CD44 and CD45 are known to be just partially glycosylated reliant on isoforms, whereas CD55 and C1-INH have limited sites of O-glycosylation. Extensive degradation of slightly glycosylated substrates by SPATEs was not seen despite prolonged incubation times or with higher SPATE concentrations. SepA, EpeA or the PicS258A did not show any protease activity under these conditions. In our previous study we found that the Pic protease activity was dependent on mucin-type O-glycosylation. In order to determine whether other glycoprotein types are also susceptible to SPATE proteolytic activity, we incubated CD97 and CD99 ; two non-mucin type glycoproteins, and three heavily Nlinked glycosylated proteins such as the fibronectin, E-selectin and the alpha beta integrin with SPATEs overnight at 37uC and analyzed them by SDS-PAGE for proteolytic cleavage. Under these conditions, none of the these molecules were susceptible to SPATEs. Furthermore, SPATE proteins had no effect on leukocyte molecules as assessed by flow cytometry. We and other groups have previously shown that Pic activity on glycoproteins relies on the saccharide nature of substrates. Pic displays reduced proteolytic activity on deglycosylated substrates, and reduced binding ability to mucin in presence of free monosaccharides. In order to determine if other class-2 members share this property, we treated CD44, CD162 and C1inh with a deglycosylation enzyme cocktail followed by incubation with class-2 SPATEs for two hours at 37uC. We observed reduced cleavage of deglycosylated proteins by all class-2 SPATEs when compared with intact glycoproteins treated for only one hour. Although Tsh/Hbp and Vat-Ex GPR39-C3 showed reduced protease activity on deglycosylated CD44 and C1-inh, they efficiently ML281 cleaved deglycosylated CD162, which seemed to be partially deglycosylated, as judged by the presence of bands with higher molecular weight similar to the glycosylated protein. Crc2sp showed lesser proteolytic activity on deglycosylated proteins than other class-2 SPATE. Pic and PicU showed similar activity on deglycosylated proteins, with some degree of degradation of higher molecular weight species of deglycosylated CD162.