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Prediction of the functional effect of novel SLC25A13 variants using a S. cerevisiae model of AGC2 deficiency

  • Original Article
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Journal of Inherited Metabolic Disease

Abstract

AGC2, a member of the mitochondrial carrier protein family, is as an aspartate-glutamate carrier and is important for urea synthesis and the maintenance of the malate-aspartate shuttle. Mutations in SLC25A13, the gene encoding AGC2, result in two age dependent disorders: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and type II citrullinemia (CTLN2). The clinical features of CTLN2 are very similar to those of other urea cycle disorders making a clear diagnosis difficult. Analysis of the SLC25A13 gene sequence can provide a definitive diagnosis, however the predictive value of DNA sequencing requires that the disease association of variants be characterized. We utilized the yeast Saccharomyces cerevisiae lacking AGC1 as a model system to study the effect on the function of AGC2 variants and confirmed that this system is capable of distinguishing between AGC2 variants with normal (p.Pro632Leu) or impaired function (p.Gly437Glu, p.Gly531Asp, p.Thr546Met, p.Leu598Arg and p.Glu601Lys). Three novel AGC2 genetic variants, p.Met1? (c.2T>C), p.Pro502Leu (c.1505C>T), and p.Arg605Gln (c.1814G>A) were investigated and our analysis revealed that p.Pro502Leu and p.Arg605Gln substitutions in the AGC2 protein were without effect and these variants were fully functional. The p.Met1? mutant is capable of expressing a truncated p.Met1_Phe34del AGC2 variant, however this protein is not functional due to disruptions in a calcium binding EF hand as well as incorrect intracellular localization. Our study demonstrates that the characterization of AGC2 expressed in yeast cells is a powerful technique to investigate AGC2 variants, and this analysis should aid in establishing the disease association of novel variants.

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Abbreviations

AGC:

aspartate-glutamate carrier

NICCD:

neonatal intrahepatic cholestasis caused by citrin deficiency

CTLN2:

type II citrullinemia

MCF:

mitochondrial carrier family

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Acknowledgements

This work was supported by a joint grant from Mahidol University Faculty of Science and Ramathibodi Hospital Faculty of Medicine (LJ and DW), Mahidol University (DW), and the Medical Scholars Program of Mahidol University (PW). DW is a recipient of Research Career Development Award, Faculty of Medicine Ramathibodi Hospital. We thank Araceli del Arco and Jeffrey Gerst for their gifts of yeast strains and plasmids. In addition we thank Mathurose Ponglikitmongkol for helpful discussions and suggestions and Amornrat Naranuntarat Jensen for assistance with fluorescence microscopy, helpful discussions, and critical reading of the manuscript.

Details of funding

This work was supported by a joint grant from Mahidol University Faculty of Science and Faculty of Medicine Ramathibodi Hospital (LJ and DW), Mahidol University (DW), and the Medical Scholars Program of Mahidol University (PW). The authors confirm that this work was independent and that the sponsors did not influence the content of the article.

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Authors and Affiliations

  1. Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, Thailand

    Parith Wongkittichote

  2. Department of Biochemistry, Faculty of Science, Mahidol University, 272 Rama 6 Road, Bangkok, 10400, Thailand

    Sumalee Tungpradabkul & Laran T. Jensen

  3. Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

    Duangrurdee Wattanasirichaigoon

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  1. Parith Wongkittichote
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  2. Sumalee Tungpradabkul
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  3. Duangrurdee Wattanasirichaigoon
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  4. Laran T. Jensen
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Correspondence to Laran T. Jensen.

Additional information

Communicated by: Gajja Salomons

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Fig. S1

Growth of yeast agc1∆ mutants and complementation by human AGC2. The agc1∆ yeast strain PW001 transformed with human AGC2 (pYX142-AGC2) or the empty vector (pRS425) were tested for growth in synthetic medium containing acetate (100 mM), galactose (2 %), glycerol (2 %) and ethanol (2 %) as carbon sources. Cell growth was monitored by measuring A600 nm and is shown as % of AGC2 expressing cells. Cells expressing human AGC2 are displayed as white bars and the empty vector is shown as black bars. Results for 72 hours of growth are shown. Yeast agc1∆ mutants without human AGC2 display poor growth in medium containing acetate but not other carbon sources tested. (JPEG 11 kb)

High resolution image file (TIFF 2908 kb)

Fig. S2

DNA sequence of the N-terminal region of AGC2 showing three potential translation initiation sites. A) The standard translation start site is indicated by M1 and the two downstream potential sites are labeled M2 (out of frame) and M3 (p.Met1_Phe34del producing). The translated amino acid sequence for this region for M2 and M3 is shown below the DNA codons in panels B and C respectively. Use of M3 will result in expression of p.Met1_Phe34del AGC2. (JPEG 9 kb)

High resolution image file (TIFF 1332 kb)

Fig. S3

Wild type and p.Met1_Phe34del AGC2 exhibit similar rates of degradation. Wild type AGC2-GFP (pPW015) (Panel A) and p.Met1_Phe34del GFP (pPW016) (Panel B) in strain PW001 (agc1∆) were monitored in crude mitochondrial extracts from cultures grown in YPR+0.5 % galactose medium for 16 hours followed by addition of glucose to 2 % to represses expression. Time points are after addition of glucose. AGC2-GFP remaining was monitored using immunoblots with an anti-GFP antibody. As a loading control the levels of porin were monitored. C) Densitometric analysis of AGC2-GFP or p.Met1_Phe34del AGC2-GFP was measured using ImageJ 1.45S software. Results are normalized based on porin levels and presented as percent of AGC2 prior to addition of glucose. White bars represent wild type AGC2 and black bars p.Met1_Phe34del AGC2. (JPEG 30 kb)

High resolution image file (TIFF 9074 kb)

Fig. S4

Homology model of the AGC2 N-terminal domain showing sequences absent in p.Met1_Phe34del AGC2. A model of the AGC2 N-terminal region was generated using porcine calbindin D9k (1cb1) (Akke, Drakenberg et al 1992) as a template. Amino acid residues 1–34 are shown in white and all other sequences are gray. A) Ribbon diagram of the N-terminal domain with the position of M35, EF1 and EF2 indicated. B) Space-filling representation showing contacts between residues 1–34 and other regions of the N-terminal domain. (JPEG 26 kb)

High resolution image file (TIFF 4344 kb)

Fig. S5

Topology model of AGC2 showing location of variants tested. Variants that result in reduced complementation of the agc1∆ strain are shown in black and those that exhibited wild type activity are shown in white. (JPEG 10 kb)

High resolution image file (TIFF 1460 kb)

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Wongkittichote, P., Tungpradabkul, S., Wattanasirichaigoon, D. et al. Prediction of the functional effect of novel SLC25A13 variants using a S. cerevisiae model of AGC2 deficiency. J Inherit Metab Dis 36, 821–830 (2013). https://doi.org/10.1007/s10545-012-9543-5

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