Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin

Evelyne Furger,Roger Schibli,Dorte Launholt Lildballe,Sergey N Fedosov,Robert Waibel,Eliane Fischer,Ebba Nexo

doi:10.1371/journal.pone.0037421

Evelyne Furger, Roger Schibli + Show 5 more

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https://doi.org/10.1371/journal.pone.0037421

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Abstract

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.

Highlights

Vitamin B12 is a water soluble vitamin which is involved in biosynthetic processes in every living cell
Purification of recombinant human HC (rhHC) from HEK293 culture supernatant We developed a mammalian cell expression system for production of rhHC, which contained a thrombin-cleavable, Cterminal fusion tag consisting of the Myc and pohlyhistidine peptides (Myc-His-tag) for affinity purification and immunological detection by anti-Myc-tag antibody (Figure 1A)
The fusion tag was removed from rhHC by cleavage with thrombin and rhHC was further purified by a Ni2+ affinity chromatography step to remove uncleaved rhHC, the cleaved tag peptides and any protein impurities with affinity for Ni2+ (Figure 1B, lane 5)

Summary

Introduction

Vitamin B12 (cobalamin, Cbl) is a water soluble vitamin which is involved in biosynthetic processes in every living cell. Its transport within the human body is mediated by an elaborate system involving three soluble binding proteins, haptocorrin (HC) (previously referred to as transcobalamin I or R-binder), intrinsic factor (IF) and transcobalamin (TC) as well as their receptors [1]. IF ensures the uptake of Cbl in the intestinal cell, and once Cbl is transferred to the blood, TC is needed in order to transport Cbl into all cells of the body. HC is degraded by pancreatic enzymes in the duodenum, whereupon the released Cbl binds to IF and is transferred to the blood. HC binds Cbl and so-called Cbl analogues, which are corrinoids without cofactor activity in mammalian cells. Up to 40% of all corrinoids bound to HC in plasma are Cbl analogues [2,4,5]

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