Isoform-specific Binding of Selenoprotein P to the β-Propeller Domain of Apolipoprotein E Receptor 2 Mediates Selenium Supply

• Published in: The Journal of biological chemistry 2014-03, Vol.289 (13), p.9195-9207
• Main Authors: Kurokawa, Suguru, Bellinger, Frederick P., Hill, Kristina E., Burk, Raymond F., Berry, Marla J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Biology; Endocytosis; Female; HEK293 Cells; Humans; Ligands; Lipoprotein Receptor; Low Density Lipoprotein Receptor-Related Protein-1 - chemistry; Low Density Lipoprotein Receptor-Related Protein-1 - metabolism; Male; Mice; Molecular Sequence Data; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Protein Structure, Tertiary; Selenium; Selenium - metabolism; Selenocysteine; Selenocysteine - metabolism; Selenoprotein; Selenoprotein P - chemistry; Selenoprotein P - metabolism; Sequence Alignment; Site-directed Mutagenesis; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.549014

Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery. Background: ApoER2 facilitates uptake of Sepp1, but the binding mechanism has not been elucidated. Results: The two longest isoforms of Sepp1 bind to the YWTD β-propeller domain of apoER2, which functions as a Sepp1 receptor. Conclusion: Only longer Sepp1 isoforms with six or more selenocysteine residues can interact with a unique binding site of apoER2. Significance: ApoER2 takes up long isoform Sepp1 through its YWTD β-propeller domain.