Proteomic analyses were performed on lamellar bodies isolated from rat lungs. Of particular interest to these analyses were the proteins of the limiting membrane, as this subpopulation is likely involved in the biogenesis and maturation of the organelle. To enrich for limiting membrane proteins, surface accessible free amines on isolated LBs were conjugated to sulfo-NHS-SS-biotin. Biotinylated LBs were subsequently washed with Na2
to strip cytoskeletal proteins, lysed in RIPA buffer and loaded onto an avidin column. The column was washed extensively and bound proteins were eluted by reduction of the biotin conjugate. Proteins not captured by avidin represented enrichment of the interior, core LB proteins, while the eluted fraction represented enrichment of LB limiting membrane proteins (Figure S1
). Lamellar bodies lysed before or during biotinylation resulted in some core proteins being labeled and co-isolated with the limiting membrane fraction.
The purity and integrity of the LB preparations was assessed by western blotting and electron microscopy. Cryo-TEM performed on LBs prior to Na2CO3 stripping detected largely intact organelles with organized lamellae and continuous limiting membranes (). The lamellar body proteins, surfactant protein B (SP-B), lysozyme and surfactant protein C (SP-C) () were progressively enriched in lung homogenates, type II cells and isolated LBs, consistent with significant purification of the LB isolate. Subsequent treatment of LBs with Na2CO3 further enhanced purity: sodium carbonate stripping greatly reduced the content of actin () and Clic5 (not shown) in the LB and limiting membrane fractions with consequent enrichment in the stripping buffer. In contrast SP-C () was not removed by stripping buffer and was detected in the limiting membrane depleted (LMD) fraction. The LB limiting membrane protein, ABCA3 (), was not removed by Na2CO3 stripping and was recovered in the limiting membrane enriched (LME) fraction, as expected. Together these results are consistent with minimal perturbation of LB structure during the isolation procedure.
Characteristics of isolated lamellar bodies.
The LB proteome was compiled from MS analyses of the three LB fractions (intact LB, LME and LMD). A total of 455 proteins were detected at least once with a Mascot score ≥75: 351 proteins were detected in the intact LB fraction and 280 proteins were detected in the LME and LMD fractions (184 and 96 proteins, respectively). Many proteins were identified in both the LMD and LME fractions, likely related to failure of some proteins to bind the avidin matrix or retention of biotinylated core proteins on the column, respectively. Proteins were therefore assigned as LB limiting membrane (157) or LB core (87 proteins) based on their Mascot scores (Table S1
); 211 proteins were not assigned to either compartment because the protein was detected only in the intact LB fraction or because the LME and LMD Mascot scores were very similar. Ribosomal proteins (13 in total, Table S2
) were excluded in subsequent tables and analyses because these proteins are contaminants of many organelle proteomes 
. As an alternative method to identify LB proteins, PCT was performed on stripped LBs to delipidate the sample prior to SDS-PAGE and MS analysis. Ninety-four of the 291 proteins detected by this method (Table S3
) were not detected by the conventional approach, bringing the total number of proteins to 562. The entire 562 protein dataset was then used to interrogate archived microarrays of isolated, mouse type II epithelial cells (n
and unpublished data, available upon request). Twenty-three proteins (4%) were not represented on the Affymetrix chip and thus expression data are unavailable for this subset, while another 65 proteins (12%) were not expressed or expressed at low levels in the type II cell. Expression of the remaining 474 proteins (88%) was confirmed in type II cells. The broad representation of type II cell proteins in the LB proteome further confirms successful isolation of LBs from lung homogenates.
Like other organelles, the limiting membrane of the LB is expected to have a substantial number of transmembrane proteins; however, because the LB is filled with tightly packed bilayer membranes, the core protein transmembrane content is also expected to be very high. Proteins in each fraction were categorized as soluble, membrane-associated or membrane using the gene ontology database (http://www.ebi.ac.uk/GOA/
). Each fraction, including the core LMD fraction, contained 33 to 42% transmembrane proteins (). These ranges are consistent with the previously reported ranges for ER and lysosomal membrane proteins 
. The content of hydrophobic proteins (transmembrane + membrane associated) was very similar for the LME (60%) and LMD (57%) fractions consistent with the membrane cargo of the LB.
