Because of the central role of hepcidin in the regulation of iron homeostasis, the posttranslational processing of the peptide is of potential importance, but has heretofore received scant attention. We therefore find the results reported by Valore and Ganz demonstrating that inhibitors of furin proprotein convertases affect processing of prohepcidin to mature hepcidin at the furin consensus sequence RXRR of special interest. We have also explored this process from two points of view. First of all, we have studied the sequence requirements for this cleavage in some detail, particularly based upon a known human mutation. Secondly, we have addressed the possibility that regulation of hepcidin production occurs not only at the transcriptional level, which has been studied extensively in several laboratories, but also at the posttranslational level, which has been almost entirely neglected. The sequence surrounding the human hepcidin cleavage site is QRRRRR↓DTHF and the mouse hepcidin cleavage site is QKRRKR↓DTNF. It is notable that there is a previously described mutation in hepcidin reported by Jacolot et al [1], R59G, the predicted arginine in the P1 site of the furin cleavage consensus sequence. Since the mutation leaves 4 intact arginines, one would predict that furin would still be able to cleave R59G mutant hepcidin provided the glycine in the P1’ site is acceptable. We have made several mutants of prohepcidin in order to examine processing of prohepcidin to mature hepcidin (Figure 1). Our studies have shown that the R59G mutation (QRRRR↓GDTHF) is not cleaved efficiently but a D60G mutation (QRRRRR↓GTHF) is cleaved efficiently. This suggests that the presence of a glycine in the P1’ site is acceptable but, possibly, the presence of four arginines is not sufficient. In fact, we found that cleavage of a hepcidin double mutant R58G/R59G that would leave only three arginines of the consensus sequence (QRRR↓GGDTHF) was indistinguishable from the R59G mutant with four arginines. We further examined the importance of the P1’, P2’, P4’ and P4’ sites. The prohepcicin mutants with deleted amino acids 60–62 (22mer) with the recognition sequence RRRRR↓FPIC and deleted 60–64 (20mer) (RRRRR↓ICIF) were inefficiently processed and not processed at all, respectively. The T61I (RRRRR↓DIHF) and the H62W (RRRRR↓DTWF) prohepcidin mutants cleavage products were different from the F63F (RRRRR↓DTHY) prohepcidin mutant, the latter being larger. The migration of the T61I (P2’) and H62W (P3’) mutants suggested that they were processed to the 20- and 22 mer forms since they comigrated with the 22mer cleavage products but the F63Y (P4’) mutant was cleaved to a larger form (possibly 25mer) of hepcidin that was resistant to further processing to the smaller 22- and 20mer forms. This would suggest that the presence of the phenylyalanine at +4 from the amino terminus of the mature hepcidin 25mer was important for degradation to the 22 and 20mer forms of hepcidin. The electrophoretic mobility of the F68G and F68A mutants is the same as that of the F63Y mutant suggesting that these mutations also produce a stable 25mer hepcidin resistant to further processing. The previously reported G71D mutant [1], and the M80Y and T84P mutants, appeared to be processed in a manner similar to wildtype prohepcidin (25-, 22- and 20mer). We have also examined whether iron had an affect on hepcidin processing. Treating prohepcidin transfected HEK293T cells with either FeNTA or desferal demonstrated that iron had no effect of the processing of prohepcidin to mature hepcidin (data not shown). These data are consistent with Valore and Ganz’s data that demonstrate that apo- and holo-transferrin had no effect on prohepcidin processing. Figure 1 Human hepcidin constructs In conclusion, our data suggests that the R59G mutation observed in some humans subjects results in inefficient cleavage of prohepcidin due to the loss of an arginine in the recognition sequence rather than the presence of a glycine in the P1’ position. Furthermore, if a furin-like prohormone convertase is responsible for the cleavage of prohepcidin, as suggested by Valore and Ganz, then the consensus cleavage site requires more than four arginine/lysine residues in the P sites and, a non-hydrophobic residue in the P1’ site. In addition, our data suggest that other mutations in hepcidin, affect the processing of the 25 amino acid protein to the 22 and 20 amino acid forms.