# Shedding of GPP130 by PC7 and Furin: Potential Implication in Lung Cancer Progression
## 3 Discussion  
GPP130 is a Golgi resident protein with a unique trafficking pathway. Most of the literature covering GPP130 is related to its role in transporting the Shiga toxin intracellularly [22]. In addition, investigations aiming at targeting GPP130 trafficking for therapeutic uses in the context of Shiga toxin infections have demonstrated that this protein is sensitive to Mn\({}^{2+}\) and can be rerouted for degradation in lysosomes following Mn\({}^{2+}\) treatment [21]. However, other biological roles of GPP130, including its cell biology, remain unknown. Notably, a group interested in Ca\({}^{2+}\) signaling during tumor growth has recently highlighted the importance of GPP130 in cell cycle progression and showed that the knockdown of GPP130 severely impacts cell proliferation in head-and-neck cancer cells [25]. Herein, we demonstrated that GPP130 is shed by PC7 and Furin at two distinct sites (S1 and S2) in its luminal domain, which would be expected to have dramatic consequences on the efficient binding and transport of the Shiga toxin. Site-directed mutagenesis revealed that the S1 site cleaved by PC7, and Furin occurs at Arg\({}_{70}\downarrow\) and Lys\({}_{73}\downarrow\), respectively (Figure 3B), and that Furin is the only conversate that cleaves at Arg\({}_{277}\downarrow\) (S2 site). Notably, site-directed mutagenesis revealed that the PC7 cleavage at S1 occurs at HESR\({}_{70}\downarrow\) and that Arg\({}_{70}\) at P1 and Arg\({}_{68}\) at P3 are critical but not His\({}_{57}\) at P4 (Figure 3B). It is unusual that a P3 Arg would be critical for PC-cleavage, but His at P4 is present in other PC-substrates such as mouse pro-epidermal growth factor (proEGF) [34]. We also demonstrated that the independent cleavages at S1 and S2 occur in distinct intracellular acidic compartments. Cleavage at S1 is inhibitable by dec-RVKR-cmk and NH\({}_{4}\)Cl, suggesting that both PC7 and Furin process GPP130 in the TGN and/or acidic endosomes, as expected from the localization of PC7 activity in endosomes [30] and of Furin activity in the TGN and endosomes [35]. In contrast, the S2 cleavage by Furin seems to occur in dec-RVKR-cmk resistant but NH\({}_{4}\)Cl-sensitive acidic compartment(s) (Figure 4). Interestingly, in the absence of overexpressed PC7 or Furin, GPP130 seems to be shed at a site closer to the transmembrane domain, possibly by an endogenous cell surface metalloprotease (more active in the presence of NH\({}_{4}\)Cl), as suggested by the upward shift in S1 on Western blots (compare EV versus PC7 or Furin in Figure 4C-E). Finally, one drawback of this study is the need to overexpress PC7 or Furin to detect GPP130 shedding by endogenous proteases in either HEK293 or A549 cells. The identification of a lung cancer cell line with high enough endogenous PC7, Furin, and GPP130 levels may support our conclusions. Indeed, analysis of the Cancer Genome Atlas database revealed that in certain tumors, PC7, Furin and GPP130/GOLIM4 are highly expressed (Supplementary Figure S2). ([[https://www.proteinatlas.org/ENSG00000160613-PCSK7/cancer](https://www.proteinatlas.org/ENSG00000160613-PCSK7/cancer)]([https://www.proteinatlas.org/ENSG00000160613-PCSK7/cancer](https://www.proteinatlas.org/ENSG00000160613-PCSK7/cancer)); [[https://www.proteinatlas.org/ENSG00000140564-FURIN](https://www.proteinatlas.org/ENSG00000140564-FURIN)]([https://www.proteinatlas.org/ENSG00000140564-FURIN](https://www.proteinatlas.org/ENSG00000140564-FURIN)); [[https://www.proteinatlas.org/ENSG00000173905-GOLIM4/cancer](https://www.proteinatlas.org/ENSG00000173905-GOLIM4/cancer)]([https://www.proteinatlas.org/ENSG00000173905-GOLIM4/cancer](https://www.proteinatlas.org/ENSG00000173905-GOLIM4/cancer)), accessed on 21 May 2025).