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

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.

Organometallic DNA–B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin‐Transport Proteins

“…[a] rhHC is the recombinant form with an additional LPVR tag at the C terminus. This tag slightly reduces the affinity for Cbls, relative to native hHC, as discussed earlier for deglycosylated rhHC . [b] This work and ref.…”

VariantASGR1Associated with a Reduced Risk of Coronary Artery Disease

“…Both alkaline phosphatase and haptocorrin, a vitamin B 12 transporter, are asialylated glycoproteins that are known to bind ASGPR and thus be cleared from the circulation. [31][32][33][34] The likely explanation for the increased levels of alkaline phosphatase among carriers of loss-of-function ASGR1 variants is that clearance of desialylated molecules from the circulation is compromised.…”

Structural Basis for Universal Corrinoid Recognition by the Cobalamin Transport Protein Haptocorrin

“…Protein Expression and Purification-Recombinant human HC and TC (referred to below as HC and TC) and murine TC (mTC) were prepared as described previously (24,25). Briefly, HC was expressed in HEK293 cells, purified on a nickel-nitrilotriacetic acid affinity column, and subjected to size exclusion chromatography using a Superdex 200 column equilibrated in 100 mM HEPES (pH 7.5) and 20 mM NaCl.…”