Abstract
In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1′ position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1′ and P2′ preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2′ is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: SCHI 871/5, SCHI 871/8, SCHI 871/9, SCHI 871/11, INST 39/900-1, and SFB850-Project Z1
Award Identifier / Grant number: 1444
Funding statement: The authors thank Ms. Annikki Löfhjelm for excellent technical assistance. HK acknowledges support from the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. O.S. acknowledges support by Deutsche Forschungsgemeinschaft (SCHI 871/5, SCHI 871/8, SCHI 871/9, SCHI 871/11, INST 39/900-1, and SFB850-Project Z1), the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS), the European Research Council (PoC 780730, ProteaseNter), and the German-Israeli Foundation for Scientific Research and Development (Funder Id: 10.13039/501100001736, grant no. 1444).
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