title: Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation.
abstract: A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide 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 expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes 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 longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution 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 includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.
authors.affiliation: Division of Pulmonary/Critical Care Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Ohio 45267, USA.
authors.forename: M
authors.initials: M
authors.lastname: Damodarasamy
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Dienger
authors.forename: F X
authors.initials: FX
authors.lastname: McCormack
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018387:Codon, Initiator; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011510:Proteolipids / Q000096:biosynthesis* / Q000235:genetics; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D018387:Codon, Initiator; D008565:Membrane Proteins; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase