“SP-G”, a Putative New Surfactant Protein – Tissue Localization and 3D Structure

a Putative New Surfactant Protein - Tissue Localization and 3D Structure. PLoS ONE 7(10): e47789. doi:10.1371/journal.pone.0047789 ''SP-G'', a Putative New Surfactant Protein - Tissue Localization and 3D Structure Felix Rausch 0 Martin Schicht 0 Friedrich Paulsen 0 Ivan Ngueya 0 Lars Bra uer 0 Wolfgang Brandt 0 Kyle Ramsey, Midwestern University, United States of America 0 1 Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry , Halle, Germany , 2 Institute of Anatomy, Department II, Friedrich Alexander University Erlangen-Nuremberg , Erlangen , Germany Surfactant proteins (SP) are well known from human lung. These proteins assist the formation of a monolayer of surfaceactive phospholipids at the liquid-air interface of the alveolar lining, play a major role in lowering the surface tension of interfaces, and have functions in innate and adaptive immune defense. During recent years it became obvious that SPs are also part of other tissues and fluids such as tear fluid, gingiva, saliva, the nasolacrimal system, and kidney. Recently, a putative new surfactant protein (SFTA2 or SP-G) was identified, which has no sequence or structural identity to the already know surfactant proteins. In this work, computational chemistry and molecular-biological methods were combined to localize and characterize SP-G. With the help of a protein structure model, specific antibodies were obtained which allowed the detection of SP-G not only on mRNA but also on protein level. The localization of this protein in different human tissues, sequence based prediction tools for posttranslational modifications and molecular dynamic simulations reveal that SP-G has physicochemical properties similar to the already known surfactant proteins B and C. This includes also the possibility of interactions with lipid systems and with that, a potential surface-regulatory feature of SP-G. In conclusion, the results indicate SP-G as a new surfactant protein which represents an until now unknown surfactant protein class. - . These authors contributed equally to this work. Surfactant proteins have been described in detail in relation with research on the lungs in which surface activity and immunological functions within both the specific and the nonspecific immune defenses are ascribed to them [1,2]. SP-A and SP-D are representatives of the C-type lectin family, in which other molecules with immunological properties can also be included. In accordance to the current understanding of the Ctype lectin mechanism, the proteins bind to specific carbohydrates of bacteria, protozoans, fungi and viruses [3,4]. This facilitates opsonization of and accelerated immune defense reactions to these microorganisms [57]. The presence of SP-A and SP-D with regard to their immunological function has been confirmed in various tissues, including human nasal mucosa, the digestive tract, tear ducts, salivary glands of the head and the gingiva [812]. In contrast to SP-A and SP-D, the small and extremely hydrophobic surfactant proteins SP-B and SP-C are essential components during formation of surfactant monolayers and the stabilization of air-fluid interfaces [1,13,14]. This extreme hydrophobicity of the surfactant proteins B and C is mostly obtained by posttranslational modifications. For example, the surfactant protein C is palmitoylated to increase its hydrophobic character [15]. Similar to SP-A and SP-D, the presence of SP-B and SP-C has already been demonstrated in a variety of tissues and humors, including tissues of the nasolacrimal apparatus and ocular surface, in tear fluid, in salivary glands, in the gingiva and in saliva [10,11,16]. While working with the four already known surfactant proteins, our attention was also attracted to another putative surfactant protein, which was identified by means of bioinformatic investigations and named surfactant protein G (SP-G) or surfactantassociated protein 2 (SFTA 2) [17]. The protein (SP-G) is encoded on the human chromosome 6, its primary theoretical translation product consist of 78 amino acid residues resulting in a molecular weight of approximately 8 kDa. This putative surfactant protein shows no sequential or structural similarities to surfactant proteins or other known proteins in general and therefore seems to represent a new group of proteins. Furthermore, there is no hard evidence or information neither on the organ or tissue distribution nor on the function of the protein. It is carrying an N-terminal signal peptide of 19 amino acid residues which is essential for protein secretion [18]. Therefore, there are probably other parts of the protein which show surface activity as well. Since there are only a few already known facts about this protein available, choosing the right experimental work for further characterization can be very difficult. In such cases, computational methods like the protein structure modeling or molecular dynamics (MD) simulations can be very helpful. The generation of a three-dimensional (3D) model of the yet unknown protein structure can give hints about the solubility of the protein or possible interactions with solutes of its environment like lipids, sugars or other proteins. Furthermore, the model can show which parts of the protein are exposed to the solvent and in that way are most likely to carry posttranslational modifications. These are probably essential for the protein function [19], as already described for the known surfactant proteins [15,20,21]. The behavior of a protein in solution and possible interactions with other nearby solutes can be investigated by MD simulations. This method can calculate the time-dependent state of a system and in that way give a hint which dynamic processes a protein could perform. There are already MD simulations described in the literature, which showed the detailed interaction of SP-B with lipid monolayers [22,23] and also demonstrated the crucial role of SP-B and SP-C for the preservation and formation of a stable lipid layer system on air-fluid surfaces [24,25]. Similarly, MD simulations with SP-G could show if this protein can also interact with single lipids or lipid layers and with that, has functions comparable to the already know surfactant proteins. The objective aim of our work was to combine both computational chemistry and experimental work to get further insights into the character and function of SP-G to show that this protein indeed has the potential to interact with lipid systems and is located in tissues where this functionality is very important (e.g. lung or ocular system). This will suggest SP-G in fact as a surfactant protein itself which represents an until now unknown surfactant protein class. Furthermore, the computational chemistry methods used in our work could assist during the development of potent antibodies to be able to investigate the tissue and organ specific distribution of the protein and therefore help to understand the function of this put (...truncated)