HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

We wanted to address whether the observed differences in ISGylation capacity was only quantitative or also qualitative. Therefore, Tandem Affinity Purification (TAP) experiments were performed to identify ISGylated substrates by Hu and AgmISG15. To this end, a tandem proteinA binding domain and FLAG-tag, separated by a Tobacco Etch Virus protease cleavage domain was fused N-terminally to Hu or AgmISG15. Human fibroblast 2fTGH cells were stably transfected with these constructs, and 2 cell clones expressing TAP-tagged HuISG15 or TAP-tagged AgmISG15 with comparable expression levels were selected (see Figure S1 ). To rule out experimental variation, this experiment was also performed in transiently transformed HekT cells with either TAP-tagged HuISG15 or AgmISG15. As shown in Table 2 , the TAP experiments identified substantially more ISGylation substrates of AgmISG15 compared to HuISG15 in human celllines. Details of the identified peptides can be found in Table S1 . In addition to an overlap with the already described ISG15 substrates [30] [31] [32] [33] [34] , we here report the Ub-Conjugating enzymes UbcH10, UbcH16 are UbcH17 as novel ISGylation substrates of AgmISG15.