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Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

This work is a structure-activity relationship study that investigates the influence of the nature and amount of negative charge in carbohydrate substrates on the affinity of b-N-acetylhexos-A C H T U N G T R E N N U N G aminidases, and on the stimulation of natural killer cells. It describes synthetic procedures yielding novel glycosides that are useful in immunoactivation. Specifically, we present a thorough study on the ability of six C-6 modified b-N-acetylhexosaminides (aldehyde, uronate, 6-O-sulfate, 6-O-phosphate) to serve as substrates for cleavage and glycosylation by a library of b-N-acetylhexosaminidases from various sources. Four novel disaccharides with one or two (negatively) charged groups were prepared in synthetic reactions in good yields. Surprisingly, the 6-Ophosphorylated substrate, although cleaved by a number of enzymes from the series, worked neither as a donor nor as an acceptor in transglycosylation reactions. The results of wet experiments were supported by molecular modeling of substrates in the active site of two representative enzymes from the screening. All ten prepared compounds were examined in terms of their immunoactivity, namely as ligands of two activation receptors of natural killer (NK) cells, NKR-P1 and CD69, both with isolated proteins and whole cells. Sulfated disaccharides in particular acted as very efficient protectants of NK cells against activation-induced apoptosis, and as stimulants of the natural killing of resistant tumor cells, which makes them good candidates for potential clinical use in cancer treatment.

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Mouse Nkrp1a receptor is a C-type lectin-like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304-1311. © 2016 Wiley Periodicals, Inc.

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

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