Mechanisms of regulated lung surfactant secretion

Abstract

Scope and Method of Study:

This project was to study the molecular mechanisms of lung surfactant secretion from three aspects. The first part was to investigate the interaction between SNARE proteins and annexin A2. GST-tagged protein pull-down assay was the major technique utilized. The physical interaction of recombinant GST-tagged SNARE proteins with annexin A2 were tested. To confirm the interaction between annexin A2 and SNAP-23, additional methods were used, including co-immunoprecipitation, mammalian two-hybrid assays. Immunocytochemistry was used to study the co-localization of annexin A2 and SNAP-23. Deletion and site-directed mutagenesis were used to identify the binding sites for annexin A2 on SNAP-23. Furthermore, an in vitro bio-membrane fusion assay was utilized to study the functional interaction between annexin A2 and SNAP-23. The second part was to identify the v-SNARE protein involved in regulated surfactant secretion. Various VAMP genes were amplified from alveolar type II cells by using RT-PCR. The expression of VAMP proteins were detected with Western blotting. Immunohistochemistry and immunocytochemistry were utilized to study the localization of VAMP in lung and type II cells. In the third part, the proteomic profile of lamellar bodies was analyzed. Lamellar bodies were isolated from rat lungs and the proteins were separated with 1-D and 2-D SDS-PAGE. The proteins were applied to MALDI-TOF MS, and identified by searching the peptide spectra against the Mass Spectrometry protein sequence Database (MSDB) using the Mascot web based search engine.

Findings and Conclusions:

I.

1. SNAP-23 specifically binds with annexin A2 in a Ca2+-dependent manner.

2. The cysteine rich domain of SNAP-23 is required for its binding with annexin A2.

3. SNAP-23 is required in annexin A2 tetramer mediated membrane fusion between isolated lamellar bodies and the plasma membrane.

II.

1. VAMP-2 and -8 are expressed in type II cells and lamellar bodies.

2. VAMP-2 is localized on lamellar bodies and VAMP-8 is partially localized on lamellar bodies.

III.

1. Forty-four proteins in lamellar bodies were identified.

2. Proteins involved in membrane trafficking and Ca2+-binding may play roles in lamellar body biogenesis and fusion with the plasma membrane.