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

Suguru Kurokawa,Frederick P Bellinger,Kristina E Hill,Raymond F Burk,Marla J Berry

doi:10.1074/jbc.m114.549014

Suguru Kurokawa, Frederick P Bellinger + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m114.549014

Copy DOI

Abstract

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.

Highlights

ApoER2 facilitates uptake of Sepp1, but the binding mechanism has not been elucidated
Sepp1 Longer Isoforms Bind to ApoER2, but Shorter Forms Do Not—We evaluated Sepp1 binding to Apolipoprotein E receptor 2 (apoER2) in vitro using serum Sepp1 from Sepp1ϩ/ϩ or Sepp1⌬240 –361 transgenic mice
GFP as a negative control or apoER2-GFP proteins were overexpressed in human embryonic kidney 293T (HEK293T) cells, which were exposed to culture media containing serum from Sepp1ϩ/ϩ or Sepp1⌬240–361 mice

Summary

Background

ApoER2 facilitates uptake of Sepp, but the binding mechanism has not been elucidated. Results: The two longest isoforms of Sepp bind to the YWTD ␤-propeller domain of apoER2, which functions as a Sepp receptor. Selenoprotein P (Sepp1) is an extracellular glycoprotein that has multiple selenocysteine residues and functions as a selenium transport protein via receptor-mediated endocytosis [5, 6]. A recent study identified N-terminal Sepp fragments in megalin knockout mouse urine [22] These results suggest that apoER2 and megalin have different ligand binding properties for Sepp. It is postulated that apoER2 is selective for longer Sepp isoforms to maximize selenium uptake by tissues, whereas megalin prevents the loss of Sepp in the urine by binding N-terminal Sepp1 [23]. We investigated the potential apoER2 binding residues in Sepp and demonstrate that only the longer two isoforms can bind apoER2 via the Sepp C-terminal domain. We further demonstrate that apoER2 lacking the LBR can still function as a Sepp receptor, suggesting a unique mechanism of receptor-dependent endocytosis of Sepp

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION