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

Fernando Sánchez-Barbero,Jochen Strassner,Rafael García-Cañero,Wolfram Steinhilber,Cristina Casals

doi:10.1074/jbc.m410266200

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-A1(DeltaAVC,C6S)), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys(6) and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys(6) mutant lacked the NH(2)-terminal Ala(-3)-Val(-2)-Cys(-1) (DeltaAVC) extension present in some SP-A1 isoforms. SP-A1(DeltaAVC,C6S) was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1(DeltaAVC,C6S) was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1(DeltaAVC,C6S) showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T(m) was 32.7 degrees C for SP-A1(DeltaAVC,C6S) and 44.5 degrees C for SP-A1. Although SP-A1(DeltaAVC,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 Ca(2+). On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1(DeltaAVC,C6S) to inhibit the production of tumor necrosis factor-alpha 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.

Highlights

Surfactant protein A (SP-A)1 is a large oligomeric extracellular protein primarily found in the alveolar fluid of mammals
The trimeric SP-A1⌬AVC,C6S exhibited two major bands corresponding to two and three disulfide-linked chains, respectively, and a faint band corresponding to monomers. These results proved that part of the SP-A1⌬AVC,C6S trimers contained two polypeptide chains covalently linked by at least one disulfide bond, and part of the SP-A1⌬AVC,C6S trimers contained the three polypeptide chains cross-linked by two disulfide bonds
The aim of this study was to investigate the effect of the degree of oligomerization on the structural and functional properties of mammalian cell-derived human SP-A1

Summary

Introduction

Surfactant protein A (SP-A) is a large oligomeric extracellular protein primarily found in the alveolar fluid of mammals. The primary structure of mature SP-A is conserved among different mammals with some differences It consists of four structural domains: 1) an NH2-terminal segment involved in intermolecular disulfide bond formation; 2) a collagen-like. In human SP-A1, four cysteine residues are potentially involved in the arrangement of the disulfide bonding: two cysteine residues in the NH2-terminal segment (CysϪ1 and Cys6), another in the middle of the collagen-like sequence within the Pro-Cys-Pro-Pro interruption (Cys48), and a fourth cysteine at position 65 (Cys65) in the collagen domain near the neck. The first objective of this study was the production of a full-length mammalian cell-derived SP-A1 molecule that was not capable of forming higher oligomers than trimers To this end, the cysteine at position ϩ6 was removed by site-direct mutagenesis (Ser was substituted for Cys). The second objective was the analysis of the structure and function of SP-A1⌬AVC,C6S in comparison with wild type recombinant human SP-A1, expressed in the same system, which has a supratrimeric assembly and is functionally comparable to the protein isolated from lung lavages of healthy subjects [22]

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