Abstract
Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10–50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.
Highlights
Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein
The unpaired cysteine thiols in bead-bound fibrinogen were alkylated with 2-iodo-N-phenylacetamide (12C-IPA), the protein isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE; Fig. 1b), the disulfide bonds reduced with dithiothreitol, and the disulfide-bonded cysteine thiols alkylated with a stable carbon[13] isotope of IPA (13C-IPA)[14]
We considered two scenarios that could account for this change: (1) a subset of fibrinogen covalent states containing more oxidized disulfide bonds is preferentially used for fibrin formation, or (2) disulfide bonds form during fibrin formation
Summary
Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein We test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. Our laboratory has focused on the identification and study of allosteric disulfides, which are a subset of disulfide bonds that regulate mature protein function when cleaved or formed[1] The study of these functional bonds has been facilitated by the development of a differential cysteine alkylation and mass spectrometry method to precisely quantify the redox state of disulfides in proteins in their native environments[12]. We report that fibrinogen exists in multiple disulfide-bonded states in the circulation that are important for the function of the protein
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