Abstract
Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide containing the alpha-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examined the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain association. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective molecular assembly. The secreted proteins eluted in the position of monomers 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-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assembly and secretion of functional dodecamers.
Highlights
Pulmonary surfactant protein D (SP-D),1 like other members of the collectin subfamily of C-type lectins, is believed to play important roles in the innate defense against a variety of respiratory pathogens [1,2,3]
We examined the assembly of rSP-D in CHO-K1 transfected with selected neck domain mutants
We examined the assembly of a chimeric protein consisting of the amino-terminal propeptide domain of type IIA procollagen [25] linked to the neck and carbohydrate recognition domain (CRD) domains of SP-D to determine whether the carboxyl-terminal domains are sufficient to mediate trimerization of a heterologous amino-terminal, collagen-containing sequence
Summary
Oligo (5Ј-ggtacgaattcatgattcgcctcggg) ϩ Oligo (5Ј-cagcactgtccattggtccttgcat). Template rSP-D rSP-D rSP-D rSP-D rSP-D rSP-D rSP-D rSP-D rSP-D rSP-D IIA rSP-D rangement leading to the formation of disulfide-cross-linked dodecamers. Other studies have shown that a 35-amino acid polypeptide corresponding to the sequence immediately carboxyl-terminal to the collagen domain of human SP-D is sufficient for the stable but reversible association of the peptides to form trimeric complexes in vitro [16] This sequence, which lacks cysteine, predicts an ␣-helical coiled-coil containing four uninterrupted heptad repeats with hydrophobic residues in a 3-4-3-4 spacing. Together these observations provide strong circumstantial evidence that the coiled-coil conformation of the neck domain is sufficient for trimerization of the neck ϩ CRD domains and suggest a crucial role for the carboxyl-terminal repeat in driving trimeric assembly of SP-D monomers It is not known whether these sequences are necessary for the complete intracellular assembly of disulfide-cross-linked trimeric subunits. We examined the assembly of a chimeric protein consisting of the amino-terminal propeptide domain of type IIA procollagen [25] linked to the neck and CRD domains of SP-D to determine whether the carboxyl-terminal domains are sufficient to mediate trimerization of a heterologous amino-terminal, collagen-containing sequence
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