Tsuyoshi Katoh

Identification and Characterization of a Novel Human Collectin CL‐K1

Collectins are a family of C‐type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host‐defense. Here we report the cloning and characterization of a novel collectin CL‐K1. RT‐PCR analyses showed CL‐K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL‐K1 cDNA expressing CHO cells revealed that CL‐K1 is expressed as a secreted protein. CL‐K1 is found in blood by immunoblotting and partial amino acid analyses. CL‐K1 showed Ca2+‐dependent sugar binding activity of fucose and weakly mannose but not N‐acetyl‐galactosamine, N‐acetyl‐glucosamine, or maltose, though mannose‐binding lectin (MBL) containing similar amino acid motif. CL‐K1 can recognize specially several bacterial saccharides due to specific sugar‐binding character. Elucidation of the role of two ancestor collectins of CL‐K1 and CL‐L1 could lead to see the biological function of collectin family.

Pulmonary Surfactant Protein D Inhibits Lipopolysaccharide (LPS)-induced Inflammatory Cell Responses by Altering LPS Binding to Its Receptors

Pulmonary surfactant protein D (SP-D) is a member of the collectin family that plays an important role in regulating innate immunity of the lung. We examined the mechanisms by which SP-D modulates lipopolysaccharide (LPS)-elicited inflammatory cell responses. SP-D bound to a complex of recombinant soluble forms of Toll-like receptor 4 (TLR4) and MD-2 with high affinity and down-regulated tumor necrosis factor-α secretion and NF-κB activation elicited by rough and smooth LPS, in alveolar macrophages and TLR4/MD-2-transfected HEK293 cells. Cell surface binding of both serotypes of LPS to TLR4/MD-2-expressing cells was attenuated by SP-D. In addition, SP-D significantly reduced MD-2 binding to both serotypes of LPS. A chimera containing the N-terminal region and the collagenous domain of surfactant protein A, and the coiled-coil neck and lectin domains of SP-D, was a weak inhibitor of LPS-induced cell responses and MD-2 binding to LPS, compared with native SP-D. The collagenase-resistant fragment consisting of the neck plus the carbohydrate recognition domain of SP-D also was a very weak inhibitor of LPS activation. This study demonstrates that SP-D down-regulates LPS-elicited inflammatory responses by altering LPS binding to its receptors and reveals the importance of the correct oligomeric structure of the protein in this process.

Acid-labile ATP and/or ADP/Pi Binding to the Tetraprotomeric Form of Na/K-ATPase Accompanying Catalytic Phosphorylation-Dephosphorylation Cycle

The Na/K-ATPase has been shown to bind 1 and 0.5 mol of 32P/mol of α-chain in the presence [γ-32P]ATP and [α-32P]ATP, respectively, accompanied by a maximum accumulation of 0.5 mol of ADP-sensitive phosphoenzyme (NaE1P) and potassium-sensitive phosphoenzyme (E2P). The former accumulation was followed by the slow constant liberation of Pi, but the latter was accompanied with a rapid ∼0.25 mol of acid-labile Piburst. The rubidium (potassium congener)-occluded enzyme (∼1.7 mol of rubidium/mol of α-chain) completely lost rubidium on the addition of sodium + magnesium. Further addition of ∼100 μm[γ-32P]ATP and [α-32P]ATP, both induced 0.5 mol of 32P-ATP binding to the enzyme and caused accumulation of ∼1 mol of rubidium/mol of α-chain, accompanied by a rapid ∼0.5 mol of Pi burst with no detectable phosphoenzyme under steady state conditions. Electron microscopy of rotary-shadowed soluble and membrane-bound Na/K-ATPases and an antibody-Na/K-ATPase complex, indicated the presence of tetraprotomeric structures (αβ)4. These and other data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, the sequential appearance of (NaE1P:E·ATP)2, (E2P:E·ATP:E2P:E·ADP/Pi), and (KE2:E·ADP/Pi)2, each of which has been previously referred to as NaE1P,E2P, and KE2, respectively.

Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model

We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross‐reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing.

Nonmuscle myosin II folds into a 10S form via two portions of tail for dynamic subcellular localization

Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB‐SK1·2), in which S1462‐R1490 and L1551‐E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB‐SK1·2 mutant did not fold into a 10S form under physiological condition in vitro. IIB‐SK1·2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB‐SK1·2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB‐SK1·2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB‐SK1·2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.

Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450kDa subunits, resulting in a molecular mass >3.3MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form.

Action of ascorbic acid on a myosin molecule derived from carp

The influence of L-ascorbic acid at 40 °C incubation on the subfragment-1 and rod regions, prepared by chymotryptic digestion of myosin, and myosin was investigated by SDS–polyacrylamide gel electrophoresis and transmission electron microscopy respectively. It was observed that L-ascorbic acid acted more readily on the subfragment-1 region of myosin. Further, circular dichroism measurement indicated that L-ascorbic acid did not affect the structure of myosin. These results suggest that L-ascorbic acid acts more readily on the myosin subfragment-1 region and promotes the gelation of myosin without producing a conformational change in this protein.