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Truncated UDP-glucuronosyltransferase (UGT) from a Crigler-Najjar syndrome type II patient colocalizes with intact UGT in the endoplasmic reticulum.

Suzuki M, Hirata M, Takagi M, Watanabe T, Iguchi T, Koiwai K, Maezawa S, Koiwai O - J. Hum. Genet. (2014)

Bottom Line: Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells.UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively.Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan.

ABSTRACT
Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.

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Fluorescence photobleaching of EGFP, EGFP-UGT1A1 and UGT1A1-p.Q331X-Venus. HEK293T cells expressing EGFP (a), EGFP-UGT1A1 (b) or UGT1A1-p.Q331X-Venus (c) were subjected to photobleaching and the fluorescence recovery after photobleaching recoveries were determined. Figures show the mobility of the EGFP, EGFP-UGT1A1 or UGT1A1-p.Q331X-Venus molecules. Images were captured using an LSM5 33 EXCITER confocal microscope (Carl Zeiss). Excitation was performed at 488 (514) nm to visualize EGFP (Venus) using LP505 (530) nm emission filters. Fluorescence recovery after photobleaching was performed using the region of interest, 2 μm in diameter, and four bleaching iterations. Graphic imaging was processed with the Zen software. A full color version of this figure is available at the Journal of Human Genetics journal online.
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fig5: Fluorescence photobleaching of EGFP, EGFP-UGT1A1 and UGT1A1-p.Q331X-Venus. HEK293T cells expressing EGFP (a), EGFP-UGT1A1 (b) or UGT1A1-p.Q331X-Venus (c) were subjected to photobleaching and the fluorescence recovery after photobleaching recoveries were determined. Figures show the mobility of the EGFP, EGFP-UGT1A1 or UGT1A1-p.Q331X-Venus molecules. Images were captured using an LSM5 33 EXCITER confocal microscope (Carl Zeiss). Excitation was performed at 488 (514) nm to visualize EGFP (Venus) using LP505 (530) nm emission filters. Fluorescence recovery after photobleaching was performed using the region of interest, 2 μm in diameter, and four bleaching iterations. Graphic imaging was processed with the Zen software. A full color version of this figure is available at the Journal of Human Genetics journal online.

Mentions: We next asked whether EGFP-UGT1A1 and UGT1A1-p.Q331X are retained in the ER, using the fluorescence recovery after photobleaching method. As shown in Figure 5, when we used EGFP as a control, all the fluorescence was recovered within 1 s after applying laser radiation to photobleach a small area within the cytoplasm. In contrast, only 50% of the fluorescence was recovered 10 s after photobleaching when using EGFP-UGT1A1. The fluorescence after photobleaching Venus-tagged UGT1A1-p.Q331X was also more slowly recovered when compared with the control. The diffusion constants of EGFP, EGFP-UGT1A1 and Venus-tagged UGT1A1-p.Q331X were 0.73, 0.078 and 0.48 μm2s−1, respectively. The average proportion of EGFP-UGT1A1 and Venus-tagged UGT1A1-p.Q331X bound to the ER membrane was 72% and 42%, respectively. These results indicate that EGFP-UGT1A1 is not diffusible and is retained in the ER, and that UGT1A1-p.Q331X is also retained in the ER.


Truncated UDP-glucuronosyltransferase (UGT) from a Crigler-Najjar syndrome type II patient colocalizes with intact UGT in the endoplasmic reticulum.

Suzuki M, Hirata M, Takagi M, Watanabe T, Iguchi T, Koiwai K, Maezawa S, Koiwai O - J. Hum. Genet. (2014)

Fluorescence photobleaching of EGFP, EGFP-UGT1A1 and UGT1A1-p.Q331X-Venus. HEK293T cells expressing EGFP (a), EGFP-UGT1A1 (b) or UGT1A1-p.Q331X-Venus (c) were subjected to photobleaching and the fluorescence recovery after photobleaching recoveries were determined. Figures show the mobility of the EGFP, EGFP-UGT1A1 or UGT1A1-p.Q331X-Venus molecules. Images were captured using an LSM5 33 EXCITER confocal microscope (Carl Zeiss). Excitation was performed at 488 (514) nm to visualize EGFP (Venus) using LP505 (530) nm emission filters. Fluorescence recovery after photobleaching was performed using the region of interest, 2 μm in diameter, and four bleaching iterations. Graphic imaging was processed with the Zen software. A full color version of this figure is available at the Journal of Human Genetics journal online.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973126&req=5

fig5: Fluorescence photobleaching of EGFP, EGFP-UGT1A1 and UGT1A1-p.Q331X-Venus. HEK293T cells expressing EGFP (a), EGFP-UGT1A1 (b) or UGT1A1-p.Q331X-Venus (c) were subjected to photobleaching and the fluorescence recovery after photobleaching recoveries were determined. Figures show the mobility of the EGFP, EGFP-UGT1A1 or UGT1A1-p.Q331X-Venus molecules. Images were captured using an LSM5 33 EXCITER confocal microscope (Carl Zeiss). Excitation was performed at 488 (514) nm to visualize EGFP (Venus) using LP505 (530) nm emission filters. Fluorescence recovery after photobleaching was performed using the region of interest, 2 μm in diameter, and four bleaching iterations. Graphic imaging was processed with the Zen software. A full color version of this figure is available at the Journal of Human Genetics journal online.
Mentions: We next asked whether EGFP-UGT1A1 and UGT1A1-p.Q331X are retained in the ER, using the fluorescence recovery after photobleaching method. As shown in Figure 5, when we used EGFP as a control, all the fluorescence was recovered within 1 s after applying laser radiation to photobleach a small area within the cytoplasm. In contrast, only 50% of the fluorescence was recovered 10 s after photobleaching when using EGFP-UGT1A1. The fluorescence after photobleaching Venus-tagged UGT1A1-p.Q331X was also more slowly recovered when compared with the control. The diffusion constants of EGFP, EGFP-UGT1A1 and Venus-tagged UGT1A1-p.Q331X were 0.73, 0.078 and 0.48 μm2s−1, respectively. The average proportion of EGFP-UGT1A1 and Venus-tagged UGT1A1-p.Q331X bound to the ER membrane was 72% and 42%, respectively. These results indicate that EGFP-UGT1A1 is not diffusible and is retained in the ER, and that UGT1A1-p.Q331X is also retained in the ER.

Bottom Line: Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells.UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively.Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan.

ABSTRACT
Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.

Show MeSH
Related in: MedlinePlus