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.

• Klíčová slova
• C-type lectin-like ligand, LLT1,
• MeSH
• glykosylace MeSH
• kvarterní struktura proteinů MeSH
• lektinové receptory NK-buněk - podrodina B chemie   genetika   metabolismus   MeSH
• lektiny typu C chemie   genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu * MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• receptory buněčného povrchu MeSH

Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.

• Klíčová slova
• C-type lectin-like, Glycosylation, HEK293S GnTI(−), LLT1, NK cell, Transfection,
• MeSH
• buňky NK metabolismus   MeSH
• disulfidy metabolismus   MeSH
• DNA metabolismus   MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• krystalizace MeSH
• lektiny typu C chemie   izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• N-acetylglukosaminyltransferasy metabolismus   MeSH
• polyethylenimin chemie   MeSH
• polysacharidy metabolismus   MeSH
• rozpustnost MeSH
• roztoky MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• terciární struktura proteinů MeSH
• transfekce metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• DNA MeSH
• lektiny typu C MeSH
• N-acetylglukosaminyltransferasy MeSH
• polyethylenimin MeSH
• polysacharidy MeSH
• roztoky MeSH

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
• antigeny povrchové MeSH
• buňky NK * MeSH
• difrakce rentgenového záření MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• lidé MeSH
• ligandy MeSH
• maloúhlový rozptyl MeSH
• receptory buněčného povrchu * MeSH
• shluková analýza MeSH
• synapse MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu * MeSH

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI- cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.

• Klíčová slova
• CD161, HEK293, LLT1, NK cells, NKR-P1, klrb1,
• MeSH
• bioreaktory MeSH
• buňky NK metabolismus   MeSH
• buňky Th17 metabolismus   MeSH
• Escherichia coli genetika   MeSH
• HEK293 buňky MeSH
• lektinové receptory NK-buněk - podrodina B biosyntéza   genetika   izolace a purifikace   MeSH
• lektiny typu C metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• receptory buněčného povrchu metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu MeSH