Saposin-like Protein Family Research Articles

Characterization of NK-lysin A, a potent antimicrobial peptide from the zebrafish Danio rerio

Antimicrobial peptides (AMPs) are important players of the innate immune system with a major role in the defense against invading pathogens. AMPs belonging to the family of saposin-like proteins (SAPLIPs) include the porcine NK-lysin and the human granulysin. In the zebrafish Danio rerio, transcript analyses of NK-lysin encoding genes have been reported, but biochemical characterizations at the protein level are missing so far. Here, we present the recombinant expression, purification, and characterization of one of these homologs, namely of NK-lysin A (DaNKlA). To remove the affinity tag from DaNKlA, we made use of a self-splicing intein. Recombinant DaNKlA depolarized liposomes over a broad pH range and showed a preference for negatively charged lipids. DaNKlA inhibited the growth of and killed different Gram-positive and Gram-negative bacteria, including the fish pathogenic bacterium Vibrio anguillarum, by membrane permeabilization but displayed substantially lower activity against yeast cells. Structural modelling and bioinformatic comparison of DaNKlA with characterized SAPLIPs suggest membrane destabilization accompanied by strong electrostatic interactions as the mode of action.

SPP-5 affects larval arrest via insulin signaling pathway in Caenorhabditis elegans.

Diapause is an endocrine-mediated metabolic and growth arrest state in response to unfavorable external environments. The nematode Caenorhabditis elegans can enter diapause/arrest during embryonic, larval, or adult stages when subjected to detrimental external environments.Larval stage 1 (L1) arrest happens when animals hatch without food. Previous work has shown that the insulin pathway plays a prominent role in regulating L1 arrest. However, the downstream signal molecular mechanisms and biomarkers are still missing. In this study, we showed that SaPosin-like Protein family member SPP-5 is significantly upregulated during L1 arrest, suggesting that it could act as an L1 arrest biomarker. Using RNA interference we demonstrated that spp-5 knockdown accelerated larval development, while the overexpression resulted in L1 arrest. Consistently,SPP-5 level was significantly up-regulated in the L1 arrest daf-2(e1370) mutants, and spp-5(RNAi) suppressed the daf-2(e1370) induced L1 arrest. These results suggest that SPP-5 can serve as an L1 arrest biomarker and promote the arrest probably via the insulin signaling pathway.

Identification of a linear B-cell epitope on the Schistosoma japonicum saposin protein, SjSAP4: Potential as a component of a multi-epitope diagnostic assay.

BackgroundSchistosoma japonicum is one of three major species of blood flukes causing schistosomiasis, a disease, which continues to be a major public health issue in the Philippines. SjSAP4, a member of a multigene family of saposin-like proteins, is a recognized immunodiagnostic biomarker for schistosomiasis japonica. This study aimed to identify linear B-cell epitopes on SjSAP4 and to validate their potential as components of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica.MethodologySjSAP4-derived peptides were expressed as GST-peptide-fused proteins and these were Western blot probed with human serum samples from S. japonicum Kato-Katz (KK)-positive individuals and uninfected controls. A core epitope was further identified by Western blotting through probing a series of truncated peptides with the schistosomiasis patient sera. The diagnostic performance of the core epitope-containing peptides and the full-length SjSAP4 was evaluated by enzyme-linked immunosorbent assay (ELISA) using a panel of sera collected from subjects resident in a schistosomiasis-endemic area of the Philippines.Main findingsAs a result of the peptide mapping, one peptide (P15) was found to be highly immunogenic in the KK-positive individuals. We subsequently showed that -S163QCSLVGDIFVDKYLD178- is a core B-cell epitope of P15. Subsequent ELISAs incorporating SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide showed a sensitivity of 94.0%, 46.0% and 74.0%, respectively, and a specificity of 97.1%, 100% and 100%, respectively. Notably, complementary recognition of the B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of the KK-positive individuals. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a diagnostic sensitivity of 84.0% and a specificity of 100%.Conclusions/SignificanceIn this study, -S163QCSLVGDIFVDKYLD178- was identified as a dominant linear B-cell epitope on SjSAP4. This peptide and the complementary recognition of other B-cell epitopes using sera from different KK-positive individuals can provide the basis of developing a multi-epitope assay for the serological diagnosis of schistosomiasis.

Mechanistic insights into the lipid interaction of an ancient saposin-like protein.

