A SURVEY OF FURIN SUBSTRATE-SPECIFICITY USING SUBSTRATE PHAGE DISPLAY

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was stu died using substrate phage display. In this method, a multitude of sub strate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chrom atography. The cleaved phage are propagated and submitted to additiona l rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprisi ng 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatas e fusion protein system. All were cleaved after the RxxR, and some sub strates with Pro or Thr in P2 were also found to be cleaved as efficie ntly as RxKR or RxRR. To further elaborate surrounding determinants, w e constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Althoug h no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, su ggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity o f furin and suggest the substrate phage method may be useful for ident ifying consensus substrate motifs in other protein processing enzymes.