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

Abstract

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.

Identification of copper metabolism-related biomarkers and exploration of mechanisms based on osteoarthritis transcriptomics data

“…Additionally, because it is an upstream factor of IL-1β, it can further in uence the OA development process by either activating iron death or upregulating IL-1β [42,43,44] . As HEPH plays a crucial role in the intestinal transport of iron ions, it keeps cellular iron from building up and shields cells from iron-induced oxidative stress by inhibiting the overexpression of iron ions and their excretion through the intestines [45] ,and copper ion de ciency can lead to a decrease in HEPH protein oxidase activity thereby reducing the binding of iron ions [46] .…”

Association between iron metabolism and SARS-COV-2 infection, determined by ferritin, hephaestin and hypoxia-induced factor-1 alpha levels in COVID-19 patients

“…Hephaestin is a multi-copper ferroxidase (MCF) expressed in enterocytes that facilitates the absorption of dietary iron from the intestine [ 7 – 9 ] by facilitating export of iron through the basolateral membrane to the circulation. Iron is exported from enterocytes as Fe 2+ via the basolateral transporter ferroportin and hephaestin converts Fe 2+ to ferric Fe 3+ for loading onto transferrin.…”

“…It was discovered as the protein mutated in the sla locus of the sex-linked anemia (sla) mice. The microcytic anemia of the sla mouse was attributed to defective export of iron from enterocytes, indicating that decreased or defective hephaestin can lead to iron deficiency [ 7 – 9 ]. Infection of enterocytes with SARS-CoV-2 can induce down-regulation of ferroportin and hephaestin [ 10 ] and it was of interest to see if changes in serum hephaestin could correlate with COVID-19.…”