A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.
Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.
Department of Medicine, Monash Medical School, Box Hill Hospital, Box Hill 3128, Australia. philb@boxhill.med.monash.edu.au
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001853:Bone Marrow / Q000378:metabolism; D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D017931:DNA Primers; D053804:Granzymes; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D015843:Serpins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D011994:Recombinant Proteins; C102316:SERPINB9 protein, human; D015843:Serpins; D014764:Viral Proteins; C074641:interleukin-1beta-converting enzyme inhibitor; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases