d suggesting that either the context of the primary cleavage site is Subopt imal or that cotranslational modifications affect cleavage. Preventing N-te rminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-1) by substi tution with serine markedly enhanced cleavage at the Gly(-4)-Ile(-3) bond, but substitution with alanine enhanced cleavage at the Cys(-1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that include s both alternative translation initiation at two in-frame AUGs and a subopt imal context for signal peptidase hydrolysis at the primary cleavage site.