X-ray structure and mutagenesis analyses of Clostridioides difficile endolysin Ecd09610 glucosaminidase domain.

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove.

Crystal structure and conformational stability of a galectin-1 tandem-repeat mutant with a short linker.

Modification of the domain architecture of galectins has been attempted to analyze their biological functions and to develop medical applications. Several types of galectin-1 repeat mutants were previously reported but, however, it was not clear whether the native structure of the wild type was retained. In this study, we determined the crystal structure of a galectin-1 tandem-repeat mutant with a short linker peptide, and compared the unfolding profiles of the wild type and mutant by chemical denaturation. The structure of the mutant was consistent with that of the dimer of the wild type, and both carbohydrate-binding sites were retained. The unfolding curve of the wild type with lactose suggested that the dimer dissociation and the tertiary structure unfolding was concomitant at micromolar protein concentrations. The midpoint denaturant concentration of the wild type was dependent on the protein concentration and lower than that of the mutant. Linking the two subunits significantly stabilized the tertiary structure. The mutant exhibited higher T-cell growth-inhibition activity and comparable hemagglutinating activity. Structural stabilization may prevent the oxidation of the internal cysteine residue.

Modulation of the carbohydrate-binding specificity of two Xenopus proto-type galectins by site-directed mutagenesis.

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 ß-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.

Structures of human galectin-10/monosaccharide complexes demonstrate potential of monosaccharides as effectors in forming Charcot-Leyden crystals.

The galectins are a family of ß-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Galectin-9 induces atypical ubiquitination leading to cell death in PC-3 prostate cancer cells.

Galectin-9 is the most potent inducer of cell death in lymphomas and other malignant cell types among the members of the galectin family. We investigated the mechanism of galectin-9-induced cell death in PC-3 prostate cancer cells in comparison with in Jurkat T cells. Galectin-9 induced apoptotic cell death in Jurkat cells, as typically revealed by DNA ladder formation. On the other hand, DNA ladder formation and other features of apoptosis were not apparent in PC-3 cells undergoing galectin-9-induced death. Exogenous galectin-9 was endocytosed and destined to the lysosomal compartment in PC-3 cells. The internalized galectin-9 was resistant to detergent solubilization but was solubilized with lactose. Agents inhibiting actin filament dynamics abolished the internalization and cytocidal effect of galectin-9 in PC-3 but not Jurkat cells. Galectin-9 induced accumulation of ubiquitinated proteins, possibly heterogeneously ubiquitinated and/or monoubiquitinated proteins, in PC-3 cells. PYR-41, an inhibitor of the ubiquitin-activating E1 enzyme, suppressed the cytocidal effect of galectin-9. Although ubiquitination was upregulated also in Jurkat cells by galectin-9, PYR-41 was ineffective against galectin-9-induced cell death. Colocalization of ubiquitinated proteins and LAMP-1 was detectable in PC-3 cells treated with galectin-9. The ubiquitinated proteins were recovered in the insoluble fraction upon cell fractionation. In contrast, ubiquitinated proteins that accumulated after treatment with proteasome inhibitors did not co-localize with LAMP-1 and were mainly recovered in soluble fraction. The results suggest that atypical ubiquitination and accumulation of ubiquitinated proteins in lysosomes play a pivotal role in galectin-9-induced non-apoptotic death in PC-3 cells.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9.

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.

Crystal structure of a Xenopus laevis skin proto-type galectin, close to but distinct from galectin-1.

Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer-tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer-dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galß1-3GalNAc and not Galß1-4GlcNAc, while xgalectin-Ib preferred Galß1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines.

BACKGROUND: There is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients. METHODS: Galectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state. RESULTS: Galectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80~10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by~90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth. CONCLUSIONS: Among the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact. GENERAL SIGNIFICANCE: The study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.

Optimization of the inter-domain structure of galectin-9 for recombinant production.

We previously developed a stable form of galectin-9, an immunomodulatory animal lectin with a truncated linker peptide (G9Null), to overcome the protease sensitivity of wild-type galectin-9. G9Null is highly resistant to proteolysis, while the modification marginally improved the low solubility of the wild-type protein. To increase its solubility, we further modified the remaining linker region of G9Null. A 10-amino acid deletion with a single amino acid substitution resulted in an ∼400% increase in solubility and yield without an adverse effect on its biological activity. This mutant protein might be useful for large-scale recombinant production needed for evaluation of the therapeutic potential of galectin-9.