The members of the expanding family of saposin-like proteins (SAPLIPs) have various biological functions in plants, animals, and humans. In addition to a similar protein backbone, these proteins have in common the fact that they interact with lipid membranes. According to their phylogenetic position, it has long been thought that amoeboid protozoans produce archetypes of SAPLIPs and that these are lytic proteins that can perforate membranes of prokaryotic and eukaryotic target cells. Here, we show that an amoebic SAPLIP from Entamoeba invadens does not form lytic pores in membranes but displays several characteristics that are known from human saposins. The protein named invaposin changes the conformation from a closed to an open form in the presence of lipid membranes, acts in a pH-dependent manner, selectively binds anionic lipids, aggregates lipid vesicles of the preferred composition, and dimerizes upon acidification. Our data indicate that the principal features of the lipid-binding saposins evolved long before the appearance of the vertebrate lineage and push the origin of saposins even deeper down the phylogenetic tree to unicellular organisms.

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

Antimicrobial Activity of Chicken NK-Lysin Against Eimeria Sporozoites

NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role in innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we examined its ability to reduce the viability of various bacterial strains and two species of Eimeria parasites. Culture supernatants from COS7 cells transfected with a chicken NK-lysin cDNA and His-tagged purified NK-lysin from the transfected cells both showed high cytotoxic activity against Eimeria acervulina and Eimeria maxima sporozoites. In contrast, no bactericidal activity was observed. Further studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry.

Novel Antiinflammatory and Antiinfective Agents

Despite the availability of antibiotics, infectious diseases become an even increasing threatening for human health, in particular due to resistance development, for example in animal husbandry. Furthermore, the sepsis shock syndrome, which in many cases is a result of the fact that antibiotics may kill bacteria, but do not inactivate bacterial virulence factors such as endotoxins (lipopolysaccharides), is worldwide of increasing importance regarding the very high death rate (40 to 50 %). Therefore, the development of suitable antimicrobial drugs is urgently requested. In recent years, various groups focus on the use of antimicrobial peptides (AMP) derived from natural, innate immunity proteins, which in vivo bind to the virulence factors. Such binding proteins are, for example, lactoferrin, the Limulus anti-LPS-factor, and the family of saposin-like proteins (NK-lysins, granulysins), which all have particular binding domains for example for bacterial endotoxin. Furthermore, an alternative approach to combat infections is the use of cyclooxygenase and lipoxygenase inhibitors. From the reviewed literature the mechanisms of action of antiinfective compounds on bacteria, on pathogenicity factors of the bacteria, and on viruses are summarized. In the last years, considerable progresses have been made in the fight against infections, specially also to overcome bacterial resistance. Keywords: Antimicrobial peptides (AMP), lipopolysaccharide (LPS), endotoxins, tumor-necrosis-factor-α, cyclooxygenase (COX), lipoxygenase (LOX)

Mapping of B-cell epitopes on a novel 11.5-kilodalton Fasciola hepatica-Schistosoma mansoni cross-reactive antigen belonging to a member of the F. hepatica saposin-like protein family.

FhSAP-2 is a novel member of the Fasciola hepatica saposin-like protein family that induces protection in rabbits against a challenge infection. We investigated the presence of lineal B-cell epitopes within this protein using a set of overlapping synthetic peptides. Peptides were tested in enzyme-linked immunosorbent assays against sera from rabbits infected with F. hepatica. Two dominant epitopes were identified, which were also highly reactive with sera from mice infected with Schistosoma mansoni. These peptides may be suitable to incorporate into a polyepitope-based vaccine formulation against F. hepatica.

The saposin-like proteins 1, 2, and 3 of Fasciola gigantica

The SAP genes of Fasciola encode proteins belonging to the saposin-like protein family. The saposin signature, a compact domain of mainly α-helical character, contains six conserved cysteine residues and has been implicated in membrane-binding, pore formation, and subsequent cell lysis in several family members. Recombinant SAP-2 of F. hepatica has been shown to induce lysis of human erythrocytes and peripheral blood mononuclear cells. This suggests that the SAPs are involved in the nutrition of Fasciola as the released content of lysed host cells is available for further enzymatic processing and uptake by the parasite. In the present study a new SAP-3 cDNA was obtained in an immunoscreen of an adult stage F. gigantica cDNA library with an antiserum against the parasite's excretion/secretion antigens. SAP-1 and SAP-2 cDNAs were isolated from F. gigantica cDNA libraries using oligonucleotide primers specific to the SAP-1 and SAP-2 DNA sequences from F. hepatica. Transcripts of the three SAPs are present from the metacercarial to the adult stage and are located to the gut epithelium. In immatures SAP-1 RNA is the predominant product whereas in adults SAP-2 and -3 are the more abundant products. Polyclonal anti-SAP-1 and SAP-2 antisera confirmed the tissue-specificity and revealed the subcellular localization of SAPs in large granules concentrated in the apical part of the gut epithelial cells of the parasite. Interestingly, evolutionary conservation of the Fasciola SAP sequences among other trematodes is low at 20–30% sequence identity comparable to the Entamoeba amoebapore sequences.

