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

A key finding of this report is the significant difference in ISGylation capacity between ISG15 orthologues. In human cells, protein modification by ISG15 of OWm greatly outperforms that of Hu and MoISG15. Mutation of the residues in HuISG15 to the corresponding residues in the OWm orthologue mapped amino acid 89 as a critical residue for ISGylation. The occurrence of an acidic residue (preferably an Asp in human cells and Glu in mouse cells) at this position greatly enhances its ISGylation capacity. The Asp residue at position 89 in OWmISG15 also occurs in sheep and cows. In rodents and fish, this residue is a Glu residue. The incidence of the unfavorable Asn residue at position 89 for ISG15 modification in HuISG15 is also found in chimpanzee and dog ISG15. However, notwithstanding the greatly enhanced ISGylation capacity by mutation of the single Asn 89 residue in HuISG15 to an Asp residue, the overall ISGylation pattern in human HekT cells was still intermediary compared to AgmISG15. Additional mutations were created in the HuISG15 N89D mutant. Mutation of D133N and QIT31-33KIA further increased its ISGylation capacity. The triple HuISG15 mutant N89D D133N QIT31-33KIA proved to be as effective as AgmISG15 in conjugating target proteins in HekT cells. The use of this hyperefficient ISG15 variant may help to unravel the physiological function of ISG15.