C Sun

D Belletti

J Fisher

K Fan

L Conti

L Li

L Mendes-Jorge

M Liang

PM Harrison

S Geninatti Crich

Y Zhao

Z Chen

Z Heger

Kelong Fan

Meng Zhou

Xiyun Yan

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COMMENTARYQuestions about horse spleen ferritin crossingthe blood brain barrier via mouse transferrinreceptor 1Kelong Fan1, Meng Zhou1,2, Xiyun Yan1,2&1 Key Laboratory of Protein and Peptide Pharmaceuticals, CAS-University of Tokyo Joint Laboratory of Structural Virology andImmunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China2 University of Chinese Academy of Sciences, Beijing 100049, China& Correspondence: yanxy@ibp.ac.cn (X. Yan)Ferritin, an iron storage protein naturally occurring in thebody, has emerged as a promising nanocarrier thanks to itsunique architecture, excellent biocompatibility, and ability toself-assemble/disassemble (Fan et al., 2013). More speciﬁ-cally, the ﬁnding that human H-ferritin intrinsically targetstumor cells via binding to its receptor transferrin receptor 1(TfR1) (Li et al., 2010; Fan et al., 2012; Liang et al., 2014;Zhao et al., 2016) inspired research into using ferritins fortumor target therapy.Recently, a paper published in Molecular Pharmaceuticsshowed that horse spleen ferritin (HosFn) binds to andcrosses the mouse BBB because of binding to mouse TfR1(Chen et al., 2017), which raises great concerns to us.To the best of our knowledge, HosFn lacks the ability tocross the mouse BBB, due to its lack of binding to mouseTfR1. Below we brieﬂy describe others’ and our own evi-dence supporting such a conclusion.Firstly, in our own experiments, we failed to detect anyspeciﬁc binding sites for HosFn on mouse BBB endothelialbEnd.3 cells.In their paper, Chen et al. employed antibody-blockingexperiments to show that the speciﬁc binding ofHosFn to TfR1on mouse bEnd.3 cells (Chen et al., 2017). However, theantibody used was mouse anti-human TfR1 (Clone M-A712;BD Bioscience), which was developed to speciﬁcally detecthuman TfR1 according to the Product Information provided bythe manufacturer (BD Bioscience). Our ELISA assay con-ﬁrmed that this antibody recognizes human TfR1, but notmouse TfR1 (Fig. 1A). We also demonstrated that althoughmouse TfR1 was abundantly expressed on bEnd.3 cells(Fig. 1C), mouse anti-human TfR1 antibody fail to interact withthese cells (Fig. 1B), further indicating the idea that this anti-body does not bind to mouse TfR1. Therefore, it appearsimpossible to block the binding of HosFn to mouse TfR1 withthe antibody used in Chen et al.’s paper.In our experiments, the pre-incubation of bEnd.3 cells withexcessamount (up to100-foldmoleexcess) of unlabeledHosFnexhibited little effect on the binding of FITC-labeled HosFn tothese cells (Fig. 1D). These results suggest that no speciﬁcbinding sites for HosFn were present on bEnd.3 cells, whichfurther conﬁrmed that HosFn does not bind to mouse TfR1.Secondly, the conclusion that HosFn cannot bind to andcross the BBB in mice was also drawn by Connor and col-leagues (Fisher et al., 2007). The receptor of HosFn wasreported to be the mouse L-ferritin receptor—mouse Scara 5by different groups (Sun et al., 2011; Mendes-Jorge et al.,2014; Conti et al., 2016), which is also consistent with thefact that nearly 92% (22/24) composition of HosFn is Lsubunits (Harrison, 1986; Sun et al., 2011), and HosFn istypically regarded as L-ferritin in most previous research(Sun et al., 2011; Mendes-Jorge et al., 2014; Geninatti Crichet al., 2015; Conti et al., 2016).Taken together, the conclusions of Chen et al.’s workneed to be reconsidered, as they are misleading at best.Here, we provide more information about ferritins and theirreceptors. Importantly, we hope that this commentary willclarify the precise nature of the interactions of ferritins withtheir receptors.To date, several ferritin receptors have been identiﬁed (Fanet al., 2013; Heger et al., 2014; Belletti et al., 2017). Althoughferritins are highly conserved in various species, their recep-tors could be very different, e.g., the receptor of humanH-ferritin is TfR1, while that of mouse H-ferritin is TIM-2. Thus,we cannot infer that different ferritin receptors function simi-larly. As amatter of fact, ferritins from different species do playcontrasting roles beyond iron storage. We hope to point outElectronic supplementary material The online version of thisarticle (doi:10.1007/s13238-017-0481-8) contains supplementarymaterial, which is available to authorized users.