A novel saposin-like protein of Entamoeba histolytica with membrane-fusogenic activity

Amoebapores, the pore-forming proteins of Entamoeba histolytica, are major pathogenicity factors of the parasite. Upon a comprehensive survey in the recently completed genome data sets for the protozoon, we identified in addition to the three amoebapore genes, 16 genes which are constitutively expressed and code for structurally similar proteins, all belonging to the family of saposin-like proteins. Here, we recombinantly expressed in bacteria a defined single entity of this expansive amoebic protein family, namely SAPLIP 3. The protein consists of the saposin-like domain only, comparable to amoebapores, and we characterized its interactions with membranes using different assays. In contrast to amoebapores, SAPLIP 3 neither forms pores in liposomes nor permeabilizes bacterial membranes. However, SAPLIP 3 induces leaky fusion of lipid vesicles as evidenced by fluorescence microscopic analysis and by using a fusion assay that monitors the dequenching of a lipophilic dye. The membrane-fusogenic activity of SAPLIP 3 which is dependent on the presence of negatively charged lipids and on acidic pH resembles in combination with the negative surface charge of the protein characteristics of human saposin C. Beside its function as a cofactor of sphingolipid hydrolysing enzymes, the human protein is considered to be involved in the reorganization of lysosomal compartments due to its fusogenic activity. We hypothesize that in the amoeba, SAPLIP 3 fulfils a similar function in the multifarious endo- and exocytotic transport processes.

Fasciola hepatica: Humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG 1, IgG 2, and IgE as well as high levels of IL-10 and IFNγ indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG 2a isotype antibody as well as a dominant IFNγ cytokine, with significant IgE, IgG 1, and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.

Molecular Characterization of a Saposin-Like Protein Family Member Isolated from Bovine Lymphocytes

Human and porcine T lymphocytes and natural killer (NK) cells produce antibacterial proteins that belong to the saposin-like family of proteins (SAPLIP). The objective of this study was to determine if a bovine homolog of SAPLIP exists in lymphocytes that exhibit antibacterial activity. Following stimulation with IL-2, bactericidal activity against Staphylococcus aureus was detected to some extent in most major subpopulations of T lymphocytes including CD4+, CD8+, CD3+, and WC1+ γδ T lymphocytes. However, the majority of antibacterial activity was observed in the CD2+CD3− lymphocytes, which are similar phenotypically to NK cells. A partial sequence of a bovine SAPLIP was generated using low specificity primers designed from regions of homology between other SAPLIP including porcine NK-lysin and human granulysin. Enhanced expression of the bovine lysin gene was detected in mRNA isolated from IL-2–stimulated CD2+CD3− lymphocytes. The partial cDNA sequence was then used to make gene specific primers for a rapid amplification of cDNA ends (RACE) procedure that provided repeatable 5′ and 3′ cDNA ends. By examining overlapping regions from the RACE procedure, full-length sequence information was obtained for the bovine lysin homologue. Conceptual translation of the cDNA demonstrated conserved similarities to known SAPLIP members. Further characterization of the bovine lysin may facilitate its use in protecting dairy cattle against bacterial infections.

A novel Fasciola hepatica saposinlike recombinant protein with immunoprophylactic potential.

A member of the Fasciola hepatica saposinlike/NK-lysin protein family with lytic activity on human peripheral blood mononuclear cells and erythrocytes was recently described. The current study was designed to test the immunoprophylactic potential of this protein termed FhSAP-2 against infection with F. hepatica in rabbits. Two doses of 50 microg of recombinant FhSAP-2 (rFhSAP-2) emulsified in TiterMax were injected subcutaneously on the dorsal surface of 4 rabbits at 2-wk intervals. Four weeks after the second immunization, the rabbits were infected orally with 25 F. hepatica metacercariae. Four non-immunized-infected rabbits were used as controls. An enzyme-linked immunosorbent assay revealed high levels of antibodies to both rFhSAP-2 and F. hepatica excretory-secretory antigens by 2 wk after the first immunization, which were always significantly higher in immunized-infected rabbits than in control-infected rabbits. On the completion of the trial, vaccinated rabbits had 81.2% less flukes than controls. Moreover, F. hepatica egg counts in feces, as well as in bile collected from the gall bladders from vaccinated animals, were lower, 83.8 and 73%, respectively, compared with controls. The vaccinated rabbits also had significantly lower amounts of parasite antigen in stool and bile samples than controls. Last, evaluation of macroscopic liver lesions revealed that the rabbits vaccinated with rFhSAP-2 had milder lesions than the infected-control rabbits. These findings support the hypothesis that this novel rFhSAP-2 protein has immunoprophylactic potential against fascioliasis in rabbits including antifecundity and antipathology effects. This is the first report on experimental vaccination of rabbits against F. hepatica with a purified, defined, recombinant protein related to a member of the saposinlike protein family.