Self-association of the galectin-9 C-terminal domain via the opposite surface of the sugar-binding site.

Galectin-9 is a lectin, which has various biological functions such as T-cell differentiation and apoptosis. Multivalency of carbohydrate binding is required for galectin-9 to function. Although galectin-1 (a proto-type galectin) forms an oligomer to obtain its multivalency, galectin-9 (a tandem-repeat-type one) has two carbohydrate recognition domains (CRD) in one polypeptide. However, a single CRD of galectin-9, especially the C-terminal one, exhibited pro-apoptotic activity suggesting oligomer formation capability. In this study, we monitored the nuclear magnetic resonance (NMR) signals of the backbone atoms of the galectin-9 C-terminal CRD (G9CCRD). Protein concentration dependence of the signals suggested that a region (F1-F4 strands) opposite to the ligand-binding site was involved in the self-association of G9CCRD. Site-directed mutagenesis in this region (Leu210, Trp277 and Leu279 to Thr; G9CCRD-3T) inhibited the self-association of G9CCRD, and improved the solubility, whereas it reduced its pro-apoptotic activity towards T cells. The high pro-apoptotic activity of G9CCRD seems to be due to the ability to form an oligomer. In addition, the same substitution in two-CRD-containing galectin-9 (G9Null-3T) also diminished the self-association and improved its solubility, although it hardly reduced the anti-proliferative and pro-apoptotic activities. G9CCRD contributes the self-association of full-length galectin-9 at high protein concentrations.

STPR, a 23-amino acid tandem repeat domain, found in the human function-unknown protein ZNF821.

The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.

X-ray crystallography and structural stability of digestive lysozyme from cow stomach.

In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.

Novel pyromellitic diimide-based macrocycle with a linear pi-electronic system and bis(phenylethynyl)pyromellitic diimide: syntheses, structures, photophysical properties, and redox characteristics.

We report the syntheses, structures, photophysical properties, and redox characteristics of the [2 + 2] pyromellitic diimide-based macrocycle with a linear pi-electronic system 2 as well as the 3,6-bis(phenylethynyl)pyromellitic diimide derivative 3. The interesting solid state structural properties of the clathrates of 3 with pi-donors are also reported. The macrocycle 2 was synthesized by the direct cyclocondensation followed by the Sonogashira coupling reaction. X-ray crystallographic studies showed that the phenylacetylene moieties in 2 formed the intramolecular benzene dimer structures, and the bis(phenylethynyl)pyromellitic diimide moieties in both 2 and 3 were stacked in a parallel and slanted arrangement. Theoretical calculations for 2' and 3 suggested the existence of electrostatic interactions between the bis(phenylethynyl)pyromellitic diimide moieties. The UV/vis spectral measurements and TDDFT calculations of 2, 2', and/or 3 were performed to understand their electronic transitions. The fluorescence spectral measurements showed that 2 and 3 have visible fluorescence properties and 2 displays an excimer fluorescence at ca. 590 nm. The cyclic voltammetry measurements revealed that the electrostatic repulsion between the diimide moieties in 2 is greater than that in 1 according to the extension of the pi-electronic systems. X-ray crystallography of the clathrates of 3 with various pi-donors demonstrated the formation of the segregated donor-acceptor structures, indicating the strong aggregation ability of the bis(phenylethynyl)pyromellitic diimide moiety.

Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions.

Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.

[Spontaneous dislocation of the arytenoid cartilage].

Dislocation of the arytenoid cartilage occurs following medical instrumentation involving the laryngeal cavity or laryngeal injury from outside the larynx. We reported a case of spontaneously posterior dislocation of the arytenoid cartilage. A 53 year-old man suffering from suddenly recurring aphonia and its improvement many over 3 months without laryngeal injury or inducement eventually ceased to improve. Laryngoscopic findings showed that the left vocal fold was tensely prolonged and the vocal process of the arytenoid cartilage on the left side was dislocated posterolaterally. X-ray videofluorography of the larynx on repetitive phonation of /he/ showed abnormally high and diagonal displacement of the vocal fold and the upper structure of the arytenoid cartilage on the left side. Palpating the cricoarytenoid joint on the left side showed abnormal swelling with tenderness. Electomyography of the intrinsic laryngeal muscle on the left side showed normal action potential. From these findings, we diagnosed his voice disorder as spontaneously posterior dislocation of the arytenoid cartilage. We manually reduced it by pulling up a balloon inserted from the piriform sinus of the affected side to the esophagus.