Hybrid lactoferrins containing transferrin C1 or C2 subdomains bind asialoglycoprotein receptors

Abstract

The Fe‐binding proteins lactoferrin (Lf) and transferrin (Tf) are structurally similar, each Fe‐binding lobe (N, C) composed of two sub‐domains (N1/N2; C1/C2). Lf, but not Tf, binds the major subunit of the asialoglycoprotein receptor (RHL1) in a galactose‐independent manner. To identify C‐lobe regions involved in RHL1 binding, we generated recombinant Lf‐Tf hybrid proteins in which Tf C1 or C2 subdomains were positioned within a Lf background. cDNAs of human Lf and Tf encoding Lf:TfC1 and Lf:TfC2 were constructed and expressed in Sf9 insect cells. Binding constants of purified native and recombinant Lfs for RHL1 were determined by surface plasmon resonance. Binding of desialylated ligands and native and recombinant Lf proteins to immobilized RHL1 required Ca2+ and neutral pH. Tf did not bind RHL1 under any conditions. Average dissociation constants measured for the various proteins were as follows: native Lf: 138 ± 44 nM; r‐Lf: 87 ± 50 nM; Lf:TfC1: 31 ± 28 nM; Lf:TfC2: 150 ± 76 nM; and asialofetuin: 21 ± 16 nM. Binding constants for native and recombinant Lfs did not differ statistically. Thus, Tf C1 or C2 sub‐domains in a Lf background showed no loss of RHL1‐binding activity. We conclude that the RHL1 binding determinants on Lf do not reside solely in one of the C‐lobe subdomains. (Support: Research Corporation, NIH DK‐61984).