Hemolysis of Erythrocytes by Granulysin-Derived Peptides but Not by Granulysin

Abstract

Granulysin, a 9-kDa protein localized in human cytolytic T lymphoctyes and natural killer cell granules, is cytolytic against tumors and microbes but not against red blood cells. Synthetic peptides corresponding to the central region of granulysin recapitulate the lytic activity of the intact molecule, and some peptides cause hemolysis of red blood cells. Peptides in which cysteine residues were replaced by serine maintain their activity against microbes but lose activity against human cells, suggesting their potential as antibiotics. Studies were undertaken to determine the mechanism of resistance of red blood cells to granulysin and sensitivity to a subset of granulysin-derived peptides. Granulysin lyses immature reticulocytes, which have mitochondria, but not red blood cells. Granulysin lyses U937 cells but not U937 cells lacking mitochondrial DNA and a functional respiratory chain (U937°cells), further demonstrating the requirement of intact mitochondria for granulysinmediated death. Peptide G8, which corresponds to helix 2/loop 2/helix 3, lyses red blood cells, while peptide G9, which is identical except that the cysteine residues were replaced by serine, does not lyse red blood cells. Granulysin peptide-induced hemolysis is markedly inhibited by an anion transporter inhibitor and by Na ؉ , K ؉ , and Ca 2؉ channel blockers but not by Na ؉ /K ؉ pump, cotransport, or Cl ؊ channel blockers. Although recombinant granulysin and G9 peptide do not induce hemolysis, they both competitively inhibit G8-induced hemolysis. The finding that some derivatives of granulysin are hemolytic may have important implications for the design of granulysin-based antimicrobial therapeutics.

Granulysin Induces Cathepsin B Release from Lysosomes of Target Tumor Cells to Attack Mitochondria through Processing of Bid Leading to Necroptosis

“…1C). The results were in agreement with a previous observation (26). We also found the lytic effects of GNLY to human PBMC were comparable to the RBC (Fig.…”

Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin

“…Several studies have confirmed that the cytolytic activity of the 9-kDa GNLY occurs in the 50-100M range. 37 It has previously been reported that the 9-kDa recombinant human GNLY could induce migration of human peripheral blood monocytes and mature DCs, 32 but not Mo-iDCs or murine DCs. We For personal use only.…”

Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell–invasive merozoites

“…Indeed, although trophozoites and schizonts specifically triggered degranulation and granulysin release, their intracellular development was not impaired when cocultured with V␥9V␦2 T cells. Because the erythrocytic membrane is resistant to granulysin, 40 perforin does not seem to be sufficient to allow granulysin to pass this barrier and reach the intravacuolar parasite. Remaining protected from cytotoxic molecules within the erythrocyte for the duration of the intracellular development can be viewed as a Plasmodium evasion strategy that has not been appreciated previously.…”