Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.
Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.
Wilharm
E
E
Max-Planck-Institute of Neurobiology, Department of Neuroimmunology, Department of Structural Biology, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
Parry
M A
MA
Friebel
R
R
Tschesche
H
H
Matschiner
G
G
Sommerhoff
C P
CP
Jenne
D E
DE
D000510:Alpha-Globulins / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D053818:Chymases; D004792:Enzyme Precursors / Q000138:chemical synthesis / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology*; D008562:Membrane Glycoproteins; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000097:blood*; D014360:Trypsin Inhibitor, Kunitz Soybean; D053802:Tryptases
D000510:Alpha-Globulins; D004792:Enzyme Precursors; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; C011877:inter-alpha-inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases