Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris

Vashchenko, Ganna; Bleackley, Mark R.; Griffiths, Tanya A. M.; MacGillivray, Ross T. A.

doi:10.1016/j.abb.2011.07.010

Cited by 10 publications

(10 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The K m values of recombinant hephaestin for such organic substrates as p -phenylenediamine and O -dianisidine were close to values determined for ceruloplasmin [130]. However, in contrast to ceruloplasmin, hephaestin was incapable of direct oxidation of biogenic amines, such as adrenaline and dopamine [130], implying a difference in biological substrate specificities between these two homologous oxidases. In addition, kinetic studies revealed that similar to ceruloplasmin, hephaestin has two types of iron-binding sites with different affinities towards ferrous iron [131].…”

Section: Human Multi-copper Oxidasesmentioning

confidence: 90%

Multi-Copper Oxidases and Human Iron Metabolism

2013

Self Cite

View full text Add to dashboard Cite

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis.

show abstract

Section: Human Multi-copper Oxidasesmentioning

confidence: 90%

Multi-Copper Oxidases and Human Iron Metabolism

2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…Whilst protein production by BHK cells provided sufficient yields, the copper content was the lowest [ 16 ]. Conversely, expression and purification in Pichia had the highest copper content, but the yield was low and the protein generated appeared to be unstable in solution [ 14 , 17 ]. In an attempt to merge the aspects of ‘high’ copper content, high yield, and protein stability, we generated a protocol for mouse HEPH expression using the baculovirus and insect cell system.…”

Section: Resultsmentioning

confidence: 99%

“…Comparative structural modeling has indicated an analogous architecture for HEPH [ 15 ]. However, preparations of recombinant human HEPH expressed in Pichia pastoris or baby hamster kidney (BHK) cells have indicated between 3 and 4 copper ions per HEPH molecule [ 14 , 16 , 17 ]. This discrepancy could indicate that the recombinant production of HEPH leads to incomplete copper loading, or, that HEPH has a different architecture than ceruloplasmin.…”

Section: Introductionmentioning

confidence: 99%

Large scale expression and purification of secreted mouse hephaestin

Deshpande

Xin

et al. 2017

PLoS ONE

View full text Add to dashboard Cite

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.

show abstract

“…Nevertheless, the data definitely show that the SAT cells from the Ag-rats produced oxidase activity. There is a theoretical possibility that detectable oxidase activity belonged to hephaestin, an integral membrane protein from family of blue multicopper ferroxidase, which was able to oxidize o -dianisidine [31]. In rats, hephaestin is expressed at high levels throughout the small intestine and colon and at low levels in lung, spleen, placenta and embryo.…”

Section: Resultsmentioning

confidence: 99%

The role of subcutaneous adipose tissue in supporting the copper balance in rats with a chronic deficiency in holo-ceruloplasmin

2017

View full text Add to dashboard Cite

We have previously shown that (1) an acute deficiency in blood serum holo-ceruloplasmin (Cp) developed in rats that were fed fodder containing silver ions (Ag-fodder) for one month and (2) the deficiency in holo-Cp was compensated by non-hepatic holo-Cp synthesis in rats that were chronically fed Ag-fodder for 6 months (Ag-rats). The purpose of the present study is to identify the organ(s) that compensate for the hepatic holo-Cp deficiency in the circulation. This study was performed on rats that were fed Ag-fodder (40 mg Ag·kg-1 body mass daily) for 6 months. The relative expression levels of the genes responsible for copper status were measured by RT-PCR. The in vitro synthesis and secretion of [14C]Cp were analyzed using a metabolic labeling approach. Oxidase activity was determined using a gel assay with o-dianisidine. Copper status and some hematological indexes were measured. Differential centrifugation, immunoblotting, immunoelectrophoresis, and atomic absorption spectrometry were included in the investigation. In the Ag-rats, silver accumulation was tissue-specific. Skeletal muscles and internal (IAT) and subcutaneous (SAT) adipose tissues did not accumulate silver significantly. In SAT, the mRNAs for the soluble and glycosylphosphatidylinositol-anchored ceruloplasmin isoforms were expressed, and their relative levels were increased two-fold in the Ag-rats. In parallel, the levels of the genes responsible for Cp metallation (Ctr1 and Atp7a/b) increased correspondingly. In the SAT of the Ag-rats, Cp oxidase activity was observed in the Golgi complex and plasma membrane. Moreover, full-length [14C]Cp polypeptides were released into the medium by slices of SAT. The possibilities that SAT is part of a system that controls the copper balance in mammals, and it plays a significant role in supporting copper homeostasis throughout the body are discussed.

show abstract

Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris

Cited by 10 publications

References 41 publications

Multi-Copper Oxidases and Human Iron Metabolism

Multi-Copper Oxidases and Human Iron Metabolism

Large scale expression and purification of secreted mouse hephaestin

The role of subcutaneous adipose tissue in supporting the copper balance in rats with a chronic deficiency in holo-ceruloplasmin

Contact Info

Product

Resources

About