Chih-Min Kam

Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors

Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.

The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.

Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic (“Asp-ase”) or at long unbranched amino acids such as Met (“Met-ase”). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v= 185 nm/s at [S] = 0.217 mm) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mm 3,4-dichloroisocoumarin and 84% by 1.0 mmphenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.

Substrate and inhibitor studies on proteinase 3

Various amino acid and peptide thioesters were tested as substrates for human proteinase 3 and the best substrate is Boc‐Ala‐Ala‐Nva‐SBzl with a k cal/K m value of 1.0 × 106nMit‐1sit‐1 Boc‐Ala‐Ala‐AA‐SBzl (AA = Val, Ala, or Met) are also good substrates with k cal/K m values of (1‐4) × 105 M−1 s−1. Substituted isocoumarins are potent inhibitors of proteinase 3 and the best inhibitors are 7‐amino‐4‐chloro‐3‐(2‐bromoethoxy)isocoumarin and 3.4‐dichloroisocoumarin (DCI) with k obs/[I] values of 4700 and 2600 M−1 s−1, respectively. Substituted isocoumarins. peptide phosphonates and chloromethyl ketones inhibited proteinase 3 less potently than human neutrophil elastase (HNE) by 1‐2 orders of magnitude.

Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity

The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all progranzymes (zymogens of lymphocyte serine proteases) to form mature granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing progranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI (k(cat)/K(m)=9,000,000M(-1)s(-1)). The charged N-terminal amino group of the substrate was essential and replacement of the NH(2) group with OH or NH(CH(3)) in Gly-Phe-AMC reduced the k(cat)/K(m) value by two to three orders of magnitude. A hydrazide azaglycine analog, NH(2)NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH(2)COCH(2)-Phe-AMC, where the NH(2) group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block granzyme maturation and granzyme-dependent apoptosis.

Lymphocyte granule-mediated cytolysis requires serine protease activity

We show that chymotrypsin-like, as well as trypsin-like, proteases are in granules isolated from cytolytic lymphocytes by the capacity of the granules to hydrolyze the peptide substrates Z-Phe-Leu-Phe-SBzl and Z-Ala-Gly-Arg-SBzl, respectively. We report protease inhibitors that can abrogate or delay granule-mediated cytolysis. Two mechanism-based isocoumarin serine protease inhibitors and Z-Gly-Leu-Phe-CH2Cl completely abrogated granule cytolysis. Lima bean and soybean trypsin inhibitors and chymostatin delayed but did not prevent this cytolysis. These data represent the first use of the powerful isocoumarin inhibitors as biological probes and indicate that lymphocyte serine proteases participate in the granule cytolytic process.

Determination of proteinase 3—α1-antitrypsin complexes in inflammatory fluids

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is α1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3—α1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3—α1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)—α1AT complexes. Thus, in vivo α1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

Cutaneous protease activity in the mouse ear vesicant model

Tissue homogenates from mouse ear skin exposed to sulfur mustard (HD, which is a military designation and probably originated from a World War I slang term ‘Hun Stuff’) were assayed for serine and cysteine protease activities. Enzyme activity was measured using synthetic chromogenic thioester and fluorogenic 7‐amino‐4‐methylcoumarin (AMC) substrates. The tissue samples were obtained from animals (n = 6) at 3, 6, 12 and 24 h post‐exposure from the right ear (HD exposed), whereas control samples were obtained from the left ear (treated only with dichloromethane vehicle). The samples of naive control (left and right ear) were obtained from animals that received no HD treatment (n = 3). Elastase activity was assayed with t‐butyloxycarbonyl‐Ala‐Ala‐Ala‐thiobenzylester, tryptase activity with benzyloxycarbonyl‐Arg‐AMC and benzyloxycarbonyl‐Arg‐thiobenzylester, chymase activity with succinyl‐Ala‐Ala‐Pro‐Phe‐thiobenzylester and succinyl‐Ala‐Ala‐Pro‐Phe‐AMC, cathepsin B activity with benzyloxycarbonyl‐Arg‐Arg‐AMC, cathepsin H activity with Arg‐AMC and calpain activity with succinyl‐Leu‐Tyr‐AMC. The HD‐exposed skin homogenates obtained at 12 and 24 h post‐exposure had higher elastase activity (670% and 1900% increase) than control samples. For tryptase and calpain activities, only HD‐exposed skin homogenates at 24 h post‐exposure showed higher activities (220% and 170% increase) when compared to the control. No differences from control were observed for HD‐exposed skin obtained at 3 and 6 h post‐exposure for elastase, tryptase and calpain activities. Generally, both unexposed and HD‐exposed skin had distinct cathepsin B and cathepsin H enzyme activities and small chymase activity. Enzymatic assays were also performed for other serine, cysteine and metalloproteases. These data document that proteases are involved in HD skin injury and continued assessment of proteolytic activity should be useful for identifying effective antiproteases with therapeutic use in reducing or eliminating tissue injury caused by HD cutaneous exposure. Copyright

Dipeptide vinyl sulfones suitable for intracellular inhibition of dipeptidyl peptidase I.

In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I] > 72 M-1 s-1) and found six that were nontoxic. Four affected intracellular DPPI at < 25 microM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu-Phe-VS-CH3, inactivated DPPI in cells with an ID50 of approximately 5 microM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu-Phe-VS-CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly-Phe-CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.

Enhanced Serine Protease Activities in the Sulfur Mustard-Exposed Homogenates of Hairless Guinea Pig Skin

Skin homogenates of hairless guinea pigs exposed percutaneously to sulfur mustard (SM) were investigated by measuring four serine protease activities (elastase, tryptase, chymase, and Asp-ase) using sensitive chromogenic substrates. The homogenate samples from skin area directly exposed to S M showed enhanced elastase activities. The tryptase (trypsin-like enzyme) activity also increased slightly; however, the chymase (chymotrypsin-like enzyme) and Aspase (cleave after aspartic acid) activities did not show any increase. The enhanced elastase activities after SM exposure indicate that inflammation is present in the SM lesions. The inhibitory potency of MeO-Suc-Ala-Ala-Pro-Val-CH2Cl (an elastase inhibitor) and two amidine derivatives (inhibitors of trypsin-like enzyme) was tested against the activities present in samples from both exposed and control tissues. The elastase inhibitor decreased the hydrolysis of elastase substrate in the samples from SM-exposed skin to a greater extent than the samples from control tissues. The two trypsin inhibitors decreased the activity in samples from exposed and control tissues equally well. These substrate and inhibitor studies facilitate the characterization of various proteases affected by SM and may be useful for elucidating the mechanism of SM-induced vesication.

Synthetic Substrates and Inhibitors of Thrombin

Thrombin has a central regulatory role in hemostasis and is formed by both the intrinsic and extrinsic pathways of blood coagulation (Fenton, 1986, 1981). The major function of this serine protease is the cleavage of fibrinogen to form fibrin clots, but thrombin also activates factors V, VIII, XIII and protein C which are important in the control of hemostasis and thrombosis. In addition, thrombin can stimulate platelet secretion and aggregation in blood, and mediate other nonhemostatic cellular events. Our understanding of the various biological roles of thrombin has been facilitated by the development of convenient chromogenic and fluorogenic assays for the measurement of thrombin activity; and synthetic, low-molecular-weight inhibitors which are important probes for the study of thrombin’s mechanism, specificity, and function in the coagulation system.