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 cDNAs of the Ub-Conjugating enzymes UbcH6, 7, 8, 10, 13, 16 and 17 were isolated by RT-PCR from 2fTGH cells and linked to a C-terminal V5-tag. These constructs were transfected in HekT cells together with either a mock construct, HuISG15 or AgmISG15. UbcH7 was included as a control, as it was neither in our experiments nor in others identified as a substrate for ISGylation. Western blot analysis on total cell lysates containing b-ME confirmed ISGylation of UbcH10, H13 and H17 with AgmISG15 but not with HuISG15, as seen by a 15 kDa shift upon staining with anti-V5 Ab detecting the ectopic expressed UbcH proteins (Figure 3a ,b and Figure S2a ). In addition, co-immunoprecipitation experiments were performed by binding ISGylated proteins through ISG15's FLAG-tag to an anti-FLAG affinity gel. Also here, the ISGylation of UbcH10, H13 and H17 by AgmISG15 could be established ( Figure 3c ). Worth mentioning, when the co-immunoprecipitated samples were obtained in a more concentrated way, a faint band of co-immunoprecipitated UbcH13 and UbcH17 could be seen with HuISG15 ( Figure S2b ), hinting at a qualitative rather than quantitative difference in ISGylation between Hu and AgmISG15. Of note, all the experiments were performed without cotransfection of UbE1L and/or UbcH8 or IFN stimulus. Previous studies using Western blot analysis in HekT cells revealed Hu or MoISG15 conjugation to substrates such as UbcH13 only upon co-transfection of at least UbE1L -and generally also UbcH/M8or upon IFN stimulation. Moreover, in most cases an additional immunoprecipitation or pull-down purification step was required [27, 28, [30] [31] [32] [33] . Alternatively, experiments were performed in USP18-deficient Murine Embryonic Fibroblasts [30, 34] .