ALTERNATE AMINO-TERMINAL PROCESSING OF SURFACTANT PROTEIN-A RESULTS IN CYSTEINYL ISOFORMS REQUIRED FOR MULTIMER FORMATION

The biological functions of rat surfactant protein A (SP-A), an oligom er composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-revers ible covalent linkages of as many as six or more subunits in the molec ule indicate the presence of at least two NH2-terminal interchain disu lfide bonds. However, the reported primary structure of rat SP-A predi cts that only Cys(6) in this region is available for interchain disulf ide formation. Direct evidence for a second disulfide bridge was obtai ned by analyses of a set of three mutant SP-As with telescoping deleti ons from the reported NH2-terminus, Two of the truncated recombinant p roteins formed reducible dimers despite deletion of the domain contain ing Cys(6). Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH- terminal end of the reported signal peptide. Large variations in the a bundance of the IKC isoforms between truncated SP-As suggested that th e amino acid sequences located downstream from the signal peptide modu lated alternate-site cleavage by signal peptidase. Elution of the newl y identified cysteine in the position of DiPTH-Cys indicated participa tion in disulfide linkage, which was interchain based on the direct co rrelation between prevalence of the IKC variant and the extent of dime rization for each truncated protein. Sequencing of both native rat SP- A and human SP-A also revealed isoforms with disulfide-forming NH2-ter minal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-P AGE gels. Thus, a novel post translational modification results in nat urally occurring cysteinyl isoforms of rat SP-A, which are essential f or multimer formation.