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 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.
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
P
P
Zhang
A
A
McAlinden
S
S
Li
T
T
Schumacher
H
H
Wang
S
S
Hu
L
L
Sandell
E
E
Crouch
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D016466:CHO Cells; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
D001704:Biopolymers; D017931:DNA Primers; D006023:Glycoproteins; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D003094:Collagen