Binding of Nanoparticles Harboring Recombinant Large Surface Protein of Hepatitis B Virus to Scavenger Receptor Class B Type 1

Abstract

(1) Background: As nanoparticles containing the hepatitis B virus (HBV) large (L) surface protein produced in yeast are expected to be useful as a carrier for targeting hepatocytes, they are also referred to as bio-nanocapsules (BNCs). However, a definitive cell membrane receptor for BNC binding has not yet been identified. (2) Methods: By utilizing fluorescence-labeled BNCs, we examined BNC binding to the scavenger receptor class B type 1 (SR-B1) expressed in HEK293T cells. (3) Results: Analyses employing SR-B1 siRNA and expression of SR-B1 fused with a green fluorescent protein (SR-B1-GFP) indicated that BNCs bind to SR-B1. As mutagenesis induced in the SR-B1 extracellular domain abrogates or attenuates BNC binding and endocytosis via SR-B1 in HEK293T cells, it was suggested that the ligand-binding site of SR-B1 is similar or close among high-density lipoprotein (HDL), silica, liposomes, and BNCs. On the other hand, L protein was suggested to attenuate an interaction between phospholipids and SR-B1. (4) Conclusions: SR-B1 can function as a receptor for binding and endocytosis of BNCs in HEK293T cells. Being expressed various types of cells, it is suggested that functions as a receptor for BNCs not only in HEK293T cells but also in other types of cells.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide

“…NTCP-GFP, as well as SR-B1-GFP, failed to bind aa2-48, which suggests that the myristoylation of the pre-S1 peptide is important for both Myr47 bindings of SR-B1 and NTCP. BNCs bound SR-B1-GFP, whereas they failed to bind NTCP-GFP, which was consistent with the results of the previous reports [10,21]. Figure 1c shows quantified ligand binding (i.e., FI) to the surfaces of GFP + subsets in cells expressing GFP (control), SR-B1-GFP, and NTCP-GFP.…”

“…The preparation of BNCs (i.e., emBNCs were used in this study) and their fluorescence labeling with CellVue Claret (CV) (Molecular Targeting Technologies Inc., West Chester, PA, USA) were previously described [20,21].…”

“…Myr47 uptake in HEK293T, SR-B1-GFP-HEK, and GFP-HEK cells was examined according to the methods as previously described [15,16,21]. Briefly, cells were cultured in the presence or absence of Alexa 647-labeled Myr47 (final Myr47 concentration of 20 nM) in a 24-well plate for 24 h. After cells were harvested via trypsinization, they were subjected to flow cytometry.…”

“…We demonstrated that flow cytometric analysis using a fluorescence-labeled ligand and SR-B1-GFP fusion protein is useful to elucidate the specific binding of a ligand to SR-B1 [14][15][16]21]. Therefore, we applied this method to analysis for the binding of Alexa 647-labeled Myr47 and its analogs to SR-B1-GFP or NTCP-GFP.…”

“…Although HSPG was assumed to be a candidate receptor for BNCs, it was ambiguous as to which receptor contributes to the cellular binding of BNCs [10]. As BNCs can be regarded as a sort of PNPs, we examined the binding of BNCs to SR-B1 and found that they can bind SR-B1 in HEK293T cells [21]. BNCs and Myr47 share commonalities because both are composed of a part of HBV structural components.…”