© The Author(s) 2017. This article is an open access publicationProtein Cell 2017, 8(11):788–790DOI 10.1007/s13238-017-0481-8 Protein&CellProtein&Cellhere that when employing ferritin as nanocarriers to developanti-disease system, people must clearly recognize whichferritin and the corresponding receptor are suitable for theirpurpose. When HosFn is chosen, its corresponding cross-interactive receptor is Scara 5 in mouse. Other suitable re-ceptors for this purpose are yet to be determined.COMPLIANCE WITH ETHICS GUIDELINESKelong Fan, Meng Zhou, and Xiyun Yan declare that there is noconﬂict of interest that could be perceived as prejudicing theimpartiality of the research reported.This article does not contain any studies with human or animalsubjects performed by any of the authors.OPEN ACCESSThis article is distributed under the terms of the Creative CommonsAttribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit tothe original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made.BADC***NSOD450 nmHuman TfR1Mouse TfR1BSA1.51.00.50.0MFILog [Concentration of unlabled HosFn (μmol/L)100806040200mlgGAnti - human TfR1 mAbFluorescence intensityCounts100806040200100 101 102 103 104RlgGAnti-mouse TfR1 RAbFluorescence intensityCounts100806040200100 101 102 103 104 0.001 0.01 0.1 1 10 100Figure 1. Lack of interaction between HosFn and mouse BBB endothelial bEnd.3 cells. (A) ELISA analysis of the binding ofmouse anti-human TfR1 mAbs (Clone M-A712; BD Bioscience) to human TfR1 (11020-H07H, Sino Biological) and mouse TfR1(50741-M07H, Sino Biological). (B) Flow cytometry analysis of the binding of mouse anti-human TfR1 antibody (Clone M-A712; BDBioscience) to bEnd.3 cells. (C) Flow cytometry analysis of the binding of rabbit anti-mouse TfR1 antibody (50741-T16; SinoBiological) to bEnd.3 cells. (D) Competition for the binding sites of FITC-labeled HosFn (0.1 μmol/L) on bEnd.3 cells by unlabeledHosFn.Does horse spleen ferritin cross the BBB via mouse TfR1? COMMENTARY© The Author(s) 2017. This article is an open access publication 789Protein&CellREFERENCESBelletti D, Pederzoli F, Forni F, Vandelli MA, Tosi G, Ruozi B (2017)Protein cage nanostructure as drug delivery system: magnifyingglass on apoferritin. 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Proc NatlAcad Sci USA 111:14900–14905Mendes-Jorge L, Ramos D, Valença A, López-Luppo M, Pires VMR,Catita J, Nacher V, Navarro M, Carretero A, Rodriguez-Baeza Aet al (2014) L-Ferritin binding to Scara5: a new iron trafﬁc pathwaypotentially implicated in retinopathy. PLoS ONE 9:e106974Sun C, Yang H, Yuan Y, Tian X, Wang L, Guo Y, Xu L, Lei J, Gao N,Anderson GJ et al (2011) Controlling assembly of paired goldclusters within apoferritin nanoreactor for in vivo kidney targetingand biomedical imaging. J Am Chem Soc 133:8617–8624Zhao Y, Liang M, Li X, Fan K, Xiao J, Li Y, Shi H, Wang F, Choi HS,Cheng D et al (2016) Bioengineered magnetoferritin nanoprobesfor single-dose nuclear-magnetic resonance tumor imaging. ACSNano 10:4184–4191COMMENTARY Kelong Fan et al.790 © The Author(s) 2017. This article is an open access publicationProtein&Cell

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Questions about horse spleen ferritin crossing the blood brain barrier via mouse transferrin receptor 1

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Questions about horse spleen ferritin crossing the blood brain barrier via mouse transferrin receptor 1

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