HMGCS2 deficiency in Japan: Characterization of wild-type and 5 variant proteins in vitro.


Presented By:

Yasuhiko Ago M.D.,PhD Student1, Hiroki Otsuka M.D.2, Elsayed Abdelkreem M.D.,Ph.D.3, Hideo Sasai
M.D.,Ph.D.1,4, Mina Nakama Ph.D.1,4, Yuka Aoyama Ph.D.1, Yoriko Watanabe M.D.,Ph.D.5, Kaori Fukui
M.D.5, Kazuteru Kitsuda M.D.6, Tomoko Lee M.D.,Ph.D.7, Yoko Nakajima M.D.,Ph.D.8, Tetsuya Ito
M.D.,Ph.D.8, Hidenori Ohnishi M.D.,Ph.D.1, Toshiyuki Fukao M.D.,Ph.D.1,4

1. Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan
2. Gifu Prefectural General Medical Center, Gifu, Japan
3. Department of Pediatrics, Faculty of Medicine, Sohag University, Sohag, Egypt
4. Division of Clinical Genetics, Gifu University Hospital, Gifu, Japan
5. Department of Pediatrics, Kurume University, Kurume, Fukuoka, Japan
6. Department of Pediatrics, Kitazato University, Sagamihara, Kanagawa, Japan
7. Department of Pediatrics, Hyogo Collage of Medicine, Nishinomiya, Hyogo, Japan
8. Department of Pediatrics, Fujita Health University, Toyoake, Aichi, Japan

Corresponding author: Yasuhiko Ago +81-58-230-6386

3-Hydroxy-3-Methylglutaryl-CoA Synthase 2(HMGCS2) deficiency is a rare defect in ketone body synthesis. We recently identified 5 patients in Japan based on probable disease-causing variants (p.G219E, p.M235T, p.V253A, p.S392L, p.R500C). Their pathogenicity was only evaluated in silico, and HMGCS2 activity in the patients’ hepatocytes was not measured. In this study, we evaluated HMGCS2 activity of wild-type and variant proteins expressed in bacterial expression system and human fibroblasts.

We subcloned wild-type HMGCS2 cDNA into both bacterial expression vector pGEX-6P-1 and mammalian expression vector pCAGGS. Variant constructs were made by in-vitro mutagenesis. These constructs were transformed into an E.coli strain BL21(DE3), or transfected into human SV40 transformed fibroblasts.

Transformed E.coli were grown in Lysogeny broth medium to an A600 of 0.8~1.0 at 37°C. Protein expression was induced with 0.5mM IPTG at 18°C for 16 hours. Cells were disrupted by sonication. GSTtagged proteins were purified by affinity chromatography. GST was separated from HMGCS2 by PreScission Protease. The activity of each enzyme was measured by spectrophotometric method. Transfected fibroblasts were disrupted by sonication, and soluble fraction were used for immunoblot analysis.

The amount of purified wild-type enzyme obtained from 1g of E.coli was 262μg. In case of p.G219E, p.M235T, p.V253A, p.S392L, and p.R500C, it was 3, 41.8, 105, 66.5 and 11.9μg, respectively. The activity of wild-type, p.M235T, p.V253A, p.S392L, p.R500C were 259±68, 0, 15.6±1.3, 3.09±2.9, 0.4±5.6nmol/min/mg protein, respectively. p.G219E variant could not be extracted enough to be evaluated. In immunoblot visible HMGCS2 proteins were identified in case of wild-type, p.M235T, p.R500C in fibroblasts. In case of p.G219E, no HMGCS2 protein was detected. Discussion The reproducibility should be confirmed. We are now confirming these results several times with independently-purified mutant enzymes.

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