Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence

Abstract

Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.

C-proSP-B: A Possible Biomarker for Pulmonary Diseases?

“…Although the route by which proteins leak through the alveolar capillary membrane is not yet fully understood, this could be associated with higher membrane permeability in ILDs due to regions of alveolar epithelial cell injury and hyperplasia, basal membrane disruption, as well as capillary leakage and subsequent aberrant wound healing known to be part of the disease mechanism in this disease entity. This is consistent with the already published data associating mature SP-B with acute lung injury [17]. …”

“…Our findings with C-proSP-B underscore the conclusions of previous and recent literature that the precursor proteins of SP-B are particularly robust and adequate as potential prospective biomarkers for pulmonary diseases damaging the alveolocapillary barrier [17, 20, 21, 23, 28]. …”

“…SP-B precursor proteins are present in the endoplasmic reticulum, Golgi complex, and lamellar bodies, whereas mature SP-B is found only in lamellar bodies [16]. Proteolytic processing of the SP-B precursor into mature protein takes place intra- and extracellularly [16], resulting in the occurrence of the mature protein as well as the SP-B precursor and the processing intermediates in the alveolar epithelial lining fluid [17]. Because of differences in hydrophobici ty, mature SP-B is intimately associated with the phospholipid-rich surfactant [18], whereas the less hydro­phobic SP-B precursor and its processing intermediates (C-proSP-B and proSP-B) are less tightly bound to phospholipids and can therefore predominantly be recovered in the hydrophilic parts of, e.g., bronchoalveolar lavage fluid supernatant [17].…”

“…Proteolytic processing of the SP-B precursor into mature protein takes place intra- and extracellularly [16], resulting in the occurrence of the mature protein as well as the SP-B precursor and the processing intermediates in the alveolar epithelial lining fluid [17]. Because of differences in hydrophobici ty, mature SP-B is intimately associated with the phospholipid-rich surfactant [18], whereas the less hydro­phobic SP-B precursor and its processing intermediates (C-proSP-B and proSP-B) are less tightly bound to phospholipids and can therefore predominantly be recovered in the hydrophilic parts of, e.g., bronchoalveolar lavage fluid supernatant [17]. In addition, Agostoni et al [19] stated that the immature form of SP-B seems to remain high in the blood for a longer time in patients with acute lung injury compared with mature SP-B.…”

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

“…The isoelectric point of BSA is about 4.1, and at a measured pH of 7 for the aqueous samples, the protein is anionic. The hydrophobic surfactant protein, SP-B has been shown to have positively charged amphiphatic alpha-helices that can interact strongly with the anionic phosphatidylglycerol (PG) lipid head-groups in model membrane bilayers (Longo et al, 1992). SP-C also has positive charges in its N-terminal region which is associated with the lipid head-groups in bilayers (Morrow et al, 1993).…”