Site-directed Mutagenesis of Human Ceruloplasmin

Abstract

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

Ceruloplasmin: Macromolecular Assemblies with Iron-Containing Acute Phase Proteins

“…The gradual disjunction of the fragments diminish, and ultimately interrupts the electron transfer between the copper ions of the catalytic center in Cp. Copper ions acting independently are likely to acquire prooxidant features observed in the cleaved Cp, in contrast to the intact protein [41]. Thus, Cp-Mpo interaction favors the antioxidant activity of Cp.…”

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

“…This would naturally happen when expressing membrane-bound Cp-GPI, but it could also occur with secreted Cp. In fact, although Fet3p is firmly bound to the plasma membrane, it has been reported that a fraction of Fet3p can be released into the medium by spontaneous limited proteolysis (14), and this was shown to happen also in the strain expressing recombinant Cp (9). The FLAG-tagged Cp-GPI isoform produced in yeast could be easily purified (supplemental Fig.…”

X-ray diffraction study of highly purified human ceruloplasmin

“…The limited proteolysis with trypsin affords the same fragments; i.e., the cleavage always proceeds at the particular peptide bonds located after R481, R701, and K887 [13,15]. The replacement of these amino-acid residues by site-directed mutagenesis produced human CP resistant to proteolysis [28].…”