Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins

Abstract

The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).

A quarter century of granzymes

“…18,19 A pivotal piece of information that led to the discovery of the cytotoxic mechanism of GrB was its ability to cleave aspartic acid residues. 8 This cleavage specificity is unique among eukaryotic serine proteases. Up to that point, only caspases were known to have this unusual specificity.…”

“…GrB is the most extensively studied Gr, characterized by the unusual capacity to cleave substrates at aspartic acid residues that is dependent on the presence of an arginine residue within the binding pocket ( Figure 2). 8 Grs H and GrC-G, are believed to be chymases, and cleave synthetic substrates at hydrophobic residues. 5 These proteins are also highly similar at the amino acid level, from 57-61% identity using BLAST alignment analysis.…”

Activated CD4+CD25+ T cells selectively kill B lymphocytes

“…Because perforin deficiency only partially abrogated CD4 ϩ CD25 ϩ T-cell-triggered B-cell death, we directly examined the role of granzyme B in this process. DCI is a serine proteinase inhibitor that efficiently inhibits granzyme B but not granzyme A, 35 and has been used as a specific granzyme B inhibitor in assays with intact cytotoxic T lymphocytes (CTLs). 36 DCI pretreatment of WT CD4 ϩ CD25 ϩ T cells partially prevented B-cell death ( Figure 5C).…”

Caspase substrates

“…Of the currently known human and mouse proteases only the caspase activator granzyme B, a serine protease, shares this primary specificity. 2,3 The primary specificity pockets of caspases are almost identical, being formed by the side chains of the strictly conserved residues, Arg-179, Arg-341 and Gln-283 (caspase-1 numbering convention). This deep, highly basic pocket ( Figure 1) is perfectly shaped to accommodate an Asp side chain, accounting for the up to four orders of magnitude lower catalytic efficiency for cleavage of peptides with a P 1 Glu residue in most caspases, 4 with the notable exception of the Drosophila caspase Dronc, which seems to have an equal specificity for Asp and Glu.…”