Interaction of surfactant protein A with cellular myosin.
ABSTRACT The goal of the current investigation was to characterize, purify, and identify the proteins that bind surfactant protein A (SP-A). Several polypeptides were purified by SP-A affinity chromatography, and the 200 kD major polypeptide that reacted with SP-A on ligand blots was purified further by preparative SDS-PAGE. Protein sequencing of proteolytically derived subfragments of this polypeptide gave sequences that corresponded completely with nonmuscle (cellular) myosin heavy chain. The 200 kD polypeptide was then found to be immunoreactive with antibodies against cellular myosin. A smaller polypeptide of 135 kD also binds SP-A and appears to be a proteolytic fragment of the 200 kD peptide. The ability of SP-A to bind myosin was confirmed in a microtiter well assay and was found to be concentration dependent. We speculated that the physiologic relevance of the interaction of SP-A with myosin might be to facilitate clearance of myosin from the alveolar subphase following its release during lung injury. In support of this hypothesis, we found that there were detectable levels of myosin in lavage fluid and that SP-A could indeed enhance uptake and degradation of myosin by alveolar macrophages.
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ABSTRACT: TGolgin-1 is a large, predominantly coiled coil peripheral membrane protein that associates with the trans Golgi network (TGN) by virtue of a C-terminal GRIP domain. We showed that overexpression of isolated GRIP domains from tGolgin- 1 or related proteins results in disruption of the TGN by inhibiting recycling from endosomes, suggesting that tGolgin-l and other GRIP domain proteins function in this pathway. This is important because signalling molecules required for initiating transformation and for metastasis rely on sorting and processing in the TGN. In collaboration with Dr. Chris Burd, we showed that GRIP domain localization to the TGN in yeast is regulated by a small GTPase cascade, in which the Arf-like protein Ar13 recruits the Arf-like protein Ar11, and activated Ar11 in turn binds directly to and recruits GRIP domains. Orthologues of these proteins function similarly in mammalian cells. Using RNAi to ablate expression of tGolgin-1 from mammalian cells, we have shown that tGolgin-1 also participates in the association of the Golgi complex to the dynein/dynactin complex. This has important implications for a role of tGolgin-l in cell motility and polarization required for tumor development, metastasis, and immunity. We are now probing for the mechanism of this function of tGolgin- 1.
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ABSTRACT: Surfactant proteins A (SP-A) and D (SP-D), both members of the collectin family, play a well established role in apoptotic cell recognition and clearance. Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner. SP-A and SP-D bind in a Ca(2+)-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca(2+)-independent. Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules. Myeloperoxidase (MPO), a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis, was identified by affinity purification, mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D. To confirm its role in recognition, it was shown that purified immobilised MPO binds SP-A and SP-D, and that MPO is surface-exposed on late apoptotic neutrophils. SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells. Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils. Desmoplakin was identified as a further potential ligand for SP-A, and neutrophil defensin as a target for both proteins.Protein & Cell 06/2010; 1(6):563-72. DOI:10.1007/s13238-010-0076-0 · 2.85 Impact Factor
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ABSTRACT: Surfactant protein A (SP-A) has been shown to bind to and regulate the functions of both alveolar type II cells and immune cells including alveolar macrophages. The interaction of SP-A with type II cells has been shown in vitro to inhibit lipid secretion and to promote the uptake of lipid by these cells and these observations led to the hypothesis that SP-A plays an important role in regulating surfactant turnover and metabolism. The finding that mice made deficient in SP-A by homologous recombination (SP-A -/- mice) have relatively normal surfactant pool sizes has raised the possibility that either redundant mechanisms function in vivo to keep pool sizes normal in the absence of SP-A or that the in vitro findings are not significant in the context of the whole, unstressed animal. The interaction of SP-A with immune cells has been shown to affect a variety of responses which, in general, function to promote host defense against infection. Although SP-A receptors have been identified, additional studies will be required to elucidate the mechanism of interaction of SP-A with these cells and the relative importance of the different receptors in SP-A mediated regulation of cell function.Biochimica et Biophysica Acta 12/1998; 1408(2-3):241-63. DOI:10.1016/S0925-4439(98)00071-4 · 4.66 Impact Factor