Paralog specificity of mammalian SENPs for SUMO‐1 and SUMO‐2

Abstract

There are four SUMO paralogs; SUMO‐2, ‐3 and ‐4 are closely related and about 40% identical to SUMO‐1. SENPs are SUMO specific proteases responsible for deSUMOylation. Using a panel of SUMO‐1 and ‐2 derived substrates and inhibitors we examined the distribution and paralog specificity of SENPs. The total deSUMOylation activity in tissues and mammalian cell lysates examined is 15‐fold higher using SUMO‐2 than SUMO‐1 aminomethylcoumarin (AMC). Mammalian cell lysates showed more reactivity with SUMO‐2 vinyl sulfone (VS) than with SUMO‐1VS. Only SENP1 reacts significantly with SUMO‐1VS. To confirm these observations we examined the paralog specificity of the recombinant catalytic domains (cSENPs). The cSENPs acted on SUMO‐2 with Km and Ki values from 50–200nM. cSENP1 and cSENP2 showed a 2–4 fold preference for SUMO‐1. All other cSENPs strongly preferred SUMO‐2. Because cSENP2 reacted with SUMO‐2 derivatives while full length SENP2 in lysates did not, we asked whether specificity was dependent on the N‐terminal non‐catalytic domain. Full length recombinant his6‐SENP1 showed the expected paralog specificity. In contrast the catalytic domain alone, his6‐SENP2 cleaved SUMO‐2‐AMC much faster than SUMO‐1‐AMC. Thus the non‐catalytic N‐terminal domain of SENP2 participates in determining paralog specificity. In summary, SUMO‐2/‐3 modification in vivo may play a more significant role than previously appreciated.