N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase.

beta-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse-chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line.

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

Lymphocyte activation receptors: new structural paradigms in group V of C-type animal lectins.

The structure-function relationship in group V of C-type animal lectins remains incompletely understood despite the new structures of NK (natural killer) cell receptors that have been solved recently. Recombinant, soluble forms of rat and human NKR-P1 and CD69 that we obtained after in vitro refolding were analysed by Fourier transform-ion cyclotron resonance MS and heteronuclear NMR ((1)H-(15)N correlation). In NKR-P1, calcium may not be removed by chelating agents because of the very high affinity of binding. In CD69, incorporation of calcium causes a structural shift in several amino acids important for the interaction with carbohydrates. Structural studies have also allowed us to understand an interesting preference of these receptors for either linear (NKR-P1) or branched (CD69) carbohydrate sequences.

Toward an optimal oligosaccharide ligand for rat natural killer cell activation receptor NKR-P1.

Aminosugars have a good affinity for the NKR-P1A protein, the major activating receptor at the surface of rat natural killer cells. We have systematically investigated the structural requirements of the recombinant soluble dimeric form of the receptor for its optimal carbohydrate ligands. While N-acetylD-mannosamine was the best neutral monosaccharide ligand, its participation in the context of an extended oligosaccharide sequence was equally important. The IC(50) value for the GalNAcbeta1 --> ManNAc disaccharide was nearly 10(-10) M with a further possible increase depending on the type of the glycosidic linkage and the aglycon nature. From the point of view of its availability, stability, and affinity for the receptor and a potential in vivo use, these studies are pivotal for the design of an oligosaccharide or glycomimetics suitable for further clustering into the multivalent glycodendrimers.

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4.

The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2-(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5-15. The enzyme functioned in the presence of 5-98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of n-hexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1.

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography.

We describe a 5-year-old boy with a unique congenital cataract caused by deposition of numerous birefringent, pleiochroic and macroscopically prismatic crystals. Crystal analysis with subsequent automatic Edman degradation and matrix-associated laser desorption ionization time-of-flight mass spectrometry have identified the crystal-forming protein as gammaD-crystallin (CRYGD) lacking the N-terminal methionine. Sequencing of the CRYGD gene has shown a heterozygous C-->A transversion in position 109 of the inferred cDNA (36R-->S transversion of the processed, N-terminal methionine-lacking CRYGD). The lens protein crystals were X-ray diffracting, and our crystal structure solution at 2.25 A suggests that mutant R36S CRYGD has an unaltered protein fold. In contrast, the observed crystal packing is possible only with the mutant protein molecules that lack the bulky Arg36 side chain. This is the first described case of human cataract caused by crystallization of a protein in the lens. It involves the third known mutation in the CRYGD gene but offers, for the first time, a causative explanation of the phenotype.

Cancer immunomodulation by carbohydrate ligands for the lymphocyte receptor NKR-P1.

Natural killer (NK) and T cells express a superfamily of proteins with structural features of C-type lectins. Recombinantly prepared soluble form of rat NKR-P1 (CD161) recognized carbohydrate GalNac and GlcNac moieties. Ganglioside GM2 and heparin related-IS oligosaccharides representing the high affinity ligands for this receptor, increased the sensitivity of targets for killing by the rat effectors isolated from blood and spleen in vitro. Based on these results, we investigated in vivo the possible therapeutic effect of GM2 and IS carried by liposomes during induced rat colorectal carcinogenesis. The reduction of cancer incidence versus the controls (50% vs 88.88%), approached the 5-fluorouracil treatment (41.66%).

Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin-resistant boar seminal plasma O-glycoprotein related to pB1 protein.

The complete covalent structure of a novel boar DQH sperm surface protein resistant to many classical procedures of enzymatic fragmentation was determined. The relative molecular mass of the major form of this protein determined by ESI-MS and MALDI-MS was 13,065.2+/-1.0 and 13,065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N-acetylhexosamine), 146 Da (i.e., plus deoxyhexose), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protein was determined using automated Edman degradation, MALDI-MS, and post-source decay (PSD) MALDI-MS, and shown to consist of N-terminal O-glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O-glycosidically linked to threonine 10, as confirmed by PSD MALDI-MS of the isolated N-terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALDI-MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent structure of proteins, especially those that are refractory to standard approaches of protein chemistry.

