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Identification of the Free Cysteinyl Residue and Characterization of the N-glycan Structure in the C-terminal Domain of PCSK9.

Elevated plasma low density lipoprotein cholesterol (LDL-C) level is a risk factor for the development of atherosclerosis and associated cardiovascular disorders. Studies by Brown and Goldstein have firmly demonstrated the pivotal function played by the LDL receptor (LDLR) in cholesterol metabolism. The recently discovered proprotein convertase subtilisin/kexin type 9 (PCSK9) is another important regulator of LDL-C level. The protein promotes degradation of the LDLR, thus abolishing the receptor function. Specific inhibition of PCSK9 may therefore provide another novel therapeutic approach in the treatment of hypercholesterolemia. To evaluate human PCSK9 as a therapeutic target for monoclonal antibody development, the recombinant protein has been expressed in CHO system. The mature protein consists of 2 tightly associated subunits: a pro-domain of ~14 kD containing a single cysteine residue and a C-terminal catalytic domain of ~62 kD. The latter contains a total of 25 cysteinyl residues and a single N-linked glycosylation site. To show that the recombinant protein has been properly folded, we attempted to identify the location of the free cysteinyl residue(s) in the C-terminal domain. LC-MS analysis of the intact material after incubation with iodoacetamide in the presence of denaturant showed only a single site was alkylated. The labeled protein was completely reduced and pyridyethylated prior to proteolytic degradation. Analysis by LC-MS/MS showed that Cys301 was the only residue S-carboxamidomethylated, indicating this is the single free cysteine. The data therefore confirms the prior structural information obtained by X-ray crystallography. Peptide mapping also shows Asn533 is glycosylated. The predominant glycoforms are biantennary glycans, followed by triantennary and with very low amount of tetraantennary oligosaccharides. It is interesting to note that Asn533 is located in a cysteine rich area and is proximal to a cysteine residue that is disulfide cross linked (NCS). Thus, N-glycosylation was probably complete prior to cystine bridges formation in the maturation of PCSK9.