The amino-terminal heptad repeats of the coiled-coil neck domain of pulmonary surfactant protein D are necessary for the assembly of trimeric subunits and dodecamers

Pulmonary surfactant protein D (SP-D), a lung host defense protein, is asse mbled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of t rimers is required for many of its functions. A peptide containing the alph a -helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular ass embly of disulfide-cross-linked, trimeric subunits and dodecamers, For the present studies, me synthesized mutant cDNAs with deletions or site-directe d substitutions in the neck domain of rat SP-D, and examined the assembly o f the newly synthesized proteins after transfection of CHO-K1 cells. The ne ck domain contains three "classical" heptad repeat motifs with leucine resi dues at the "d position," and a distinctive C-terminal repeat previously su ggested to drive trimeric chain association, Deletion of the highly conserv ed core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. Ey contrast, deletion of the entire ne ck domain or deletion of one or two amino-terminal repeats resulted in defe ctive molecular assembly. The secreted proteins eluted in the position of m onomers by gel filtration under nondenaturing conditions, In addition, the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-te rminal to the trimeric coiled-coil. These studies provide strong evidence t hat the amino-terminal heptad repeats of the neck. domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assem bly and secretion of functional dodecamers.