Substrate cleavage analysis of furin and related proprotein convertases. A comparative study

Abstract

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.

FIGURE 1.

Multiplexed oligonucleotide-peptide cleavage assay. A, synthesis of peptide-oligonucleotide conjugates. Peptides were synthesized using Fmoc chemistry. The N terminus of each peptide molecule contained a (His)₅ tag and hydroxyaminoacetic acid, while the C-terminal end of the peptides was linked to biotin. Synthetic oligonucleotides were modified by coupling a phosphoramidite containing a formylindole group. Hydroxylamine-modified peptides and aldehyde-modified oligonucleotides were co-incubated to form an oxime linkage. B, purification of the peptide-oligonucleotide conjugates using polyacrylamide gel electrophoresis. C, peptide cleavage assay and hybridization. The purified peptide-oligonucleotide conjugates were incubated with the individual PCs. After an exhaustive proteolysis, the biotin-labeled cleavage products and the residual intact peptides were pulled-down from the cleavage reactions using streptavidin-coated magnetic beads. The reaction supernatants were hybridized using SAMs containing unique oligonucleotide sequences that were complementary to the respective oligonucleotide of the peptide-oligonucleotide conjugates. The hybridized samples were incubated with a murine (His)₅ antibody followed by incubation with Alexa 555-conjugated goat anti-mouse IgG. The resulting fluorescence signal was measured using an Illumina Bead Array Reader. Each sample was analyzed in triplicate.

FIGURE 2.

Frequency plot of the cleavage sequence of the individual PCs in a Weblogo format. The size of the symbol indicates the frequency of the individual residue occurrence at individual substrate positions relative to the P1-P1′ scissile bond.

FIGURE 3.

Analysis of the cleavage preferences of the individual PCs in a radar-plot format. The plots represent the differences between the effective cleavage efficiency values calculated for each amino acid type at a given position of the peptide substrate and the average cleavage efficiency global value calculated for the entire set of substrates for a given PC. The 0 number in the plots denotes that the effective cleavage efficiency value of an amino acid residue is equal to the global value of the entire set of substrates for a given PC.

FIGURE 4.

Mass spectrometry analysis of the cleavage peptides. The human MT6-MMP peptide VRRRRR↓YALS and the mouse MT6-MMP peptide VRRRRR↓YSLS were incubated for 2 h at 37 °C with furin and PC2 (5 activity units each). The additional furin reactions, in which 2 units of furin were used, are indicated in the panels. The molecular mass of the peptides was determined by MALDI-TOF MS. There was no difference between the calculated and the estimated molecular mass of the peptides. Where indicated, dec-RVKR-cmk was added to the cleavage reactions. Insets, in the results of furin reactions, which contained dec-RVKR-cmk are presented as insets. The molecular mass of the intact human and mouse peptide is 1332 and 1348 Da; respectively (the numbers are underlined in the figure panels). The molecular mass of the N-terminal cleavage product VRRRRR is 898 Da.

FIGURE 5.

Cleavage of HA and PA83 by PCs. A, individual PCs cleave the HA precursor. The HA precursor (1 μm) was incubated for 1 h at 37 °C with the individual PCs (1 activity unit each). Top panel, digest reactions were analyzed by SDS-PAGE. Bottom panel, the gels were scanned, the band density was digitized and the conversion of the precursor into the mature HA was expressed in percent. B, furin efficiently cleaves PA83. PA83 and the HA precursor were each incubated for 1 h at 37 °C with the indicated amounts of furin. The asterisks indicate the amounts of furin that were required for accomplishing a 50% conversion of the precursor into the mature species. DEC, dec-RVKR-cmk.

FIGURE 6.

The amounts of the individual PCs required for accomplishing a 50% conversion of PA83 into PA63. PA83 was incubated for 1 h at 37 °C with increasing amounts of the individual PCs. The digest reactions were analyzed by SDS-PAGE, the gels were scanned, and the band density was digitized to determine the amount of each PC that is required for accomplishing a 50% conversion of PA83 into PA63. Inset, PC2 was omitted to demonstrate the differences among furin, PC1/3, PC4, PC5/6, PC7, and PACE4.

FIGURE 7.

Direct comparison of the peptide and protein cleavage efficiency by the individual PCs. A, efficiency of the cleavage of the peptides (sequences of the peptides are shown on the right) derived from PA83, HA, and PEx. The cleavage efficiency of the NSRKKR↓STSA PA83 peptide by each PC was taken as 100%. B, efficiency of the cleavage of the PA83, HA, and PEx proteins. The cleavage efficiency of PA83 by each PC was taken as 100%.