Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

• MeSH
• Antigens, Surface MeSH
• Killer Cells, Natural * MeSH
• X-Ray Diffraction MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Humans MeSH
• Ligands MeSH
• Scattering, Small Angle MeSH
• Receptors, Cell Surface * MeSH
• Cluster Analysis MeSH
• Synapses MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface * MeSH

BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.

• Keywords
• Protein functionality, Protein quality control, Purification strategies, Recombinant proteins,
• MeSH
• Chromatography, Affinity MeSH
• Recombinant Proteins chemistry   genetics   MeSH
• Reproducibility of Results * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Recombinant Proteins MeSH

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

• Keywords
• B7-H6, HPMA polymer, NK cell, NKp30, coiled coil, immunotherapy,
• Publication type
• Journal Article MeSH

Phlebotomus argentipes is a predominant vector of Leishmania donovani, the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes, sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti-P. argentipes saliva IgG responses. The anti-rPagSP06 and anti-P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti-P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area.

• Keywords
• Bangladesh, IgG antibodies, Leishmania donovani, Marker of exposure, Phlebotomus argentipes, Salivary glands,
• MeSH
• Insect Proteins * immunology   MeSH
• Bites and Stings epidemiology   MeSH
• Leishmania donovani MeSH
• Humans MeSH
• Phlebotomus * MeSH
• Salivary Proteins and Peptides * immunology   MeSH
• Saliva MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Bangladesh epidemiology   MeSH
• Names of Substances
• Insect Proteins * MeSH
• Salivary Proteins and Peptides * MeSH

Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect vector. It transmits the unicellular parasite Leishmania infantum that multiplies intracellularly in macrophages causing life-threatening visceral diseases. Leishmania establishment in the vertebrate host is substantially influenced by immunomodulatory properties of vector saliva that are obligatorily co-injected into the feeding site. The repertoire of P. perniciosus salivary molecules has already been revealed and, subsequently, several salivary proteins have been expressed. However, their immunogenic properties have never been studied. In our study, we tested three P. perniciosus recombinant salivary proteins-an apyrase rSP01 and yellow-related proteins rSP03 and rSP03B-and showed their anti-inflammatory nature on the murine bone-marrow derived macrophages. Even in the presence of pro-inflammatory stimuli (IFN-γ and bacterial lipopolysaccharide, LPS), all three recombinant proteins inhibited nitric oxide production. Moreover, rSP03 seems to have a very strong anti-inflammatory effect since it enhanced arginase activity, increased the production of IL-10, and inhibited the production of TNF-α even in macrophages stimulated with IFN-γ and LPS. These results suggest that P. perniciosus apyrase and yellow-related proteins may serve as enhancing factors in sand fly saliva, facilitating the development of Leishmania infection along with their anti-haemostatic properties. Additionally, rSP03 and rSP03B did not elicit the delayed-type hypersensitivity response in mice pre-exposed to P. perniciosus bites (measured as visible skin reaction). The results of our study may help to understand the potential function of recombinant's native counterparts and their role in Leishmania transmission and establishment within the host.

• Keywords
• Phlebotomus, apyrase, immunogenicity, macrophage polarization, sand fly saliva, yellow-related proteins,
• MeSH
• Anti-Inflammatory Agents MeSH
• Phenotype MeSH
• Macrophages MeSH
• Mice MeSH
• Phlebotomus * MeSH
• Dogs MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Inflammatory Agents MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

• Keywords
• B7-H6, NK cell, NKp30, glycosylation, oligomerization,
• Publication type
• Journal Article MeSH

BACKGROUND: During blood feeding, sand flies inoculate salivary proteins that interact with the host haemostatic system. The blocking of biogenic amines such as serotonin and histamine helps to limit vasodilatation and clot formation, and thus enables the insect to finish the blood-feeding process. In sand flies, an amine-binding ability is known only for the yellow-related proteins of Phlebotomus and Lutzomyia vectors, but not yet for members of the genus Sergentomyia. METHODS: The ability of Phlebotomus argentipes and Sergentomyia schwetzi recombinant yellow-related salivary proteins to bind histamine and serotonin was measured by microscale thermophoresis. Both sand fly species were also fed through a chicken-skin membrane on blood mixed with histamine or serotonin in order to check the effects of biogenic amines on sand fly fitness. Additionally, fecundity and mortality were compared in two groups of P. argentipes females fed on repeatedly-bitten and naive hamsters, respectively. RESULTS: The P. argentipes recombinant yellow-related protein PagSP04 showed high binding affinity to serotonin and low affinity to histamine. No binding activity was detected for two yellow-related proteins of S. schwetzi. Elevated concentrations of serotonin significantly reduced the amount of eggs laid by P. argentipes when compared to the control. The fecundity of S. schwetzi and the mortality of both sand fly species were not impaired after the experimental membrane feeding. Additionally, there were no differences in oviposition or mortality between P. argentipes females fed on immunized or naive hamsters. CONCLUSIONS: Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or anti-saliva antibodies without any influence on their fitness. The serotonin binding by salivary yellow-related proteins may play an important role in Phlebotomus species feeding on mammalian hosts, but not in S. schwetzi, which is adapted to reptiles.

• Keywords
• Anti-saliva antibodies, Histamine, Mortality, Oviposition, Phlebotomus argentipes, Sergentomyia schwetzi, Serotonin, Yellow-related proteins,
• MeSH
• Biogenic Amines * blood   pharmacology   MeSH
• Fertility drug effects   MeSH
• Histamine blood   MeSH
• Insect Proteins chemistry   metabolism   MeSH
• Insect Bites and Stings immunology   MeSH
• Cricetinae MeSH
• Blood metabolism   MeSH
• Evolution, Molecular MeSH
• Mortality MeSH
• Phlebotomus metabolism   MeSH
• Reptiles MeSH
• Antibodies MeSH
• Psychodidae metabolism   MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Mammals MeSH
• Serotonin blood   MeSH
• Salivary Proteins and Peptides * chemistry   metabolism   MeSH
• Saliva immunology   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biogenic Amines * MeSH
• Histamine MeSH
• Insect Proteins MeSH
• Antibodies MeSH
• Recombinant Proteins MeSH
• Serotonin MeSH
• Salivary Proteins and Peptides * MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field.

• MeSH
• Insect Vectors parasitology   physiology   MeSH
• Immunoassay methods   MeSH
• Leishmania infantum physiology   MeSH
• Leishmaniasis blood   diagnosis   parasitology   veterinary   MeSH
• Mice, Inbred BALB C MeSH
• Dog Diseases blood   diagnosis   parasitology   MeSH
• Phlebotomus parasitology   physiology   MeSH
• Dogs MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• Keywords
• C-type lectin-like ligand, LLT1,
• MeSH
• Glycosylation MeSH
• Protein Structure, Quaternary MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Lectins, C-Type chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization * MeSH
• Receptors, Cell Surface chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Receptors, Cell Surface MeSH