We read with interest the article by Guillemot et al. evaluating the implication of the proprotein convertases in iron homeostasis. The authors based their work on our recent genome-wide association study establishing a strong link between plasma levels of the soluble human transferrin receptor 1 (s-hTfR1) and the PCSK7 gene. We suggested three possible mechanisms of PCSK7 acting on iron homeostasis. One may speculate that PCSK7, similar to furin, modulates hepcidin expression by directly influencing soluble Hemojuvelin (sHJV) levels. Alternatively, PCSK7 may be involved in iron homeostasis either by direct shedding of the hTfR1 or indirectly by activating hepcidin. Exploring the latter two hypotheses, Guillemot et al. found that among the PC family members PCSK7 is unique in directly shedding hTfR1 by cleavage at an atypical site KTECER100↓LA, and that furin alone activates hepcidin.
To fully explore the potential roles of PCSK7 in iron homeostasis, we also tested the first hypothesis, which is the involvement of PCSK7 in generating sHJV. As PCSK7 has the ability to cut, at least partially, peptides containing an RX(R/K)R motif, it can be potentially involved in hepcidin regulation by direct cleavage of HJV at the polybasic segment RNRR (amino acid 332-335).
The possible PCSK7-mediated proteolytic activity on HJV was investigated using transient cotransfections of HeLa or LoVo cells with different combinations of PCSK7/HJV expression constructs and suitable empty vector controls followed by western blot (WB) analyses. Cotransfection of furin and HJV was used as positive control for HJV cleavage, and a transfected ADAM10 substrate was used as positive control for the proteolytic activity of PCSK7 (Fig. 1).
As shown (Fig. 1), despite the RNRR motif, we detected no PCSK7 cleavage activity directly on HJV, demonstrating that PCSK7 is not involved in hepcidin regulation by influencing s-HJV levels. Our data therefore contribute to the functional characterization of PCSK7 at different levels in iron regulation and support the shedding of TfR1 as its unique mechanism of involvement in the regulation of systemic iron homeostasis, as reported.
Acknowledgment: We thank Laura Silvestri for human-full-length HJV and Furin pcDNA3-constructs, Paolo Arosio for the anti-HJV antibody, and Rolf Postina for the pcDNA3-bovine-ADAM10 construct. The study was supported by the Ministry of Health and Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano and the South Tyrolean Sparkasse Foundation.
Christine Schwienbacher, Ph.D.1
Alice Serafin, M.Sc.1
Alessandra Zanon, M.Sc.1
Peter P. Pramstaller, M.D.1-3
Irene Pichler, Ph.D.1
Andrew A. Hicks, Ph.D.1
1Center for BiomedicineEuropean Academy Bozen/Bolzano (EURAC)Bolzano, Italy; Affiliated Institute of the University of Lübeck, Lübeck, Germany2Department of NeurologyGeneral Central HospitalBolzano, Italy3Department of NeurologyUniversity of LübeckLübeck, Germany