Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells Wataru Tsunezawa a , Hitomi Sano a , Hitoshi Sohma a , Francis X. McCormack b , Dennis R. Voelker c , Yoshio Kuroki a; * a Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060, Japan b Division of Pulmonary and Critical Care Medicine, University of Cincinnati School of Medicine, Cincinnati, OH, USA c Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, USA Received 17 February 1998; accepted 11 May 1998 Abstract Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195CGln (rE195Q), Lys201CAla (rK201A) and Lys203CAla (rK203A) for rat SP-A, Arg199CAla (hR199A) and Lys201CAla (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203CAla (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [ 125 I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : Pulmonary surfactant protein A; Alveolar type II cell; Site-directed mutagenesis; Lipid uptake; Liposome aggregation 1. Introduction Pulmonary surfactant is a complex of lipids and proteins that prevents alveolar collapse at the end of expiration. Surfactant protein A (SP-A) is an abun- dant glycoprotein present in surfactant. SP-A has been implicated as an important regulator of surfac- tant homeostasis [1]. It has also been identi¢ed as a potent inhibitor of surfactant lipid secretion by al- veolar type II cells [2,3], and this activity results from 0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII:S0167-4838(98)00159-9 Abbreviations : SP-A, surfactant protein A ; SP-D, surfactant protein D; MBP-A, mannose-binding protein A; CRD, carbohy- drate recognition domain; rSP-A, rat SP-A; hSP-A, human SP- A ; wt, wild-type ; DPPC, dipalmitoylphosphatidylcholine ; ELI- SA, enzyme-linked immunosorbent assay; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis * Corresponding author. Fax: +81-11-611-2236; E-mail : kurokiy@sapmed.ac.jp Biochimica et Biophysica Acta 1387 (1998) 433^446