CHIMERAS OF SURFACTANT PROTEIN-A AND PROTEIN-D IDENTIFY THE CARBOHYDRATE-RECOGNITION DOMAINS AS ESSENTIAL FOR PHOSPHOLIPID INTERACTION

Pulmonary surfactant proteins A (SP-A) and D (SP-D) possess similar st ructure as members of the mammalian C-type lectin superfamily. Both pr oteins are composed of four characteristic domains which are: 1) an NH 2-terminal domain involved in interchain disulfide formation (denoted Al domain for SP-A or D1 for SP-D); 2) a collagenous domain (denoted A 2 or D2); 3) a neck domain (denoted A3 or D3); and 4) a carbohydrate r ecognition domain (denoted A4 or D4). SP-A specifically binds to dipal mitoylphosphatidylcholine, the major lipid component of surfactant, an d can regulate the secretion and recycling of this lipid by alveolar t ype II cells. SP-D binds to phosphatidylinositol (PI) and glucosylcera mide (GlcCer), and its role in alveolar lipid metabolism remains to be clarified. To understand the relationship between the structure and t he function of both proteins with respect to their interaction with li pids, we expressed recombinant wild type rat SP-D (rSP-D) and chimeric molecules of SP-A and SP-D (A1A2A3D4, A1A2D3D4, and D1D2A3A4) using a baculovirus expression system, and performed lipid binding and aggreg ation assays. The rSP-D effectively competed with I-125-labeIed native rat SP-D in a solid phase binding assay to PI and GIcCer in a manner nearly identical to native SP-D. The rSP-D also bound to PI liposomes with approximately half the affinity of native rat SP-D. Chimera A1A2D 3D4 competed with iodinated SP-D in the solid phase binding assay to b oth PI and GlcCer. This chimera did not bind to dipalmitoylphosphatidy Icholine (DPPC) liposomes or induce their aggregation. Chimera A1A2A3D 4 did not bind solid phase PI or GlcCer but was equivalent to rSP-D in binding to PI liposomes. This chimera exhibited weak binding to DPPC but failed to aggregate DPPC liposomes. Chimera D1D2A3A4 failed to bin d PI and GlcCer and bound weakly to DPPC liposomes but was quite effec tive at inducing aggregation of DPPC Iiposomes. These findings demonst rate that the D3 plus D4 domains of SP-D play a role in lipid binding and that the D4 domain is essential for PI binding. Furthermore, the A 3 domain of SP-A cannot account for all the lipid binding activity of this protein. In addition, the results implicate the A4 domain of SP-A as an important structural domain in lipid aggregation phenomena.