Abstract
A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.
Highlights
Cytotoxic lymphocytes (CLs)1 play a central role in cellmediated immunity by destroying virus-infected, foreign, or tumor cells [1]
Granzyme deficiency has not been associated with familial hemophagocytic lymphohistiocytosis [9], it was recently reported that 25–30% of the population carries a triple-mutated (Q48R, P88A, Y245H (RAH)) allele of granzyme B (GrB) and that the product of this allele is incapable of inducing apoptosis [10]
There are no human GrB mutants, and the in vitro data are not supported by the mild phenotype of GrB knockout mice [5], which are defective in CL-mediated DNA degradation in target cells but otherwise are not significantly immunocompromised
Summary
Single Strand Conformation Polymorphism Analysis—DNA was isolated from 50 ml of whole blood using a PUREGENE kit from Gentra Systems. PCR-single strand conformation polymorphism analysis, described previously [14], was carried out using human GrB exon-specific primers. Activity of GrB—The activity of wild type and RAH GrB on peptide and protein substrates and their interactions with the serpin PI-9 were analyzed as described [17]. A sublytic concentration of perforin was determined empirically by diluting perforin in HE buffer (150 mM NaCl, 10 mM Hepes, pH 7.2) containing 4 mM CaCl2 and adding it to an equal volume of the cells in modified Hanks’ balanced salt solution. The amount of each granzyme required to kill 50% of the cells (EC50) was determined by preparing serial 2-fold dilutions of the granzyme in 50-l aliquots of the cells in modified Hanks’ balanced salt solution. The results (A595) were plotted against log GrB units/ml using the Prism 3.0 computer program
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