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 (SP-D) 3 is a member of the collectin protein family that also includes surfactant protein A (SP-A) and mannose binding lectin (1, 2). The structure of the collectins is characterized by four domains consisting of: 1) an N terminus involved in interchain disulfide bonding, 2) a collagen-like domain, 3) a coiled-coil neck domain, and 4) a carbohydrate recognition domain (CRD) (3). SP-A and mannose binding lectin contain collagenous domains consisting of 23 and 19 repeating Gly-X-Y triplets, respectively, with an interruption at the middle of the collagenous sequence (4, 5). In contrast, SP-D possesses a longer collagenous tail composed of 59 Gly-X-Y repeats without an interruption (6). These differences cause distinct oligomeric organization, with SP-A and mannose binding lectin exhibiting bouquet-like structures consisting of either six or four trimeric subunits (7) and SP-D exhibiting cruciform structures composed of four trimeric subunits (8).Lipopolysaccharide (LPS) is a principal component of the outer membrane of Gram-negative bacteria that activates macrophages and induces a variety of inflammatory mediators, including TNF-␣, IL-1, IL-6, IL-8, and interferon (9). LPS composed of O-antigen, core oligosaccharide, and lipid A is named smooth LPS, and LPS lacking O-antigen and a part of the core oligosaccharides is named rough LPS (10). Toll-like receptor 4 (TLR4) plays a critical role in recognition and signaling by LPS (11, 12). MD-2 binds directly to LPS and is required for TLR4-mediated signaling induced by LPS (13,14). Structural examination of the TLR4-MD-2 complex revealed that MD-2 binds to the concave surface of the N-terminal and central domains of TLR4 (15). A study with recombinant soluble forms of the extracellular TLR4 domain (sTLR4) and MD-2 (sMD-2) from our laboratory indicates the importance of the N-terminal region of TLR4 in MD-2 binding (16).Engineered genetic defects in the pulmonary collectins of mice have revealed their important functions in protecting the lung from microbial infections and inflammation. SP-D-null mice infected with group B Streptococcus or Haemophilus influenza by intra-tracheal instillation show increased inflammation and inflammatory cell recruitment in the lung (17). Increased pulmonary inflammation in LPS (Escherichia coli O55:B5, smooth serotype)-instilled SP-D Ϫ/Ϫ mice and wildtype mice was decreased by intratracheal administration of SP-D and pulmonary surfactant (18). Intratracheal recombinant SP-D prevents endotoxin shock in the newborn preterm
The Na/K-ATPase has been shown to bind 1 and 0.5 mol of 32 P/mol of ␣-chain in the presence [␥-32 P]ATP and [␣-32 P]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 P i , but the latter was accompanied with a rapid ϳ0.25 mol of acid-labile P i burst. The rubidium (potassium congener)-occluded enzyme (ϳ1.7 mol of rubidium/mol of ␣-chain) completely lost rubidium on the addition of sodium ؉ magnesium. Further addition of ϳ100 M [␥-32 P]ATP and [␣-32 P]ATP, both induced 0.5 mol of 32 P-ATP binding to the enzyme and caused accumulation of ϳ1 mol of rubidium/mol of ␣-chain, accompanied by a rapid ϳ0.5 mol of P i 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 (␣) 4 . These and other data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, the sequential appearance of (NaE1P:E⅐ATP) 2 , (E2P:E⅐ATP:E2P:E⅐ADP/P i ), and (KE2:E⅐ADP/P i ) 2 , each of which has been previously referred to as NaE1P, E2P, and KE2, respectively.It is generally accepted that Na/K-dependent ATP hydrolysis (1) occurs via the sequential appearance of phosphoenzymes and dephosphoenzymes (Scheme I), accompanied by the active transport of 3 sodium and 2 potassium ions across the membranes (2-6). The maximum ligand binding or occlusion and phosphorylation capacity per catalytic subunit (␣-chain) reported are generally taken as evidence for the functional subunit being a protomer (7-9). However, the maximum phosphorylation capacity of these enzymes has been unequivocally shown to be 0.5 mol/mol of ␣-chain during sodium-dependent ATP hydrolysis (10) even though the binding stoichiometry is 1 mol of ouabain/mol. Data concerning conformational changes of Na/K-ATPase in real time (11) and a quarter site phosphorylation of the ␣-chain by ATP (10) and P i (12-14) as well as other kinetic data (15, 16) support the hypothesis (11) that four different ATP binding sites to the tetraprotomeric enzyme form, (␣) 4 , induce out of phase conformational changes in Na/K-ATPase (14, 17). Therefore, questions arise as to the stoichiometry of ATP binding to the phosphorylated or dephosphorylated (cation-occluded) enzyme states during ATP hydrolysis as well as the gross molecular structure of the enzyme. MATERIALS AND METHODSMethods for the purification of Na/K-ATPase from kidneys of pig (14) and dog (18) and for the estimation of the amount of EP from [␥-32 P]ATP (15) and bound 32 P (19) have been described previously except that the enzyme (2ϳ4 mg of protein/ml) was incubated with 50 l of a pH 7.4 solution containing 25 mM imidazole HCl, 0.1 mM EDTA-Tris, 25 mM sucrose, 2 M NaCl, or changing the concentrations of NaCl to maintain the ionic strength at 2 M with choline chloride or 16 mM sodium and ...
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-SK1Á2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1Á2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1Á2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1Á2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1Á2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1Á2, 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.
Carp dorsal myosin formed oligomers that retained ATPase activity upon heating. Cleavage of the oligomeric myosin at subfragment-1 (S-1)/rod junction released monomeric S-1 and rod, indicating that ATPase retaining myosin associated near the S-1/rod junction. The digest also contained rod oligomers. Heating a mixture of S-1 and rod generated neither ATPase retaining S-1 oligomers nor rod oligomers. Electron microscopic observation of the heated myosin revealed that some oligomers were formed by associating at the S-1/rod joining region, exhibiting a recognized double head, probably ATPase retaining oligomers. No myosin oligomers associated at the tail region were observed, thus, rod aggregation would be formed at its very restricted region near the S-1/rod junction. Based on the findings, we proposed that the neck structure is important in the thermal oligomerization process of myosin.
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