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Title: Expression of recombinant human granzyme B. A processing and activation role for dipeptidyl peptidase I.
Austin Authors: Smyth, Mark J;McGuire, M J;Thia, K Y
Affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia
Issue Date: 15-Jun-1995
Publication information: Journal of Immunology (baltimore, Md. : 1950); 154(12): 6299-305
Abstract: Human granzyme B (hGrzB) is the key member of a family of granule serine proteases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes. The proenzyme activation peptide predicted from the cDNA encoding hGrzB is composed of two residues. We have devised a PCR strategy to delete this activation dipeptide within hGrzB and express active recombinant hGrzB in mammalian COS cells. Lysates of COS cells transfected with modified hGrzB cDNA were able to hydrolyze tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (Boc-Ala-Ala-Asp-SBzl), whereas lysates transfected with unmodified hGrzB cDNA were inactive. Accordingly, active recombinant hGrzB displayed no significant activities toward substrates containing Met-, Lys-, or Phe- at P1. It has been suggested that the mechanism by which the amino-terminal dipeptide is normally cleaved to generate active GrzB involves dipeptidyl peptidase I (DPPI). Our studies demonstrated that lysates of COS cells cotransfected with unmodified hGrzB cDNA (inactive) and rat DPPI cDNA were able to hydrolyze Boc-Ala-Ala-Asp-SBzl. Similarly, lysates of COS cells transfected with unmodified hGrzB cDNA, and devoid of Asp-ase activity, were also activated upon the addition of bovine spleen DPPI in a pH and time-dependent fashion. These results suggest that the activation dipeptide, and more particularly DPPI, may play and important role in the normal post-translational processing and activation of hGrzB in cytotoxic lymphocytes.
Gov't Doc #: 7759868
Type: Journal Article
Subjects: Animals
Base Sequence
Cathepsin C
Cell Line
DNA Primers.genetics
DNA, Complementary.genetics
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases.metabolism
Enzyme Activation
Gene Expression
Molecular Sequence Data
Protein Processing, Post-Translational
Recombinant Proteins.genetics.metabolism
Serine Endopeptidases.genetics.metabolism
T-Lymphocytes, Cytotoxic.enzymology.immunology
Appears in Collections:Journal articles

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