DMD #50468 The relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro. DMD #50468 2 Running Title: Modulation of UGT activity by i2 protein content in cells

ABSTRACT Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene which represents one of the best example of a drug metabolizing gene regulated by AS. Shorter UGT1A isoforms i2 are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be determinant of global transferase activity of cells and tissues. Here, we tested whether the i2-mediated inhibition increases with greater abundance of the i2 protein relative to the i1 enzyme using the extrahepatic UGT1A7 as a model, and a series of 23 HEK293 clonal cell lines expressing variable content of i1 and i2 proteins. Upon normalization for i1, a significant reduction of SN-38 glucuronide formation was observed for i1+i2 clones (mean of 53%) compared to the reference i1 cell line. In these clones, the i2 protein content greatly varied (38% to 263% relative to i1) and exposed 2 groups; 17 clones with i2<i