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GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

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Science Advances  17 Jul 2020:
Vol. 6, no. 29, eabb3713
DOI: 10.1126/sciadv.abb3713
  • Fig. 1 Generation and characterization of GADL1 KO mice.

    (A) Targeting strategy for knocking out exon (Ex) 7 of the mouse Gadl1 locus on chromosome 9. Gadl1 coding sequences (hatched rectangles), noncoding exon portions (blue rectangle), and chromosome sequences (orange rectangles) are represented. The neomycin (neo)–positive selection cassette is indicated between loxP sites (blue triangles) and Flippase recognition target (FRT) sites (plum triangles). (B and C) Growth curves of Gadl1+/+ (n = 4 to 34) and Gadl1−/− mice (n = 4 to 40), presented as means ± SD. Differences between genotypes were significant; P = 0.0008 (64 weeks) and P = 0.0005 (70 weeks) for males and P = 0.0084 (90 weeks) and P = 0.0001 (94 weeks) for females, respectively. (D) Southern blot analysis of genomic DNA from Gadl1+/+ and Gadl1−/−. (E) Genotyping of the offspring from intercrosses of Gadl1+/− mice by polymerase chain reaction (PCR). The DNA band at 166 base pair (bp) is the KO allele (primer 3), while bands at 330 bp (primer 2 and 3) and 750 bp (primer 1) are the wild-type alleles. (F) Representative Western blots of OB samples from Gadl1+/+ and Gadl1−/− mice (34 weeks old, female) using anti-GADL1 antibody. Positive control was recombinant His-tagged GADL1 (2 ng; lane 1). (G) Western blot of recombinant His-tagged truncated Gadl1+/+ and Gadl1−/−. (H) Enzyme activity toward CSA of recombinant His-tagged truncated Gadl1+/+ and Gadl1−/−; P < 0.001. (I to K). RNA expression levels (volcano plots) in OB tissue. (I) Gadl1+/+-to-Gadl1+/− ratio, (J) Gadl1+/+-to-Gadl1−/− ratio, and (K) Gadl1−/−-to-Gadl1+/−ratio. (L to N) Quantitative reverse transcription PCR (qRT-PCR) analysis of normalized mRNA expression in OB, brain, SKM, and liver tissues from 35-week-old female Gadl1+/+ (gray), Gadl1+/− (blue), and Gadl1−/− (red) mice for (L) Gadl1 exons 7 and 8, (M) Gadl1 exons 10 and 11, and (N) CSAD. n = 3 for each genotype. Presented on an Ln y scale as mean of 2ΔCt and upper limit (95%).

  • Fig. 2 Tissue-specific effects on metabolite levels in Gadl1−/−mice.

    (A to D) Top significant features of metabolites based on VIP scores of >2 of component 1 of PLS-DA. Untargeted metabolic profiling of (A) cerebral cortex, (B) OB, (C) SKM, and (D) liver tissue samples from Gadl1+/+ (n = 20) and Gadl1−/− (n = 21) mice. WT, wild-type. (E) The relative levels of β-alanine and carnosine derivatives in Gadl1+/+ (gray) and Gadl1−/− (red) mouse tissue. nd., not determined.

  • Fig. 3 1H-NMR and MRI of mouse tissues and substrate specificity of GADL1.

    (A to C) Measurement of carnosine in intact OB tissue. (A) Chemical structure of carnosine. ppm, parts per million. (B) MAS 1H-NMR spectra of OB tissue samples from Gadl1+/+, Gadl1+/−, and Gadl1−/− male mice (12 weeks). The two hydrogens of the imidazole ring in carnosine are marked a and b. (C) Relative integral based on NMR results, presented as means ± SD. (D to F) MRI of the brain in (D) Gadl1+/+, (E) Gadl1+/−, and (F) Gadl1−/− mice. The arrow indicates the OB. (G) Chemical structure comparison of GADL1 substrates CSA, Asp, and Glu. (H) 3D substrate structures to show the size and shape differences of Glu compared to CSA and Asp. (I) Active sites of GADL1, GAD, and CSAD and the predicted mode of binding of Glu to GAD. The prediction is based on the complex between GAD and the inhibitor chelidonic acid (63).

  • Fig. 4 Relative levels of antioxidant enzymes in Gadl1−/− mouse tissues.

    (A to E) Representative Western blots (B, D, and F) and normalized protein expression levels. (A), (C), and (E) for GSR, SOD1 (CuZnSOD), and SOD2 (Mn) in (A and B) OB, (C and D) cerebral cortex, and (E and F) SKM tissue from female Gadl1+/+ and Gadl1−/− mice.

