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. 2008 Jun 18;3(6):e2427. doi: 10.1371/journal.pone.0002427

Figure 5. Mutation of residue 89 in HuISG15 to Asp greatly enhances its conjugation capacity, which could be further boosted by mutations of QIT31-33KIA and D133N.

Figure 5

(A ) HuISG15 N89D mutant significantly increases its ISGylation capacity to UbcH proteins. HekT cells were transiently transfected with a vector encoding V5-tagged UbcH10 together with either empty vector or a vector expressing HuISG15 or variants (indicated with asterisk) or AgmISG15. Total cell lysates were prepared in a buffer containing β-ME. UbcH10 protein is revealed by its V5-tag. Closed arrow indicates the unconjugated form of UbcH10, open arrow indicates the ISG15-conjugated form of UbcH10. (B) Only mutation of residue 89 in wild type HuISG15 to an acidic amino acid is able to enhance its ISGylation activity. HekT cells were transiently transfected with vectors encoding the indicated FLAG-tagged HuISG15 variants (indicated with asterisk) each holding one amino acid substitution, FLAG-tagged wt Hu- or AgmISG15. Total cell lysates were prepared in a buffer with reducing agentia. The conjugation competence of the different ISG15 proteins is exposed by their FLAG-tag. Equal loading was confirmed by Ponceau S staining (not shown). (C) Additional mutation of QIT31-33KIA or D133N further boosts ISGylation by HuISG15 N89D. Same as (B) but with HuISG15 N89D variants with an additional amino acid substitution. (D) The triple HuISG15 QIT31-33KIA N89D D133N variant is as efficient as AgmISG15 in UbcH protein ISGylation. Upper panel. HekT cells were transiently transfected with a plasmid encoding V5-tagged UbcH17 protein together with either an empty vector or a vector expressing FLAG-tagged Hu-, Agm-, MoISG15 or a HuISG15 variant (with asterisk). Western blot analysis on total cell lysates was performed under reducing conditions using anti-V5 Ab. Closed arrow indicates the unconjugated form of UbcH17, open arrow indicates the ISG15-conjugated form of UbcH17. Lower panel The blot was stripped and reprobed with anti-FLAG Ab showing the global ISGylation pattern. Equal loading was controlled by staining with Ponceau S staining (not shown).