Large scale expression and purification of secreted mouse hephaestin

Deshpande, C.N.; Xin, Vicky; Lu, Yan; Savage, Trevor N.; Anderson, Gregory J.; Jormakka, Mika

doi:10.1371/journal.pone.0184366

Cited by 10 publications

(12 citation statements)

References 38 publications

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“…We confirmed that immunoreactivity for hephaestin was observed in the cytoplasm of human brain astrocytes. Hephaestin, also known as a ferroxidase, was reported to be expressed in a secreted form . Burkhart et al reported that immunoreactivity for hephaestin was localized at both the cellular membrane and cytoplasm using primary astrocytes .…”

Section: Discussionmentioning

confidence: 99%

“…In this way, the ferroportin‐hepcidin system likely represents the main pathway for cellular iron egress and also regulates cellular iron levels in astrocytes. Hephaestin is known to be a large membrane‐anchored multicopper ferroxidase involved in iron metabolism . Dietary iron is exported across the basolateral membrane of enterocytes by the ferrous iron transporter ferroportin, and hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin.…”

Section: Introductionmentioning

confidence: 99%

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Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains

et al. 2019

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Iron plays essential roles in the central nervous system. However, how the iron level is regulated in brain cells including glia and neurons remains to be fully clarified. In this study, the localizations of hepcidin, ferroportin, and hephaestin, which are known to be involved in iron efflux, were immunohistochemically examined in autopsied human brains. Immunoreactivities for hepcidin and ferroportin were observed in granular structures within the cytoplasm of reactive astrocytes and epithelial cells of the choroid plexus. Granular structures showing immunoreactivities for hepcidin and ferroportin were also stained with antibodies for early endosome antigen 1 (EEA1). In addition, immunoreactivity for hephaestin was observed in the cytoplasm of epithelial cells of the choroid plexus as well as reactive astrocytes. Immunoreactivity for hephaestin in the cytoplasm of reactive astrocytes was occasionally colocalized with immunoreactivity for EEA1, while that of hephaestin was frequently observed in the cytoplasm showing no immunoreactivity for EEA1. These findings suggest that immunoreactivities for hepcidin and ferroportin are localized in close proximity to granular structures showing immunoreactivity for EEA1 in the cytoplasm of human brain astrocytes. They also suggest that immunoreactivity of hephaestin is localized in the cytoplasm of the choroid plexus epithelium as well as reactive astrocytes of human brains.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains

et al. 2019

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“…RBD Wuhan was produced in a system of baculovirus/insect cells and purified as reported (we thank these authors for providing the plasmid). The protein (residues Arg319-Phe541 of S Wuhan ) hosts a C-terminal 6×His-tag and is exported to the culture medium thanks to a gp67 signal peptide that is cleaved upon exportation …”

Section: Methodsmentioning

confidence: 99%

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein

Gargantilla,

Francés,

Adhav

et al. 2023

J. Med. Chem.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. Highresolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

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“…Hp has 50% sequence similarity with Cp, including the conservation of multiple copper binding sites (Vulpe et al, 1999;Griffiths et al, 2005) and is highly expressed in sla mouse intestine. Interestingly, in sla mice, this gene shows an in-frame deletion of 194 amino acids, probably resulting in a non-functional product (Ranganathan et al, 2012a;Deshpande et al, 2017). The researchers conclude that this protein is a transmembrane-bound ferroxidase that plays an important role in the iron export mechanism involving FPN and Tf (Hudson et al, 2008;Vashchenko and Macgillivray, 2012;Lee et al, 2012;Zheng et al, 2018).…”

Section: Introductionmentioning

confidence: 98%

“…

Once inside the enterocyte, the iron can be stored or exported across the enterocyte basolateral membrane by the ferrous iron exporter ferroportin (FPN) (Knutson, 2017). Hephaestin (Hp) increases the efficiency of this process by oxidising the transported iron to its ferric form and promoting its release from FPN (Crichton, 2009;Han, 2011;Lawen and Lane, 2013;Deshpande et al, 2017), from which it is rapidly bound to apo-Tf for transport in the circulation to tissues (Lawen and Lane, 2103;Knutson, 2017). In short, Hp plays a role in the uptake of iron from the diet, whereas Cp helps with the redistribution of iron to the cells and tissues; however, how Hp interacts with Tf, Cp, and FPN is still unclear.To understand this mechanism, iron metabolismdisordered animals, e.g., knockout (KO) mice and sla mice, have been studied as useful models of iron homeostasis.

…”

mentioning

confidence: 99%

Expression of soluble, active, fluorescently tagged hephaestin in COS and CHO cell lines

et al. 2020

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Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes ac-cumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper fer-roxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron trans-port protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.

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Large scale expression and purification of secreted mouse hephaestin

Cited by 10 publications

References 38 publications

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein

Expression of soluble, active, fluorescently tagged hephaestin in COS and CHO cell lines

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