Holistic View on the Extended Substrate Specificities of Orthologous Granzymes

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.