**5. Identification of apolipoprotein A-1 as a retinoic acid binding protein**

Due to its hydrophobicity, atRA cannot diffuse freely in the hydrophilic extracellular microenvironment. Therefore, the requirement for carrier proteins capable of forming a soluble complex with atRA and transporting atRA to target cells is necessary to achieve high efficiency and specificity while avoiding toxicity associated with random diffusion. Mertz and Wallman [9] and our lab [15] identified a secreted protein of *M*r = 27,000 that was the major atRA binding protein present in choroid and sclera conditioned medium. This *M*r 27,000 protein did not correspond in size to any of the previously identified atRA binding proteins [59]. We subsequently determined that the *M*r 27,000 protein was apolipoprotein a-1 (ApoA-1) [15] (**Figure 3**).

#### *Vitamin A*

We have also shown that choroidal expression of ApoA-1 is transcriptionally regulated by atRA, *and* choroidal ApoA-1 mRNA and protein synthesis are upregulated during recovery from induced myopia, suggesting the presence of a regulatory feedback mechanism to regulate atRA transport and activity [15]. We postulate that ApoA-1 functions to transport atRA from its site of synthesis by RALDH2+ cells in the proximal choroid to the sclera for the regulation of scleral ECM remodeling. This idea is supported by our observation that the chick sclera (which is avascular) also releases significant amounts of ApoA-1 into culture medium, despite undetectable *de novo*  protein synthesis [15]. These data provide further evidence that choroidally derived ApoA-1 accumulates in the sclera, presumably as a consequence of retinoid transport.

#### **Figure 3.**

*ApoA-1 is a specific atRA-binding protein. (A) Retinoic acid (atRA) lacks intrinsic fluorescence (not shown), but can quench intrinsic protein fluorescence excited at 290 nm due to energy transfer from tryptophan residues on ApoA-1. Increasing atRA concentrations cause decreased fluorescence emission following excitation at 290 nm. (B) Titration of ApoA-1 with various retinoids by measuring quenching of protein fluorescence (emission = 340 nm). Significant quenching of protein fluorescence was observed only for atRA, indicating that ApoA-1 is a specific atRA-binding protein. atRA, all-trans-retinoic acid; RAL, all-trans-retinaldehyde; 9-cis, 9-cis-retinoic acid; 13-cis, 13-cis-retinoic acid. This research was originally published in the Journal of Biological Chemistry [70]. © The American Society for Biochemistry and Molecular Biology.* 
