Abstract
Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
Highlights
Low molecular weight compounds termed ‘‘chemical chaperones’’ (CC) can inhibit aggregation and restore trafficking of intracellularly misfolded proteins
We tested members of different compound classes of CC for their effect on hFVIII-Bdomain deleted (BDD) expression in CHO cells: Polyols [32], sugars [33], methylamines [6] and trimethylamine N-oxide (TMAO) [4], free amino acids such as taurine [5] or amino acid derivatives [34] (Fig. 1A). Beside the former listed osmolytes, we tested the hydrophobic compound sodium-4-phenylbutyrate (PBA), which has already been tested as a chemical chaperone in a cystic fibrosis clinical trial [7]
We show that supplementation with CC can improve factor VIII (FVIII) secretion both in cell culture and in vivo
Summary
Low molecular weight compounds termed ‘‘chemical chaperones’’ (CC) can inhibit aggregation and restore trafficking of intracellularly misfolded proteins. It is estimated that due to high treatment costs, only 30% of affected individuals worldwide receive any kind of hemophilia therapy [9] This fact highlights the necessity for alternative strategies that can improve the clinical outcome, reduce factor consumption or decrease production costs. Swaroop et al have shown that FVIII interacts with the chaperones immunoglobulin-binding protein (BiP/GRP78) [15], calnexin, and calreticulin [16], which results in formation of nondisulfide-bonded high molecular weight aggregates in the ER. These misfolded FVIII proteins are subsequently designated for proteasomal and lysosomal degradation [17]. Similar limitations have been observed in FVIII gene transfer approaches [20,21]
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