In order to assess the potential contribution of other organelles to the LB proteome, the subcellular localization of each protein was identified using the Gene Ontology Annotation Database and by cross-referencing with proteomes of other lysosome-related organelles ( and Table S2
). Lamellar bodies are lysosomal-like organelles that communicate extensively with both the bioysynthetic and endocytic pathways. Lysosomal proteins were well-represented in all three fractions as were proteins associated with the secretory and endocytic pathways (plasma membrane, secreted, vesicle and ER). Only a small number of proteins (<6%) were identified from the nucleus, mitochondria or Golgi consistent with minimal contamination from these compartments. The high concentration of cytoskeletal proteins was expected as the LB surface is densely covered by actin (see ) 
As with other lysosome-like organelles, a large number of ER proteins were detected in the LB proteome. Persistent co-purification of ER proteins could represent microsomal contamination and/or true localization in the LB. To begin to address this issue the distribution of the abundant ER chaperone BiP (Mascot score
1415) was assessed in intact lamellar bodies. BiP was detected by western blotting of lung homogenates, isolated type II cells and isolated LB but not BALF (). Immunogold labeling of isolated LBs () and lung tissue sections (not shown) demonstrated that BiP was peripherally distributed in the LB in close approximation to the limiting membrane. Quantitative analyses of gold particles provided further support for this conclusion (). BiP counts were detected primarily at the limiting membrane (P<0.001) similar to ABCA3, a known LB limiting membrane protein: in contrast, SP-B counts were predominantly distributed over the internal membranes (P<0.001) as expected (). These findings, coupled, with lack of detection of BiP in BALF (), suggested that this ER protein accumulates in the LB but is not secreted, perhaps because of its association with the inner surface of the limiting membrane. In addition to BiP, calnexin (Mascot score
438) was also detected in LBs by immunogold labeling of isolated LBs (Figure S2A
). Further, isoforms of the ER chaperone, protein disulfide iscmerase family A, member 3 (PDIA3, Mascot score
882), were detected in LBs by confocal microscopy of lung tissue sections (Figure S2B
). Given these preliminary findings, the significant enrichment of ER proteins in the LB (Table S4
) and the presence of ER proteins in the proteomes of 7 other LROs 
, we conclude that a subset of ER proteins is present in the LB. The number of ER proteins resident in the LB and the manner in which they are transported between the two organelles remains to be established.
Lamellar body proteins not present in other lysosome related organelles (LRO) proteomes are collected in . The major function of the LB is the assembly and storage of surfactant phospholipids for regulated release into the alveolar airspaces. Consistent with this function many of the proteins unique to the LB are involved in lipid or ion transport. In addition to ABCA3 (Mascot score
673) these include ATP8A1 (Mascot score
163), ABCA8a (Mascot score
107), StarD9 (Mascot score
76), Deleted in Liver Cancer 1/StarD12 (Mascot score
75), ATP1a1 (Mascot score
672), Major Vault Protein (Mascot score
546), SLC4A1 (Mascot score
393), and ATP2B4 (Mascot score
364). ATP8A1 and ABCA8a were the focus of further study due to their potential lipid transport activities. ABCA8a is a poorly characterized member of the ABCA subfamily of lipid transporters. MS analyses detected ABCA8a exclusively in the LB limiting membrane fraction (Table S1
). Expression of ABCA8a mRNA in lung tissue increased modestly between E13-E17 with a more pronounced increased in the perinatal period (E18-PND1) and a significant increase in juvenile and adult lung (). Similarly, expression of mRNA encoding ATP8A1, increased from E16-E18 with a more pronounced increase in postnatal lung tissue (). ATP8A1 has been implicated in transport of several phospholipids species and, like ABCA8a, was exclusively detected in the LME fraction. Identification of these candidate phospholipid transport/transfer proteins is consistent with their predicted role in transport of lipid substrates across the LB limiting membrane.
Proteins unique to the LB relative to other LROs.
Relative mRNA expression levels of (A) ATP8A1 and (B) ABCA8a, as assessed by real-time polymerase chain reaction.
Pulmonary surfactant is actively recycled from the alveolar spaces likely resulting in the incorporation of some endosomal proteins in the LB. Consistent with this hypothesis a number of endocytic pathway related proteins were identified, including adaptor-related proteins AP2A2 (Mascot score
589), and AP2B1 (Mascot score
356), clathrin (CLTC, Mascot score
1798) and annexins A1, A2 and A6 (Mascot score
254, 565 and 1551 respectively). Three EH-domain (EHD) containing proteins, previously implicated in endocytic trafficking, were also identified in the LME fraction 
. EHD2 and EHD4 had high Mascot scores (1371 and 1019 respectively) and were strongly detected in mouse lung compared to other tissues by western blotting (). Although EHD2 was enriched in isolated LB, the signal was lower in type II cells compared to whole lung lysate, suggesting that EHD2 may be expressed in multiple lung cell types (). In contrast EHD4 was detected predominantly in type II cells with lower expression in the LB fraction, suggesting that EHD4 is distributed among multiple subcellular compartments in the type II cells (). Unexpectedly, EHD4 was also found in the BALF, consistent with release into the airspaces.
Western blot for LB proteome proteins.
Multiple proteins involved in airway defense were detected in the LB proteome including antimicrobial and antioxidant proteins. Previously identified proteins in this category included surfactant protein A (Mascot score
466) and lysozyme (Mascot score
232). Defense proteins not previously detected in the LB include the paraoxonase proteins PON2 and PON3 (Mascot scores 115 and 300 respectively), which are reported to have both anti-oxidant and antimicrobial activities 
. In addition the antioxidant proteins dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST, Mascot score
279) and advanced glycosylation end product-specific receptor (AGER, Mascot score
179) were also detected. AGER is highly expressed in the lung but has not previously been associated with the LB. Both membrane (Mw~50 kDa) and soluble (Mw~45 kDa) forms of AGER were detected by western blotting of lung homogenates (). Interestingly, soluble AGER was significantly enriched in the LB and was detected in BALF, suggesting that the type II cells may be an important source of this anti-oxidant molecule. The identity of the other AGER isoform (Mw~34 kDa) has not been established but may represent unglycosylated soluble isoform 
. Collectively these findings support the conclusion that, in addition to packaging/secretion of pulmonary surfactant, the LB plays an important role in airway defense.