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Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

ABSTRACT: Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.

SUBMITTER: Cabre EJ

PROVIDER: S-EPMC6005437 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

Cabré Elisa J EJ Martínez-Calle Marta M Prieto Manuel M Fedorov Alexander A Olmeda Bárbara B Loura Luís M S LMS Pérez-Gil Jesús J

The Journal of biological chemistry 20180426 24

Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this ...[more]

PMID: 29700110

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Project description:A reverse-phase HPLC protocol for analysis of strictly hydrophobic peptides and proteins was developed. Peptide aggregation is minimized by using only 25-40% water in methanol or ethanol as initial solvents and subsequent elution with a gradient of propan-2-ol. Analysis of the pulmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (1) SP-B and SP-C retain their secondary structures and separate by about 15 min over a 40 min gradient. SP-B is more hydrophilic than SP-C, which in turn behaves chromatographically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms additional to the major form characterized previously, which contains two thioester-linked palmitoyl groups. The isoforms now observed contain one or three palmitoyl moieties and constitute together 15-20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the epsilon-amino group of Lys-11, as concluded from the elution position,MS and amino acid sequence analysis. The tripalmitoylated form increases relative to the dipalmitoylated form on incubation of SP-C ina phospholipid environment. An Nepsilon-bound palmitoyl moiety constitutes a third mode of fatty acyl modification of proteins, in addition to the established Nalpha-bound myristoyl groups and S-bound palmitoyl chains. (3) The dimeric structure of SP-B, lacking covalent modifications, is confirmed by MS detection of the dimer. No SP-B isoforms were detected. (4) Denatured, non-helical SP-C can be distinguished chromatographically from the native alpha-helical peptide. (5) HPLC of SP-C at 60-75 degrees C reveals an isoform containing an extra 14 Da moiety compared with the main form. This is concluded to arise from inadvertent methyl esterification of the C-terminal carboxy group. In conclusion, this HPLC method affords a sensitive means of assessing modifications and conformations of SP-B or SP-C in different disease states and before functional studies. It might also prove useful for analysis of other strictly hydrophobic polypeptides.

Dataset Information

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

Publications

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

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