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Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

Abstract：

:Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Bakhautdin B,Goksoy Bakhautdin E,Fox PL

doi

10.1016/j.bbrc.2014.09.134

subject

Has Abstract

pub_date

2014-10-31 00:00:00

pages

722-7

issue

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(14)01777-X

journal_volume

453

pub_type

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Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.