Human and mouse granzyme M display divergent and species-specific substrate specificities

Abstract

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

“…21 Both gel-based (2D-DIGE) (Figure 2a) and complementary positional (cofradic) proteomics have identified hnRNP K as a potential GrM substrate. 21 We focused on hnRNP K because it has been shown to have a role in herpes simplex virus-1 (HSV-1) and hepatitis B virus replication. 22,23 To validate hnRNP K cleavage by GrM, cell lysates were incubated with increasing concentrations of GrM or catalytically inactive control GrM-SA.…”

“…Recently, we have employed multiple proteomic approaches to identify potential macromolecular cellular substrates of GrM. 21 Both gel-based (2D-DIGE) (Figure 2a) and complementary positional (cofradic) proteomics have identified hnRNP K as a potential GrM substrate. 21 We focused on hnRNP K because it has been shown to have a role in herpes simplex virus-1 (HSV-1) and hepatitis B virus replication.…”

“…GrM also cleaved purified recombinant His-hnRNP K with the appearance of the B45 kDa fragment (Figure 2c), indicating that GrM directly cleaves hnRNP K. Using complementary positional proteomics, we identified at least three GrM cleavage sites in hnRNP K, that is, Leu 125 , Leu 133 , and Met 359 (Figure 2d). 21 We mutated Leu 125 , Leu 133 , Met 359 , or combinations into Ala and we evaluated these mutants in cellular lysates for GrM cleavage using immunoblotting with an anti-His antibody to detect the hnRNP K N terminus (Figure 2e). Cleavage of wild-type hnRNP K resulted in a decrease of full-length hnRNP K and the appearance of a B20 kDa N-terminal cleavage fragment.…”

“…Indeed, several proteomic screens have identified hnRNP A1, A2/B1, C1/C2, E1, M, and U as potential GrM substrates. 21,34 Interestingly, many members of the hnRNP protein family have also been identified as potential substrates of other granzymes than GrM. [35][36][37][38] Further studies It remains an intriguing question why GrM targets viral pp71 17 and host cell hnRNP K to attack the initial IE events of HCMV replication rather than just killing HCMV-infected cells.…”

“…The physiological importance of GrM as an antiviral mediator has been demonstrated in GrM-deficient mice, which are more susceptible to MCMV infections. 18 However, both murine GrM and MCMV have been largely diverged from their human counterparts, 1,21 emphasizing that caution is essential when extrapolating data from mice to humans. In this study, we demonstrated for the first time that human GrM is expressed in HCMV-specific CD8 þ T cells after primary infection and during latency (Figure 1).…”