A 32 amino acid peptide called histatin-3 (H3; 22% His) and its n-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, 14, 21), The 9-24 segment of H3 with all the His at positions 15,18,19,21 replaced by Tyr was also prepared (Delta(1-8)H3Y). 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 PCl, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg(25) residue (HRGYR down arrow SN). On prolonged incubation some minor cleavage was also observed C-terminal to the first LysArg(6) pairs of basic amino acids namely at: HAKR down arrow HH which contains a P4 as well as P'1 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 down arrow HH. As expected, neither the D-amino add 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 hPC1, 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 K-i of 1.98 mu M for H3 and 2.98 mu M 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 K-i of 2.4 mu M. The partial sequence identity of H3 to the inhibitory pro-peptide of furin and PC7 provides a rationale for our observation. (C) Munksgaard 1997.
