Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody

Abstract

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

Fig. 1

Specificity of the novel monoclonal antibody EL-2B17mAb directed against the UGT2B17 protein. (A) Recombinant human UGT2Bs in supersomes (0.5 μg). (B) Pooled HLMs, HIMs, and human kidney microsomes (HKMs) (10 μg). (C) Pooled HLMs (2B17 +) and liver microsomes from an individual carrying no UGT2B17 gene copy (2B17 −). The positions of molecular weight markers are given on the left. Arrow indicates the molecular weight of UGTs.

Fig. 2

Immunohistochemical detection of UGT2B17 with EL-2B17mAb in a normal human liver. (A) Overview of labeling distribution. Bile ducts (orange arrows) are strongly labeled, whereas staining intensity of hepatocytes in the portal and central vein areas is variable. Scale bar, 500 µm. (B) Enlarged view of a periportal area. Nuclei and cytoplasm of bile duct epithelial cells (thin black arrows) are strongly stained. Hepatocytes (large blue arrows) are variably stained, with a predominant nuclear labeling. Inflammatory cells of sinusoidal spaces are also labeled (red arrows). (C) Enlarged view of the central vein area showing a weaker labeling of hepatocytes (large blue arrows). In (B and C), bars represent 100 µm. The liver shown corresponds to liver A in Supplemental Fig. 3.

Fig. 3

Correlation between UGT2B17 protein expression in human liver microsomal samples (n = 29) determined by MS and immunoblotting (WB) using EL-2B17mAb. Spearman correlation coefficient (r) and corresponding P value are given.

Fig. 4

UGT2B17 protein abundance in liver samples according to UGT2B17 gene copy number (n = 48). Quantification of UGT2B17 protein expression levels was determined by MS. P values were determined using the Kruskal Wallis one-way analysis of variance and corrected by Bonferroni. **P < 0.01; ***P < 0.001.

Fig. 5

Frequencies of UGT2B17 gene variations in diverse ethnic groups. (A) UGT2B17 gene CNV. Frequencies were taken from Xue et al. (2008). (B) Allele frequencies of rs59678213 and rs6817882; (C) Linkage disequilibrium between rs59678213 and rs6817882. For (B and C), data were obtained from the 1000 Genomes phase 3 project. Individuals from European ethnicity correspond to CEU : Utah Residents (CEPH) with Northern and Western European Ancestry, Chinese to CHB : Han Chinese in Beijing, China; Japanese to JPT : Japanese in Tokyo, Japan; and African to YRI : Yoruba in Ibadan, Nigeria.

Fig. 6

UGT2B17 protein abundance in human liver is driven by FOXA1 rs59678213A>G status. (A) UGT2B17 expression levels in liver samples (n = 32) according to UGT2B17 gene copy number and the rs59678213A>G genotype. UGT2B17 protein levels were as determined by immunoblotting (WB); (B) UGT2B17 protein levels assessed by MS in HLMs (n = 48) are stratified by UGT2B17 gene copy number and the rs59678213A>G status; (C) UGT2B17 protein levels as in (B) are grouped according to rs59678213 in individuals with at least one gene copy. Significance was determined using the Kruskal-Wallis one-way analysis of variance and were corrected by Bonferroni. **P < 0.01; ***P < 0.001.