Yasuhiro Nonaka

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.

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.

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°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. Proteins 2008.

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.

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.

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.

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 Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc 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.

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. The members of galectin family, especially galectin-9, recognize small leucine-rich repeat.