Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles

Abstract

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.

Figure 1. Characteristics of isolated lamellar bodies.

(A) Cryo-TEM of LB samples obtained before Na₂CO₃ stripping or sulfo-NHS-SS-biotin treatment. (B) Enrichment of lysozyme, mature SP-B and mature SP-C during LB isolation. (C–E) The effect of Na₂CO₃ mediated peripheral protein stripping on LB protein distribution. Start is the initial LB sample, LB is the LB sample after Na₂CO₃ treatment, Na₂CO₃ is the Na₂CO₃ solution after stripping, LME refers to limiting membrane enriched fraction and LMD refers to limiting membrane depleted fraction. (C) β-actin protein, (D) mature SP-C and (E) ABCA3 protein within these fractions. All samples were normalized to total protein content.

Figure 2. Characteristics of LB proteome.

(A) Distribution of membrane proteins in the proteome with respect to the 3 LB fractions. (B) Predicted subcellular localization of proteins in the LB, LMD and LME fractions.

Figure 3. BiP distribution in LBs.

(A) Western blot for BiP in HEK293 cells, whole lung, isolated type II cells, isolated LBs and BALF. Samples were normalized to protein content. (B) Immunogold labeling for BiP in isolated rat LB. (C) Relative labeling index (RLI) of isolated rat LBs labeled by immunogold for ABCA3, mature SP-B and BiP.

Figure 4. Relative mRNA expression levels of (A) ATP8A1 and (B) ABCA8a, as assessed by real-time polymerase chain reaction.

Data is expressed as relative quantification (RQ), which is the fold difference in expression between embryonic day 11 sample with respect to all other samples.

Figure 5. Western blot for LB proteome proteins.

Samples were normalized to protein content. Distribution of (A) EHD2 and (B) EHD4 in mouse tissues. Distribution of (C) EHD2, (D) EHD4 and (E) AGER in lung homogenates, type II cells, isolated LBs and BALF.