Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A

Abstract The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1ΔAVC,C6S), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys6 mutant lacked the NH2-terminal Ala–3-Val–2-Cys–1 (ΔAVC) extension present in some SP-A1 isoforms. SP-A1ΔAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1ΔAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1ΔAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The Tm was 32.7 °C for SP-A1ΔAVC,C6S and 44.5 °C for SP-A1. Although SP-A1ΔAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca2+. On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1ΔAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.