Expression of enzymatically active human granzyme 3 in Escherichia coli for analysis of its substrate specificity.

Human granzyme 3 (Gr3) is a serine protease contained in the granules of natural killer cells and cytotoxic T lymphocytes. To elucidate the biochemical and physiological characteristics of Gr3, we attempted to prepare an enzymatically active recombinant human Gr3 without refolding and proteolytic activation. An expression vector was constructed, in which the pre-/pro-peptide coding sequence of Gr3 was replaced with the bacterial pelB leader sequence. The resultant expression product was a fully active protease in the periplasmic fraction of Escherichia coli and was purified to homogeneity. The purified enzyme effectively hydrolyzed Z-Lys-SBzl, a conventionally used substrate of Gr3. In addition, it also hydrolyzed the peptide substrate library FRETS-25Xaa series, required basic amino acid residues, Arg or Lys, at the P1 position, and most efficiently hydrolyzed the carboxylic side of Phe-Tyr-Arg downward arrow (P3-P2-P1) sequence of the 475 tripeptide combinations.

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center.

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Cys(122), Cys(124) and Cys(132) are placed near two catalytic sites, Lys(199) and Lys(252), and coordinate a zinc ion, referred to as "a proximal zinc ion", and Cys(223) is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as "a distal zinc ion", with His(131) . When the wild-type enzymes C122A (Cys(122)-->Ala), C124A (Cys(124)-->Ala), C132A (Cys(132)-->Ala) and C223A (Cys(223)-->Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys(122), Cys(124) and Cys(132) under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys(122), Cys(124) and Cys(132) was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.

Idiopathic opacification of Berger's space.

We report a case of idiopathic opacification of Berger's space in a 68-year-old man. The opacification was in the retrolental space between the crystalline lens and the anterior vitreous in the right eye. Opaque fluid was surgically removed, and chemical analysis detected a high concentration of protein and a low concentration of mucopolysaccharides. No underlying pathology was observed.

Organophosphorus pesticides markedly inhibit the activities of natural killer, cytotoxic T lymphocyte and lymphokine-activated killer: a proposed inhibiting mechanism via granzyme inhibition.

We have previously found that diisopropyl methylphosphonate, an organophosphorus by-product generated during sarin synthesis in the Tokyo sarin disaster, significantly inhibited natural killer (NK) and cytotoxic T lymphocyte (CTL) activities. In the present study, to investigate whether organophosphorus pesticides (OPs) also affect NK and CTL activities, we firstly examined the effect of five OPs on human NK activity, and then the effect of Dimethyl 2,2-dichlorovinyl phosphate (DDVP), an OP on murine splenic NK, CTL and lymphokine-activated killer (LAK), and human LAK activities in vitro. To explore the underlying mechanism of decreased NK activity, we also investigated the effect of 4-(2-aminoethyl) benzenesulfonyl fluoride-HCl (p-ABSF), an inhibitor of serine proteases on NK, LAK and CTL activities, and the effect of DDVP on the activity of granzymes (serine proteases). We found that OPs significantly decreased human NK activity in a dose-dependent manner, but the degree of decrease in NK activity differed among the OPs investigated, and that DDVP significantly decreased NK, LAK and CTL activities in a dose-dependent manner, but the degree of decrease in these activities differed. p-ABSF showed a similar inhibitory pattern to DDVP, and had an additive inhibitory effect with DDVP on NK, LAK and CTL activities. We also found that DDVP significantly inhibited granzyme activity in a dose-dependent manner. These findings indicate that OPs significantly decrease NK, LAK and CTL activities in vitro via granzyme inhibition.