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

The effect of mutating HuISG15 residues situated near the predicted UbE1L interface and the different allelic variants on conjugation to UbcH proteins is shown in Figure 5a and S3a. Strikingly, the single HuISG15 N89D variant displayed a greatly enhanced ISGylation efficiency. No effect was seen for all other mutants, including the N83S or S94N human allelic variants. Note that also in this experiment the cell lysates were boiled in SDS loading buffer containing b-ME. The slower migrating band differs 15 kDa from the unconjugated form of the UbcHs, and is thus the result of ISG15 bound by an isopeptide linkage, not by a thiolester bond. As with AgmISG15, no co-transfection of any Activating or Conjugating enzyme was required to visualize its conjugation by Western blot analysis on total cell lysates in HekT cells.