title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B.
abstract: BACKGROUND
  Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).
  
  
  RESULTS
  In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2.
  
  
  CONCLUSIONS
  Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.
authors.affiliation: Department of Medical Protein Research, VIB, Flanders Interuniversity Institute for Biotechnology, Ghent University, A, Baertsoenkaai 3, B9000 Ghent, Belgium. Petra.VanDamme@vib-UGent.be.
authors.forename: Petra
authors.initials: P
authors.lastname: Van Damme
authors.forename: Kim
authors.initials: K
authors.lastname: Plasman
authors.forename: Giel
authors.initials: G
authors.lastname: Vandemoortele
authors.forename: Veronique
authors.initials: V
authors.lastname: Jonckheere
authors.forename: Sebastian
authors.initials: S
authors.lastname: Maurer-Stroh
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C450834:BNIPL protein, human; D010447:Peptide Hydrolases; D053804:Granzymes