Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin.
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.
Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland.
Evelyne
E
Furger
Sergey N
SN
Fedosov
Dorte Launholt
DL
Lildballe
Robert
R
Waibel
Roger
R
Schibli
Ebba
E
Nexo
Eliane
E
Fischer
D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D003038:Cobamides / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014155:Transcobalamins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D003038:Cobamides; D011994:Recombinant Proteins; D014155:Transcobalamins; C017142:cobinamide; D014805:Vitamin B 12