Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivoKatunuma, N.; Murata, E.; Kakegawa, H.; Matsui, A.; Tsuzuki, H.; Tsuge, H.; Turk, D.; Turk, V.; Fukushima, M.; Tada, Y.; Asao, T.
doi: 10.1016/S0014-5793(99)01107-2pmid: 10518923
Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.
Functional phage display of leech‐derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitorsTanaka, Aparecida S.; Silva, Melissa M.; Torquato, Ricardo J.S.; Noguti, Maria A.E.; Sampaio, Claudio A.M.; Fritz, Hans; Auerswald, Ennes A.
doi: 10.1016/S0014-5793(99)01106-0pmid: 10518924
The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech‐derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2×104 mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1–P4′ positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI‐2T (K8R, I9V, S10, K11W, P12A), LDTI‐5T (K8R, I9V, S10, K11S, P12L) and LDTI‐10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI‐2T and ‐5T, prolong the blood clotting time, inhibit thrombin (K
i 302 nM and 28 nM) and trypsin (K
i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI‐10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1′ (I9V). The data indicate further that LDTI‐5T might be a model candidate for generation of active‐site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme‐inhibitor interactions.
cDNA cloning, characterization and stable expression of novel human brain carboxylesteraseMori, Mieko; Hosokawa, Masakiyo; Ogasawara, Yuko; Tsukada, Eiko; Chiba, Kan
doi: 10.1016/S0014-5793(99)01111-4pmid: 10518925
The DNA sequence encoding a novel human brain carboxylesterase (CES) has been determined. The protein is predicted to have 567 amino acids, including conserved motifs, such as GEAGG, GXXXXFG, and GDGD which comprise a catalytic triad, and the endoplasmic reticulum retention motif (HXEL‐COOH) observed in CES families. Their gene products exhibited hydrolase activity towards temocapril, p‐nitrophenylacetate and long‐chain acyl‐CoA. Since the molecular masses of these gene products are similar to those that exist in capillary endothelial cells of the human brain [Yamamda et al. (1994) Brain Res. 658, 163–167], these CES isozymes may function as a blood‐brain barrier to protect the central nervous system from ester or amide compounds.
Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cellsTokuyasu, Ken; Kaneko, Satoshi; Hayashi, Kiyoshi; Mori, Yutaka
doi: 10.1016/S0014-5793(99)01113-8pmid: 10518926
With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide. During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD600 nm in the culture medium and a drastic decrease in viable cell number were observed. When penta‐N‐acetyl‐chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus. The addition of a C‐terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the k
cat value, and the further addition of a 12 amino acid sequence at its N‐terminus caused a further decrease in the value. This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E. coli cells.
Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosaGotoh, Naomasa; Kusumi, Toshiyuki; Tsujimoto, Hideto; Wada, Takaomi; Nishino, Takeshi
doi: 10.1016/S0014-5793(99)01116-3pmid: 10518928
The membrane topology of a resistance‐nodulation‐division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD‐PhoA (alkaline phosphatase) and 18 MexD‐Bla (β‐lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N‐ and C‐termini are located in the cytoplasm and the predicted orientation is consistent with the ‘positive‐inside rule’. This topological model can be applied to other RND proteins.
Molecular cloning of mXCR1, the murine SCM‐1/lymphotactin receptorYoshida, Tetsuya; Izawa, Dai; Nakayama, Takashi; Nakahara, Koichiro; Kakizaki, Mayumi; Imai, Toshio; Suzuki, Ryuji; Miyasaka, Masayuki; Yoshie, Osamu
doi: 10.1016/S0014-5793(99)01114-Xpmid: 10518929
Single C motif‐1 (SCM‐1)/lymphotactin is a C‐type member of the chemokine superfamily. Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM‐1 by the baculovirus expression system. B300‐19 murine pre‐B cells expressing mXCR1 responded to mSCM‐1 in chemotactic and calcium‐mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8+ cells and NK1.1+ cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity.