Abstract
Lactoferrin is a member of the transferrin family of iron-binding proteins. Several functions have been ascribed to lactoferrin, including regulation of iron homeostasis, antibacterial properties, and regulation of myelopoiesis. However, the structural features of lactoferrin that are required for most of these functions are unknown. Previously, we reported the development of an efficient fungal expression system to produce recombinant human lactoferrin. The availability of this production system demonstrated the feasibility of producing mutant lactoferrins to address the structure/function relationship of the protein. In the present study, we used a site-directed mutagenesis approach to address the contribution of the bilobal structure of lactoferrin to its unique iron-binding stability. Like transferrin, lactoferrin consists of two repeated iron-binding lobes that bind one iron atom each. However, unlike transferrin, lactoferrin retains iron over a broad pH range, a key property that contributes to the unique iron-binding functions of the protein. Using mutants that selectively ablate the iron-binding function in either lobe, we demonstrate differential iron-binding stability of the amino- and carboxyl-terminal iron-binding lobes of lactoferrin. Further, we show that the unique iron-binding stability of the protein is imparted primarily by the carboxyl-terminal domain which functions cooperatively to stabilize iron-binding to the amino-terminal domain of lactoferrin.
Highlights
We reported the development of an efficient fungal expression system to produce recombinant human lactoferrin
Expression and Purification of Iron Binding-defective Mutants in the NH2-terminal, COOH-terminal, or NH2- and COOH-terminal Domains of Lactoferrin—In order to examine the contribution of the two-domain structure to the unique iron-binding properties of lactoferrin, we used a site-directed mutagenesis approach to generate mutants in the human lactoferrin cDNA which encoded proteins which were defective in iron binding in either or both lobes of the protein
We have used a site-directed mutagenesis approach to investigate the contribution of the bilobal structure of lactoferrin to the unique iron-binding properties of this protein
Summary
Construction of pPLF-26, a Universal A. awamori Expression Vector—The construction of an expression vector, pPLF-19, for production of lactoferrin in A. awamori has previously been described [17]. In order to construct an expression vector containing unique sites for cloning lactoferrin mutant cDNAs, pPLF-26 was generated. In vitro mutagenesis using the commercially available pALTER kit (Promega) was used to introduce a NotI restriction enzyme site at the start of mature lactoferrin cDNA in pLF18Sp.Alt generating pNot.. Construction of Iron Binding-defective Mutants of Lactoferrin, MN2Y, MC-2Y, and MNC-4Y—Synthetic 5Ј-phosphorylated oligonucleotides with EcoRI/BamHI ends, were generated in order to introduce a NotI site into pALTER. PPLF-26 was digested with NotI/EcoRI, and the resulting 2.1-kb fragment containing the human lactoferrin cDNA was subcloned into NotI/ EcoRI-digested pALTLink. The samples were purified through a NAP-10 column (Pharmacia Biotech Inc.) followed by dialysis against 0.05 M Tris-Cl, 0.2 M NaCl, pH 7.0, for 12 h to remove any nonspecific iron bound to the lactoferrin mutant proteins. The pH stability of iron binding for each of the lactoferrin mutants was carried out as described previously [17]
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