Surfactant protein A accumulating in the alveoli of patients with pulmonary alveolar proteinosis: oligomeric structure and interaction with lipids

Abstract

Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease of unknown etiology in which the alveoli and terminal bronchioles of the lung fill with large amounts of surfactant-rich lipoproteinaceous materials. Its major pathologic manifestations are a small number of normal tubular myelin structures and an unusual abundance of multilamellated structures. Since surfactant protein A (SP-A) plays an important role in surfactant phospholipid homeostasis, we investigated the structural features of SP-A oligomers (alveolar proteinosis protein, APP) accumulating in the alveoli of individuals with PAP, and examined the abilities of APP to interact with lipids. Analysis of APP by Bio Gel A15m column chromatography revealed that it was composed of two protein peaks, one of which (APP-I) eluted at the position near that of blue dextran whereas the other (APP-II) eluted far behind blue dextran but ahead of thyroglobulin. These populations of APP showed almost identical amino acid compositions. Electron microscopic observations of APP molecules using the rotary shadow technique revealed that APP-II was observed as hexameric particles, presumably consisting mainly of octadecamers whose diameter was approximately 30 nm. The population seen for APP-II was similar to that seen for SP-A from healthy individuals. In contrast, APP-I was observed as multimerized larger aggregates whose diameter appeared to be about 70 to 90 nm. Both APP-I and APP-II retained the abilities to bind dipalmitoylphosphatidylcholine (DPPC). They also induced phospholipid vesicle aggregation in a concentration-dependent manner. The maximal turbidity for light scattering induced by APP-I and APP-II was almost equivalent when analyzed as a function of molar concentration. In vitro reconstitution experiments with porcine surfactant protein B (SP-B) and phospholipids revealed that the multilamellated membranes in structures formed from APP-I consisted of several layers of doubled unit membranes. APP-I failed to form tubular myelin structures. In contrast, APP-II formed well-formed lattice structures seen in tubular myelin. From these data we conclude that there exists an abnormal multimerized form of SP-A oligomer in the alveoli of patients with PAP, and that this unusual subpopulation of SP-A oligomer exhibits abnormal function on phospholipid membrane organization.