Holistic View on the Extended Substrate Specificities of Orthologous Granzymes

Abstract

As proteases sculpt the proteome in both homeostatic and pathogenic processes, unraveling their primary signaling pathways and key substrates is of utmost importance. Hence, with the development of procedures enriching for proteolysis-indicative peptides and the availability of more sensitive mass spectrometers, protease degradomics technologies are ideally suited to gain insight into a protease's substrate repertoire and substrate-specificity profile. Especially, knowledge on discriminating sequence features among closely related homologues and orthologues may aid in identifying key targets and developing protease-specific inhibitors. Although clever labeling strategies allow one to compare the substrate repertoires and critical protease-substrate recognition motifs of several proteases in a single analysis, comprehensive views of (differences in) substrate subsite occupancies of entire protease families is lacking. Therefore, we here describe a hierarchical cluster analysis of the positional proteomics determined cleavage sites of a family of serine proteases: the granzymes. We and others previously assigned clear murine orthologues for all 5 human granzymes. As such, hierarchical clustering of the sequences surrounding granzyme cleavage sites reveals detailed insight into granzyme-specific differences in substrate selection and thereby deorphanizes the substrate specificity profiles and repertoires of the human and murine orthologous granzymes A, B, H/C, M, and K.

Granzyme K‐deficient mice show no evidence of impaired antiviral immunity

“…3 Recent proteomic studies however show that the extended substrate specificity of GzmK is distinct from granzyme A, strongly suggesting it targets different substrates and has a distinct biological role. [18][19][20] Unfortunately, these studies do not identify candidate physiological substrates. 21 Here, we take an important step toward to resolving the biological role(s) of GzmK in immunity by generating and analyzing Gzmk-null mice.…”

Single‐residue posttranslational modification sites at the N‐terminus, C‐terminus or in‐between: To be or not to be exposed for enzyme access

“…This work shows that, in addition to predictor components that recognize the position-specific amino acid type preferences next to the site to be modified, the more integral properties of the surrounding sequence stretches at either side of the PTM site (namely, the requirement for a trend towards flexibility, polarity and small side chain volume) provide valuable restrictions for the sequence search space [15,37,38,142,145,147]. Similarly, this seems important for protease cleavage sites in substrate proteins [22,[169][170][171][172] and, apparently, for many other PTMs.…”

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

“…For each category, proteins are ranked alphabetically according to their protein description. Additional information on the maximum score, identity threshold, isoforms and whether these identical cleavage sites were previously found in the N-terminal COFRADIC analyses screening for efficient hGrB cleavage sites/substrates [8] and analyses on hGrB, mGrB and mGrC respectively are listed [5]. …”

“…Human granzyme B (hGrB), a protease belonging to a family of serine proteases present in the granules of cytotoxic lymphocytes (cytotoxic T lymphocytes and natural killer cells) and implicated in the induction of cell death, was shown to display distinct functional and structural characteristics as compared to its murine ortholog despite their 80% amino acid similarity and 70% amino acid identity [1-4]. Positional scanning synthetic combinatorial libraries, phage display data and proteome-wide degradome analyses all aided in the elucidation of the differential specificity profiles and substrate repertoires of these orthologous granzymes [1-5]. Next to the higher cytotoxicity of hGrB, differences in cleavage efficiencies of Bid and caspase-3, substrates known to be of critical importance for the execution of hGrB induced cell death, were found for the human and mouse orthologous granzymes B [2-4].…”