Roles of heme iron-coordinating histidine residues of human hemopexin expressed in baculovirus-infected insect cells.

Abstract

Hemopexin (Hx), the major heme-binding plasma glycoprotein, scavenges circulating heme and performs an antioxidant function. In the present study, human Hx was expressed in a baculovirus system and its presumed essential His residues were mutated to Thr as a means of investigating their participation in heme binding. The recombinant Hx proteins were purified by sequential chromatography on Con A-agarose and SP-Sepharose. The purified recombinant wild-type Hx retained its heme binding. The binding constant for heme was considerably reduced, however, suggesting that glycosylation contributes critically to the heme binding property of Hx. Mutation either at His-127 or at His-56 plus His-127, but not at His-56 per se, reduced the affinity for heme by an order of magnitude relative to wild-type Hx. It is concluded that His-127 contributes to the high affinity for heme. We recorded proton NMR spectra to investigate the possibility that the degree of high-spin content is increased by deletion of an axial His-iron coordination. 1H NMR data indicate that each of the single-mutant heme-Hx complexes is predominantly low-spin, perhaps owing to coordination of the heme iron by the Thr side-chain oxygen or water oxygen coordinating to the iron.

Environmental stress-related gene expression and blood physiological responses in olive flounder (Paralichthys olivaceus) exposed to osmotic and thermal stress

“…The oligosaccharides attached to this protein promote glycoprotein folding, contribute to proteinÁprotein interactions, regulate other ligand recognition processes, and stabilize the protein, making it resistant to proteolysis (Helenius and Aebi 2004;Ohtsubo and Marth 2006). Up to now, the role of the oligosaccharide in this glycoprotein has been unclear; however, Satoh et al (1994) reported that N-linked glycosylation is an essential part of the high affinity of human hemopexin for heme, and Hirayama et al (2004) reported that fish Wap65 binds heme.…”

Establishment of Stable Chinese Hamster Ovary Cell Line Capable of Expressing Human Recombinant Hemopexin: A Promising Therapeutic Modality Against Hemolytic Anemia

“…Finally, RT-PCR and Western blot analyses confirmed the stable expression of human rHPX at both transcriptional and translational levels. Since human rHPX bears extensive human-specific glycosylation, mammalian expression systems, such as HEK293 or CHO cell lines, could be suitable hosts to provide sufficient post-translational modifications to afford heme binding and expected rHPX bioactivity (15). The CHO cell line is the premier expression host for therapeutic protein production due to its adaptability to various culture conditions, plasticity in terms of genetic alterations (18) and human-like glycosylation (24).…”

“…Their initial attempts to express human HPX in an E. coli system produced a non-glycosylated protein with no heme-binding ability (15). Protein expression in prokaryotic system is associated with challenges such as limited eukaryotic post-translational machinery function, protein aggregation, and generation of inclusion bodies (33).…”

“…However, using baculovirus vector expression system, Satoh et al were able to express human rHPX with 55 kDa molecular weight (MW) that was less than expected (60 kDa) (15). It should be noted that protein expression in insect cells is associated with some limitations.…”

“…Recently, human rHPX was expressed in both prokaryotic and eukaryotic hosts such as Escherichia coli (13,14), Spodoptera frugiperda Sf9 insect cells (15), and Pichia pastoris (16). Moreover, commercially available human rHPX were reported to be expressed in Human Embryonic Kidney (HEK293) cells and mouse myeloma cell line (NS0).…”