Staphylococcal Enterotoxin M Exhibits Thrombin-like Enzymatic Activity

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Staphylococcal enterotoxin M (SEM) is a known toxin.
• Recombinant protein expression often involves purification tags and cleavage sites.

Purpose of the Study:

• To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC).
• To investigate an unexpected cleavage event observed during the purification of a recombinant SEM fusion protein.

Main Methods:

• Recombinant protein expression and purification using IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC).
• N-terminal sequencing and mass spectrometry to identify cleavage products.
• AlphaFold 3 structure prediction and molecular dynamics (MD) simulations.
• Enzymatic assays using chromogenic substrate S-2238.

Main Results:

• A recombinant SEM fusion protein unexpectedly cleaved at the Arg-Gly bond within the thrombin cleavage site (TCS).
• Structural analysis revealed a catalytic triad (HSD) in SEM, suggesting intrinsic thrombin-like activity (TLA).
• Mutation of a key serine residue abolished TLA, confirming its intrinsic nature.
• Purification assays showed increasing specific activity, supporting the presence of TLA.

Conclusions:

• The wild-type SEM construct possesses intrinsic thrombin-like activity (TLA) that mediates specific Arg-Gly bond cleavage.
• This intrinsic activity is independent of the thrombin enzyme and originates from the SEM protein itself.
• The findings provide a foundation for understanding the TLA of SEM and its potential implications.