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

Total ISGylation patterns were visualized by Western blot analysis on total cell lysates. As can be seen in Figure 2a , AgmISG15 and RhmISG15 outperform HOmISG15 and MoISG15 in its ISGylation capacity in human Hek293T cells. MoISG15 also conjugates more favorably to human proteins compared to HuISG15, albeit not to the same extent as OWmISG15. To rule out the possibility that conjugation of ectopic HuISG15 could be hindered by competition with endogenous ISG15, the same blot was stripped and reprobed with an antibody recognizing Hu and OWmISG15, showing essentially the same results (data not shown). Similar findings were obtained in monkey COS cells and mouse N38 cells (resp. Figure 2b and Figure 2c ). Note that all these experiments were performed without extra stimuli (e.g. IFN treatment) or co-transfection of any Ubiquitinating enzyme meaning that in all tested cell-lines, target ISGylation by AgmISG15 could be achieved by endogenously available Ubiquitinating enzymes. Figure 1 . ISG15 orthologues show a low degree of amino acid conservation. ISG15 from different species were aligned with two molecules of Ubiquitin using Clustalx [61] . Sequence conservation is visualized using the AMAS server [62] with a conservation threshold of 7. Residues with a conservation above this threshold are shown on a grey background, residues that are identical in all sequences have a black background. doi:10.1371/journal.pone.0002427.g001