Todd C. Zankel

Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier

We have sought to identify a high-capacity transport system that mediates transcytosis of proteins from the blood to the brain. The 39 kDa receptor-associated protein (RAP) functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the low-density lipoprotein (LDL) receptor family. RAP efficiently binds to these receptors and antagonizes binding of other ligands. Previous studies have shown that two large members of the LDL receptor family, LDL receptor-related protein 1 (LRP1) and LDL receptor-related protein 2 (LRP2 or megalin), possess the ability to mediate transcytosis of ligands across the brain capillary endothelium. Here, we tested whether blood-borne RAP crosses the blood-brain barrier (BBB) by LRP1- or megalin-mediated transport by studying the pharmacokinetics of [125I]-RAP transport into the brain in intact mice and across cell monolayers in vitro. Our results show that [125I]-RAP is relatively stable in blood for 30 minutes and has a mean influx constant of 0.62±0.08 μl/g-minute from blood to brain. In situ brain perfusion in blood-free buffer shows that transport of [125I]-RAP across the BBB is a saturable process. Capillary depletion of brain homogenates indicates that 70% of [125I]-RAP is localized in the parenchyma rather than in the vasculature of the brain. Results of transport in stably transfected MDCK cells are consistent with the hypothesis that megalin mediates most of the apical-to-basolateral transport across polarized epithelial cells. The inhibition of [125I]-RAP influx by excess RAP and the involvement of megalin indicate the presence of a saturable transport system at the BBB. The higher permeability of RAP compared with that of melanotransferrin and transferrin show that the LRP receptor is a high capacity transport system. These studies suggest that RAP may provide a novel means of protein-based drug delivery to the brain.

Overexpression of inactive arylsulphatase mutants and in vitro activation by light-dependent oxidation with vanadate.

Arylsulphatases B (ASB) and A (ASA) are subject to a unique post-translational modification that is required for their function. The modification reaction, conversion of an active-site cysteine into a formylglycine, becomes saturated when these enzymes are overexpressed. We have removed the possibility of in vivo modification by expressing mutants of ASB and ASA in which the active-site cysteine is substituted with a serine. These mutants are expressed much more efficiently when compared with the native enzymes under identical conditions. The purified ASB mutant can then be converted into catalytically active ASB in vitro using vanadate and light.