Peptide fragments induce a more rapid immune response than intact proteins in earthworms.

The effect of in vivo proteolytic processing of protein antigen was studied in Eisenia foetida earthworms. Parenteral administration of the protein antigen induces elevated levels of an antigen-binding protein (ABP) which recognizes the protein used for stimulation. When the protein antigen is administered simultaneously with nontoxic serine proteinase inhibitor, ABP levels remain close to background. On the other hand, the in vivo adaptive response of earthworms to peptide fragments obtained by coelomic fluid digestion of the foreign antigen occurs even in the presence of proteinase inhibitor and, moreover, is significantly faster as compared to the response to intact antigen. These findings confirm the role of proteolytic processing in earthworms. MALDI mass spectrometric analysis of the fragments after coelomic fluid digestion has revealed the presence of the peptide fragments with molecular weights in the mass range 700-1100 Da.

Structure analysis of trivalent glycoclusters by post-source decay matrix-assisted laser desorption/ionization mass spectrometry.

Post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectra were found to be useful for the structural elucidation of a series of tris [2-(glycosylthiourylene)ethyl]amines. The reported fragmentation behaviours of [M + H]+, [M + Na]+ and [M - H]- ions differ from each other significantly; however, they can be compared to tree pruning in every case. Whereas detailed structural information on unprotected glycoclusters is obtained from all PSD experiments, only the positive-ion mode can be used to gain relevant information about the acetylated glycoclusters.

GlcNAc-terminated glycodendrimers form defined precipitates with the soluble dimeric receptor of rat natural killer cells, sNKR-P1A.

Synthetic GlcNAc-terminated thiourea-bridged glycoclusters were found to be potent inhibitors of binding of the soluble dimeric receptor of rat natural killer cells, sNKR-P1A protein, to its high affinity ligand. Moreover, we have shown here that characteristic precipitation curves can be recorded upon mixing of the GlcNAc glycoclusters with sNKR-P1A. For the GlcNAc8 glycocluster the precipitation curve is biphasic, with high affinity and low affinity precipitates differing in their sensitivity towards GlcNAc-mediated inhibition of precipitation. Quantitative analyses of the precipitates indicate the occurrence of a single sugar binding site per sNKR-P1A subunit, and lead to a model of the most possible spatial arrangements of the glycocluster-receptor lattices. These results provide new tools for further studies on carbohydrate recognition by NKR-P1A.

Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma.

Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.

NKR-P1A protein, an activating receptor of rat natural killer cells, binds to the chitobiose core of uncompletely glycosylated N-linked glycans, and to linear chitooligomers.

NKR-P1 represent a family of activating receptors in rodent natural killer cells related to C-type animal lectins. We identify here the elements involved in the reactivity of the major receptor of rat, NKR-P1A, with N-linked oligosaccharides of glycoproteins. Plate inhibition assays with isolated, structurally defined N-glycans as inhibitors of binding of NKR-P1A to GlcNAc16-BSA revealed that the removal of both the external sialic acids and the penultimate galactose residues resulted in attaining of significant inhibitory activities. Surprisingly, additional plate inhibition and glycoprotein overlay experiments brought evidence that the core chitobiose, depending on its substitution, can per se support the interaction with NKR-P1A. In a series of linear chitooligomers (n = 2-7), the inhibitory activities reached a maximum for the chitotetraose. The ability of NKR-P1 to recognize both the periphery and the core region of complex type oligosaccharides may define its dual specificity towards carbohydrate components of eukaryotic (e.g., tumor) cell surfaces, but also reflect an evolutionarily conserved reactivity with microbial saccharides important in immune recognition and signaling functions.

Association of human NK cell surface receptors NKR-P1 and CD94 with Src-family protein kinases.

Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors.

beta-Glucosylation of chitooligomers by galactosyltransferase.

Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.

CD 69 antigen of human lymphocytes is a calcium-dependent carbohydrate-binding protein.

CD69 is a signal transducing molecule of hematopoietic cells. Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins. As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V. To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion. We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization. We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein. Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group V.