Identification of Specific Binding Sites for Apolipoprotein A-1/Retinoic acid Complexes on Scleral Cell Membranes

Abstract

Purpose : We are accumulating considerable evidence to suggest that postnatal eye growth is regulated by choroidally derived all-trans-retinoic acid (atRA) which is transported to the sclera to effect changes in scleral extracellular matrix remodeling, axial length and refraction. However, the presence of carrier proteins is necessary for the transportation of atRA in the hydrophilic extracellular environment to target cells in the sclera. We have recently determined that apolipoprotein a-1 (ApoA-1) is the major atRA binding protein synthesized and secreted by the chick choroid and demonstrates binding specificity for all-trans-atRA with an apparent K_d of 0.62 µM. We hypothesize that ApoA-1 is the specific atRA carrier protein for extracellular transport of atRA between the choroid and sclera. Therefore, the objective of this study is to determine if the sclera expresses specific ApoA-1 binding sites/receptors as a physiological mediator of retinoic acid uptake.

Methods : ³H-(atRA)-ApoA-1 was prepared by incubating purified chicken HDL with ³H-atRA, followed by UV- covalent crosslinking. Scleral chondrocytes were isolated and binding of ³H-(atRA)-ApoA-1 (0.2 – 8.6 µM) was carried out at 0°C and 37°C for 1.5 hrs in the presence or absence of 40 fold excess of cold (atRA)-ApoA-1 (n = 3 samples/ concentration; 100,000 cells/sample). Following incubation, samples were washed, solubilized, and specific binding assessed by liquid scintillation counting and Scatchard analyses (GraphPad Prism 5).

Results : ³H-(atRA)-ApoA-1 bound specifically to scleral chondrocyte cell membranes and binding was completely blocked by cold ApoA1. Saturation was demonstrable at 0° but not at 37° under otherwise identical conditions, indicating that ApoA-1 up-regulates the expression of its own receptor(s), and/or endocytosis is increased at 37°. Scatchard analyses of binding data at 37°C suggest a “concave up” plot when fit with a nonlinear Hill Slope (r² = 0.643).

Conclusions : Chick scleral chondrocytes express specific binding sites for ³H-(atRA)-ApoA-1. The concave-up Scatchard analyses suggests that there are either more than one class of holoApoA-1 binding sites or negative cooperativity between members of a single class of binding site. These results provide further support for the hypothesis that ApoA-1 transports atRA to the sclera for purposes of ocular growth regulation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.