Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Hitoshi Kimura,Yuki Horie,Hiroyuki Oyama,Hiroyuki Saito,Kazuchika Nishitsuji,Sissel Lund-Katz,Norihiro Kobayashi,Takashi Ohgita,Izumi Morita,Atsuko Takeuchi,Chiharu Mizuguchi,Kenichi Akaji,Shiho Mikawa

doi:10.1038/s41598-017-03208-8

Abstract

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

Highlights

high-density lipoprotein (HDL) particles are quite heterogeneous in their size, shape, and lipid and protein compositions[12, 13]
Several reports have been published from the 1980s42–45 for generation of anti-Apolipoprotein A-I (apoA-I) monoclonal antibodies (mAbs) based on the hybridoma method[46]
BALB/c mice, the most common splenocyte donor used for the hybridoma production, were immunized with recombinant apoA-I expressed in Esherichia coli (E. coli) cells

Summary

Introduction

HDL particles are quite heterogeneous in their size, shape, and lipid and protein compositions[12, 13]. In mature spherical HDL particles, the apoA-I molecules in the double-belt conformation bend and form a stabilizing trefoil scaffold structure[16, 23]. Such conformational plasticity and flexibility of apoA-I are thought to be associated with its strong amyloidogenic property[24, 25]. We previously developed a novel method for assessing HDL production from cells based on the lipidation-induced hydrophobicity change in apoA-I during HDL formation[39]. The present results suggest that these mAbs may provide useful tools for monitoring formation and remodeling of HDL particles

Methods

Results

Conclusion