  • Fig. 5 Behavioral phenotypes associated with Gadl1−/− mice, carnosine homeostasis and human phenotypes.

    (A) Three-chamber task: Time spent at each cylinder was not different between the genotypes. Both Gadl1+/+ and Gadl1−/− mice prefer the social cylinder, indicating that sociability remains similar. (B) Open field task: The cumulative time spent in the center was not different between the genotypes. On this measure, no antianxiety effect was observed. However, on the latency to enter the center (another anxiety measure), Gadl1−/− was quicker to enter the center, which may suggest some anxiolytic effects of the Gadl1−/− phenotype that would require confirmation in additional studies. (C) Open field task: The exploration velocity was not different between the genotypes, indicating that there is no effect on motor function. Similar observations were made with the total distance moved data. Together, this suggests no effect of genotype on activity metrics. (D) Elevated plus maze: The ratio of the time spent (s) on the open and closed arms was found not to be different between the genotypes. Both genotypes prefer the closed (sheltered arm), suggesting no difference in anxiety on this measure. (E) Resident intruder paradigm: The attack latency against an intruder from the first to the fifth day (tests 1 to 5) was not different between the genotypes. Both genotypes attack faster on the second day compared to the first day after which the attack latency remains constant. This suggests no effect of genotype on aggression. (F) A summary overview of different pathways involving β-alanine. Genes analyzed in this study are marked in blue. (G) Association analyses of genes involved in the carnosine metabolism. Asterisk indicates statistical significance after correction for multiple testing. ADHD, Attention deficiency hyperactivity disorder; ALS, Amyotrophic lateral sclerosis; SWB, subjective well being; AMD, age-related macular degeneration; UACR, Urine Albumin-to-Creatinine Ratio; NA, not applicable.

  • Table 1 Pathway enrichment analysis comparing Gadl1−/− to Gadl1+/+.

    Green, up-regulated; yellow, down-regulated.

    Drug
    metabolism
    Retinol
    metabolism
    Xenobiotics
    metabolism
    Tryptophan
    metabolism
    KEGG IDmmu00983mmu00830mmu00980mmu00380
    Gene IDAbbr.NameLog2 foldP value0.000160.000220.000250.00045
    14869Gstp2Glutathione S-
    transferase, pi 2
    −4.28190.0374
    13897Ces1eCarboxylesterase 1E4.90870.0040
    104158Ces1dCarboxylesterase 1D3.87230.0131
    234564Ces1fCarboxylesterase 1F2.60560.0193
    17523MpoMyeloperoxidase2.42470.0001
    394432Ugt1a7curidine 5′-diphospho
    glucuronosyltransferase
    1A7C
    1.05240.0019
    103149Upb1β-Ureidopropionase1.01210.0154
    27400Hsd17b6Hydroxysteroid (17-β)
    dehydrogenase 6
    4.08290.0103
    13087Cyp2a5Cytochrome P450 2A53.99850.0129
    213043Aox2Aldehyde oxidase 23.49650.0260
    13086Cyp2a4Cytochrome P450 2A43.19730.0157
    13076Cyp1a1Cytochrome P450 1A13.08000.0010
    11522Adh1Alcohol dehydrogenase
    1, class 1
    1.72080.0144
    13107Cyp2f2Cytochrome P450 2F22.96640.0024
    12409Cbr2Carbonyl reductase 21.26410.0012
    11298AanatArylalkylamine
    N-acetyltransferase
    −1.36320.0008
    15930Ido1Indoleamine
    2,3-dioxygenase 1
    2.40390.0192
    21743InmtIndolethylamine
    N-methyltransferase
    1.20430.0401
  • Table 2 Male and female mice used in the metabolomic study.

    GenotypeSexNumber (n)Age
    (weeks ± SD)
    Gadl1+/+Female1114.64 ± 7.85
    Gadl1−/−Female1114.27 ± 7.85
    Gadl1+/+Male98.88 ± 3.98
    Gadl1−/−Male108.40 ± 3.81

Supplementary Materials

  • Supplementary Materials

    GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

    Elaheh Mahootchi, Selina Cannon Homaei, Rune Kleppe, Ingeborg Winge, Tor-Arne Hegvik, Roberto Megias-Perez, Christian Totland, Floriana Mogavero, Anne Baumann, Jeffrey Colm Glennon, Hrvoje Miletic, Petri Kursula, Jan Haavik

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