Saposin Family Research Articles

Structure and regulation of the NK-lysin (1–4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss)

Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128–133 aa in length, and mature peptides of 109–111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a &b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2–3 fold) of five (nkl 2–4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading.

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work strongly indicates that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work strongly indicates that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

The Role of Domains and Proteins in the Function of Lung Surfactant

Lung surfactant forms a thin film at the gas exchange interface in alveoli. The film reduces the surface tension which is necessary for breathing. The film consists of a monolayer at the air/water interface connected with bilayer reservoirs in water. Lung surfactant is composed of a mixture of saturated and unsaturated, zwitterionic and anionic lipids; surfactant proteins B (SP-B) and C are associated with the interface. Surfactant proteins facilitate adsorption of lipids to the interface likely via stalk-like intermediates. The proteins are also believed to induce monolayer collapse by creating nucleation sites/fluidizing effect. Lipid components in monolayer segregate into domains of coexisting phases, which is suggested to increase monolayer stability. However, the exact mechanisms of these effects are still not fully understood.We study the role of phase coexistence and proteins in the function of lung surfactant. Molecular dynamics simulations with the coarse-grained model MARTINI are employed. We simulate mixtures of saturated and unsaturated phosphatidylcholine and phosphatidylglycerol lipids, cholesterol and SP-B proteins. The model of SP-B is built based on homology with the saposin family and was shown previously to mediate early stages of vesicle fusion [S Baoukina, DP Tieleman, Biophys J, 2010]. We reproduce phase separation into liquid-expanded (LE) and liquid-condensed (LC) or liquid ordered (Lo) phases in monolayers. SP-B partitions into the LE phase and prevents formation of the LC phase at conditions close to the phase transition. The protein also enhances lipid de-mixing. SP-B dimers induce bilayer folds in monolayers at positive surface tensions below the equilibrium spreading value. Monolayer collapse is initiated from the LE phase, and the composition of bilayer folds differs from the monolayer. SP-B anchors monolayers and bilayers and promotes lipid transfer.

IR Spectroscopy of Protein and Peptide-Membrane Interactions: In Situ Strategies for Tracking Fusion, Remodelling, and Refolding

Attenuated total reflection-infrared spectroscopy is a powerful technique for characterizing protein and peptide-membrane interactions. Using a real-time, flow-through design and a single bounce diamond ATR element, we have examined how three different families of proteins interact with model supported lipid bilayers. These include (1) a lipid-remodelling protein from the saposin family; (2) the dynamin-related GTP-ase Mgm1p; (3) a series of model membranolytic peptides designed to adopt a helical motif in bacterial membranes. Our studies provided compelling evidence of the action of these proteins, including membrane remodeling, GTP-ase activity, and peptide aggregation. We will describe the integration of laser-based fluorescence spectroscopy with ATR-IR, which, when coupled with correlated in situ atomic force microscopy, will afford a uniquely capable multimodal imaging and characterization platform.

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.

Pulmonary surfactant protein B: a structural model and a functional analogue

Surfactant proteins B and C (SP-B and SP-C), together with phospholipids, are important constituents of pulmonary surfactant and of preparations used for treatment of respiratory distress syndrome (RDS). SP-B belongs to the saposin family of homologous proteins, which include other lipid-interacting proteins, like the membranolytic NK-lysin. SP-B, in contrast to other saposins, is hydrophobic and a disulfide-linked dimer, and its mechanism of action is not known. A model of the three-dimensional structure of one SP-B subunit was generated from the structure of monomeric NK-lysin determined by nuclear magnetic resonance, and the SP-B dimer was formed by joining two subunits via the intersubunit disulfide bond Cys48–Cys48′. After energy minimization, intersubunit hydrogen bonds/ion pairs were formed between the strictly conserved residues Glu51 and Arg52, which creates a central non-polar region located in between two clusters of positively charged residues. The structural features support a function of SP-B in cross-linking of lipid membranes. Mixtures of phospholipids, an SP-C analogue and polymyxin B (which cross-links lipid vesicles but is structurally unrelated to SP-B) exhibit in vitro surface activity which is indistinguishable from that of analogous mixtures containing SP-B instead of polymyxin B. This suggests an avenue for identification of SP-B analogues that can be used in synthetic surfactants for treatment of RDS.