Interaction of amoebapores and NK-lysin with symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers.

Amoebapores from protozoan parasite Entamoeba histolytica and NK-lysin of porcine cytotoxic lymphocytes belong to the same family of saposin-like proteins. In addition to the structural similarity, amoebapores and NK-lysin are both highly effective against prokaryotic and eukaryotic target cells in that they permeabilize the target cell membranes. Here, we have investigated in detail the protein/lipid interaction for the three isoforms of amoebapore and NK-lysin. Results obtained from electrical measurements on planar bilayer membranes, including reconstitution models of the lipid matrix of the outer membrane of Escherichia coli and phospholipid membranes, fluorescence energy transfer spectroscopy with liposomes, and monolayer measurements on a Langmuir trough, provided information on lipid preferences, pH dependences, and membrane interaction mechanisms. The three amoebapores led to the formation of transient pores with similar characteristics in conductance, sublevels, and lifetime for the different isoforms. The conductance of the pores was dependent on the polarity of the applied clamp voltage, and the distribution of the sublevels was affected by the value of the clamp voltage. The size of the pores and distribution of conductance sublevels differed between symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers. Notably, NK-lysin caused the formation of well-defined pores, which were lipid- and voltage-dependent, and their characteristics differed from those induced by amoebapores; e.g., the protein concentration necessary to induce pore formation was 20 times higher. The biophysical data give important information on the mode of action of these small effector proteins, which may further lead to a better understanding of peptide-membrane interactions in general.

Novel putative saposin-like proteins of Entamoeba histolytica different from amoebapores

Amoebapores, the pore-forming proteins of Entamoeba histolytica, have been shown to play a pivotal role in the pathogenicity of the protozoan parasite. They belong to the functionally diverse family of saposin-like proteins (SAPLIPs) characterized by a conserved pattern of cysteine residues and the ability to interact with lipids. Here, we report the identification of genomic sequences encoding presumably novel SAPLIPs in E. histolytica and classify them in the structural and functional context provided by known family members. The genes of altogether 15 SAPLIPs are transcribed in the axenically cultured trophozoites as evidenced by reverse transcriptase–polymerase chain reaction. Interestingly, a remarkable sequence variety with a strong resemblance to that of known, functionally diverse SAPLIPs is present in this archaic, unicellular organism.

Amoebapore homologs of Caenorhabditis elegans

It is shown that the Caenorhabditis elegans genome contains several distantly related members of the gene family of saposin-like proteins. The putative products of genes T07C4.4, T08A9.7A, T08A9.7B, T08A9.8, T08A9.9, T08A9.10 are similar to the amoebapores of Entamoeba histolytica, granulysin of cytotoxic T lymphocytes and a putative amoebapore-related protein of the liver fluke Fasciola hepatica inasmuch as they consist of only a single saposin-like domain and a secretory signal peptide. The saposin-like domain of protein T07C4.4, which is most closely related to NK-lysin and granulysin, has been expressed in Escherichia coli and the recombinant protein was shown to have a circular dichroism spectrum consistent with the helix bundle structure characteristic of saposin-like domains. Recombinant T07C4.4 protein was found to have antibacterial activity, suggesting that these amoebapore homologs may play a role in antibacterial mechanisms of C. elegans.

Secondary structure and limited proteolysis give experimental evidence that the precursor of pulmonary surfactant protein B contains three saposin-like domains

The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP-B polypeptide, which belongs to the family of saposin-like proteins and has unique functional roles in pulmonary surfactant. From sequence comparisons it has been suggested that proSP-B, in addition to SP-B, contains two saposin-like domains, but their existence has until now not been experimentally verified. The 381 residue human proSP-B was now fused to an N-terminal poly-His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography. Recombinant proSP-B thus obtained exhibits about 35% α-helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin-like domains. These results experimentally support that proSP contains, in addition to SP-B, two further saposin-like domains.