Histidine‐rich human salivary peptides are inhibitors of proprotein convertases furin and PC7 but act as substrates for PC1

Publisher Website
Google Scholar
Abstract
A 32 amino acid peptide called histatin‐3 (H3; 22% His) and its TV‐terminal 24 amino acid fragment histatin‐5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His‐rich peptides have been synthesized by Fmoc‐based solid‐phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9–24 segment of H3 with all the His at positions 15, 18, 19, 21 replaced by Tyr was also prepared (Δ1–8H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PCl, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PCI, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR↓SN). On prolonged incubation some minor cleavage was also observed C‐terminal to the first Lys Arg6 pairs of basic amino acids namely at: HAKR↓HH, which contains a P4 as well as P'l and P'2 His residues. The second potential site YKRK12‐FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PCI only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR↓HH. As expected, neither the D‐amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all. In contrast to the above findings for hPCl, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin‐mediated cleavage of the pentapeptide pGlu‐Arg‐Thr‐Lys‐Arg‐MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 μM for H3 and 2.98 μM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 μM. The partial sequence identity of H3 to the inhibitory pro‐peptide of furin and PC7 provides a rationale for our observation. © Munksgaard 1997.