Atsushi Takeda

Squamous cell carcinoma antigen is a potent inhibitor of cysteine proteinase cathepsin L

A squamous cell carcinoma antigen (SSCA), which is a member of the serpin family of proteinase inhibitors, was purified from sera of cancer patients. It did not inhibit serine proteinases. However, it non‐competitively inhibited human cathepsin L with a K i of 0.064 nM, but not cathepsins B and H among cysteine proteinases. These results indicated that SCCA is a non‐functional serpin that inhibits cathepsin L in cancer cells.

Immunoreactivity of antibodies raised against synthetic peptide fragments predicted from mid portions of dystrophin cDNA

We synthesized 3 peptide fragments predicted by residues 2354-2368 (peptide I), 2310-2324 (peptide II) and 2255-2269 (peptide III) on the mid-portion of the human dystrophin cDNA map where the most frequent intragenic deletions occurred in Duchenne muscular dystrophy. Rabbit antibodies against these peptides were raised and cryosections of 47 biopsied muscles were studied immunohistochemically. The 47 biopsied muscles included the quadriceps femoris muscles of 8 Duchenne muscular dystrophy patients, 8 child and 5 adult normal controls, 1 facioscapulohumeral dystrophy, 2 limb girdle dystrophy, 3 myotonic dystrophy, 3 polymyositis, 1 mitochondrial myopathy, 1 nemaline myopathy, 3 amyotrophic lateral sclerosis and the extensor digitorum longus muscles of 6 mdx mice (C57BL/10ScSn-mdx) and 6 normal control mice (C57BL/10ScSn). The peptide I antiserum continuously stained the myofiber surface membranes in 8 child and 5 adult normal control muscles, and in 14 other muscles from various neuromuscular diseases, but failed to stain the surface membranes in normal control mice. The surface membranes of 8 Duchenne muscles were not stained by the peptide I antiserum except for a few myofibers. Although the ELISA titers of peptide I, II and III antibodies were high, immunostaining by peptide II antiserum showed no reaction in the myofibers of any of the biopsied muscles, and immunostaining by peptide III antiserum revealed faint reactions on the myofiber surface membranes of all biopsied muscles, including the mdx control mouse muscles except for the Duchenne and mdx myofibers.

Purification and Characterization of Metallo-β-Lactamase from Serratia marcescens

Carbapenem‐hydrolyzing β‐lactamase from Serratia marcescens FHSM4055 was purified 926‐fold by means of carboxylmethyl Sephadex C‐50, Sephacryl S‐200, and Mono S column chromatography. The molecular weight was 30,000 by SDS‐PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p‐chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo‐β‐lactamase and that the SH‐group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (kcat/Km) showed that the enzyme hydrolyzed various β‐lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino‐ and imino‐groups, ceftazidime with a carboxypropyloxyimino‐group, and cefclidin with a carbamoylquinuclidine‐group were poor substrates among the β‐lactam antibiotics other than the monobactams tested. The plots of the turnover number (kcat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK1, 5.9; pK2, 9.0; pK3, 10.8), whereas those of kcat/Km gave a bell‐shaped curve (pK1, 5.8; pK2, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N‐terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71‐78), the above enzymatic characteristics seem to coincide.

Size and localization of dystrophin molecule: immunoelectron microscopic and freeze etching studies of muscle plasma membranes of murine skeletal myofibers.

The ultrastructure and mode of existence of the dystrophin molecule and its relations to actin filaments were examined in murine skeletal myofibers. Electron microscopy of freeze-etched replicas of goldlabelled dystrophin molecules in quick-freeze, deepetch, rotary-shadow preparations revealed rod-like structures 108.2±16.3 nm long and 3.1±1.5 nm thick. Some dystrophin molecules appeared to link their ends to form anastomosing networks; others were separate from each other. The dystrophin molecules were parallel or nearly parallel to the inner surface of the muscle plasma membrane. Double immuno-labelling transmission electron microscopy using N- and C-terminal dystrophin antibodies showed that the group mean distances of the N- and C-terminal signals from the muscle plasma membrane were 52.7±8.1 nm and 45.9±11.3 nm, respectively, which were not significantly different. Histograms of the distribution of the N- and C-terminal distances from the muscle plasma membrane had similar patterns with peaks 10∼20 nm from the membrane. This was consistent with the findings of the mode of existence of dystrophin molecules seen in freeze-etched replicas. Finally, the dystrophin molecules were linked with the most peripheral sarcoplasmic actin like filaments, end to side as well as end to end.

Enzyme-linked immunosorbent assay for the detection of cathepsin-kininogen complexes in human plasma.

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of cathepsins B-, H- and L-kininogen (B-KG, H-KG, and L-KG) complexes was constructed using a microtiter plate based sandwich technique. The assay range was 7.5-480 ng/ml with three cross-linked cathepsin-kinin-free low molecular weight KG (LMWKGf) complexes. The average within-run coefficients of variation (CV) using three concentrations were 7.5, 7.2 and 9.9% for B-LMWKGf, H-LMWKGf, and L-LMWKGf complexes, respectively. The between-run CV values indicated satisfactory reproducibility for the method. Recoveries of cross-linked B-LMWKGf, H-LMWKGf, and L-LMWKGf complexes from negative control plasma were 127 +/- 7.1, 112 +/- 5.8 and 102 +/- 8.8% (n = 10, mean +/- SD), respectively, whereas the average recovery from phosphate buffer (pH 7.2) containing 0.15 M NaCl, 0.05% Tween-20 and 0.1% bovine serum albumin was 93.1 +/- 8.2% (n = 10, mean +/- SD). It was possible to detect B-KG and H-KG complexes in the plasma of 5/280 and 14/280 patients manifesting acute phase responses. Levels in plasma from healthy individuals were negligible. This ELISA should permit the study of cathepsin metabolism in acute phase disorders.

Monoclonal antibodies as probes to detect conformational changes in the rat cysteine proteinase inhibitor cystatin A

Five monoclonal antibodies (MAbs), 77, 114, 138, 175 and 187, were established for rat cystatin A. MAbs 77, 114, 138 and 175 were shown to belong to the IgG1 subclass, whereas MAb 187 was an IgM. These MAbs partially suppressed inhibitory activity of rat cystatin A to papain. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs with NH2-terminally truncated forms and fragments of rat cystatin A by an enzyme-linked immunosorbent assay (ELISA), and by reactivity with the inhibitor on immunoblotting. In competitive binding assays the MAbs did not compete with each other, indicating that the epitopes recognized by these MAbs were substantially different. The conformational epitope recognized by the three MAbs 114, 138 and 175 belonged to one group that was highly sensitive to denaturation, but those epitopes were unchanged by NH2-terminal truncation. MAb 187 was able to recognize a linear epitope present in amino acid residues 15-50 in the NH2-terminal region. MAbs 77 and 114 reacted weakly with mouse cystatin A but not at all with human cystatin A, whereas MAb 187 reacted similarly with mouse cystatin A but at about half that level with human. The MAbs produced in this study should be useful tools for detecting conformational changes in the rat cystatin A molecule.

Molecular Cloning, Enhancement of Expression Efficiency and Site-Directed Mutagenesis of Rat Epidermal Cystatin A

A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.