Activation of recombinant murine cytotoxic cell proteinase-1 requires deletion of an amino-terminal dipeptide.
Murine cytotoxic cell proteinase-1 (granzyme B) is a member of a family of novel serine proteinases that have been implicated to participate in destruction of target cells by cytotoxic T lymphocytes. Comparison of the sequence of the cDNA with the sequence of the protein isolated from cytoplasmic granules of cytotoxic T lymphocytes suggested that this protein may be synthesized as a preproenzyme containing an amino-terminal activation dipeptide. Here we show that this activation dipeptide regulates the activity of the enzyme in hydrolysis of its preferred substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester. Lysates of COS cells transfected with a vector expressing the unmodified cytotoxic cell proteinase-1 cDNA were unable to hydrolyze this substrate, whereas lysates of cells transfected with a construct in which the activation dipeptide codons has been deleted were able to hydrolyze the substrate. In each case Western blotting of the lysates revealed a form of the proteinase with an apparent molecular weight of 27,000. We conclude that the activation dipeptide regulates activity of the enzyme. This is the first report of production of an enzymatically active recombinant cytotoxic T cell serine proteinase. The strategy for successful expression of an activated form of cytotoxic cell proteinase-1 may be applicable to other members of this proteinase family.
Caputo
A
A
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Garner
R S
RS
Winkler
U
U
Hudig
D
D
Bleackley
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA; D004151:Dipeptides; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D053804:Granzymes; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014162:Transfection
D000906:Antibodies; D004151:Dipeptides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases