Abstract
In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.
Highlights
In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids
These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes
We examined the specific role of amino acid Arg197 of the SP-A in the interaction of the protein with the type II cell receptor and lipids
Summary
Tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. The simultaneous substitutions E195Q and R197D of the CRD alter carbohydrate binding specificity, prevent SP-A-mediated phospholipid uptake and aggregation of phospholipid, and destroy the ability of SP-A to compete with its radiolabeled counterpart for surface receptors on alveolar type II cells. These results implicate the Glu195 and Arg197 as essential amino acids in SP-A function. We examined the specific role of amino acid Arg197 of the SP-A in the interaction of the protein with the type II cell receptor and lipids. We show that mutant R197H is defective exclusively in the lipid uptake process
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