Protein S-Leu17Pro disrupts the hydrophobicity of its signal peptide causing a proteasome-dependent degradation

• Published in: Thrombosis research 2022-02, Vol.210, p.26-32
• Main Authors: Okada, Kentaro, Tamura, Shogo, Suzuki, Nobuaki, Odaira, Koya, Mukaide, Masato, Fujii, Wataru, Katsuragi, Yumi, Suzuki, Atsuo, Kanematsu, Takeshi, Okamoto, Shuichi, Suzuki, Naruko, Katsumi, Akira, Matsushita, Tadashi, Kojima, Tetsuhito, Hayakawa, Fumihiko
• Format: Article
• Language: English
• Subjects: Animals; Chlorocebus aethiops; COS Cells; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; PROS1; Proteasome Endopeptidase Complex; Proteasome-dependent degradation; Protein S - genetics; Protein S deficiency; Protein Sorting Signals; Signal peptide
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/j.thromres.2021.12.014

Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T > C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency. The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry. In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol. PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation. •We identified a variation in protein S gene, translating into protein S deficiency.•The variation was named protein S gene-L17P (c.50 T > C, p.Leu17Pro).•The resultant protein S has reduced hydrophobicity in the h-region.•Hydrophobicity in the h-region causes protein S cytosolic mislocalization.•Mislocalized protein S-L17P is degraded in a proteasome-dependent manner.