However, all deglycosy lated GSK2256098 proteins were degraded after overnight incubation with class2 SPATEs except SepA and EpeA. Alignment of the amino acid sequence of domain-1 of SepA, with all other Avastin class-2 SPATEs with glycoprotease activity using Clustal-Omega, revealed very conserved residues among class members which were not shared with SepA. To determine the relevance of those residues in glycoprotease activity, we mutated seven randomly selected residues in Pic, spanning the catalytic triad, to the residues naturally occurring in SepA by site directed mutagenesis. Surprisingly, we found that even though secretion of the constructs harboring site mutations was not affected, mutation of conserved residues greatly reduced the glycoprotease activity of Pic on glycoproteins. They have been classified into pathotypes based in the clinical, pathological and epidemiological features of the disease they cause. Despite pathotypes harbor many diverse virulence factors, they all have in common the SPATEs, whose contributions have remained until recently obscure. We observed that most members of the class-2 SPATE family have the ability to agglutinate leukocytes, here evidenced in Jurkat T cells. The agglutination phenotype resembled that observed in a number of lectins produced by plants and microorganisms. In addition, Tsh/Hbp and Pic have been previously shown to agglutinate red cells. Interestingly, we observed that the agglutinating activity appears rapidly upon contact with leukocytes, and increased over the time of exposure, but was dependent on the serine protease activity given that denatured Pic protease or the protease-deficient PicS258A failed to agglutinate leukocytes. Likewise, Sat, a class-1 SPATE, did not exhibit this phenotype. We observed direct binding of most class-2 SPATEs to all leukocyte populations including granulocytes, monocytes, T, B and natural killer lymphocytes. Interestingly, binding efficiency was higher in granulocytes and monocytes than lymphocytes.We have previously shown that Pic protease activity against glycoproteins is reliant on the saccharide modification of the substrates since treatment of these substrates with neuraminidase greatly reduced or inhibited Pic protease activity.