Endopeptidase Activity of SeMac against Horse IgG1 and Human IgG 
GAS Mac can cleave the heavy chain of human IgG at the lower hinge region between Fab and Fc fragments.
A catalytic triad of Cys94, His262, and Asp284 residues is critical for the enzymatic activity of GAS Mac [7, 8].
SeMac possesses putative catalytic residues of Cys102, His272, and Asp294 (Fig. 1).
To determine whether SeMac also is a cysteine endopeptidase targeting IgG, Cys102 and His272 or Asp294 of SeMac were replaced with Ser and Ala, respectively, by site-directed mutagenesis, and wild-type and mutant (SeMacCys102Ser, SeMacHis272Ala and SeMacAsp294Ala) SeMac proteins expressed in E. coli were tested for IgG endopeptidase activity using human IgG.
Wild-type SeMac cleaved the heavy chain of human IgG, while SeMacCys102Ser and SeMacHis272Ala completely lost the IgG endopeptidase activity, and SeMacAsp294Ala had dramatically lower enzymatic activity than the wild-type protein (Fig. 4A).
The results indicate that SeMac is a cysteine endopeptidase, which uses Cys102, His272 and Asp294 as its catalytic triad to cleave human IgG.
Purified recombinant SeMac and GAS Mac were then tested for activity to cleave horse IgG.
While both proteins cleaved human IgG efficiently, only a small fraction of horse IgG was cleaved by either protein (Fig. 4B), suggesting that SeMac or GAS Mac cannot digest all subgroups of horse IgG.
To test this possibility, available horse IgG1 and a mixture of IgG1 and IgG4 were treated with SeMac or GAS Mac.
SeMac or GAS Mac cleaved a small portion of the IgG1/IgG4 mixture, but not IgG4.
Therefore, the Mac proteins can cleave IgG1 but not IgG4.
Due to the unavailability of other purified subgroup IgG, it is not known whether SeMac cleaves the other IgG subgroups.
