|Mutation Type||frame shift|
|Coordinate||96,140,629 bp (GRCm38)|
|Base Change||GCC ⇒ GC (forward strand)|
|Gene Name||glycerol kinase 5 (putative)|
|Chromosomal Location||96,119,362-96,184,608 bp (+)|
|MGI Phenotype||PHENOTYPE: Homozygous knockout does not result in an obvious skin phenotype and does not lead to alopecia. [provided by MGI curators]|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000082313 †] [ENSMUSP00000112717 †] [ENSMUSP00000123594 †] † probably from a misspliced transcript|
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Predicted Effect||probably benign|
|Meta Mutation Damage Score||0.9755|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; human score: 0; ML prob: 0.5252|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Sperm, gDNA|
|Last Updated||2019-08-15 3:05 PM by External Program|
|Record Created||2011-02-22 10:20 PM by Wataru Tomisato|
|Other Mutations in This Stock||
Stock #: I1329 Run Code: HSQ01003
Coding Region Coverage: 10x: 96.9% 20x: 95.2%
Validation Efficiency: 42/47
The toku phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized mice as a secondary phenotype of the Kokuten mice (I1329). Toku homozygous mice exhibited alopecia (Figure 1A). Homozygous toku mice exhibited delayed eruption of hair from the skin surface (Figure 1B).
|Nature of Mutation|
The toku mutation was mapped to a 57.4 Mbp region of chromosome 9 by bulk segregation analysis (Figure 2). Sanger sequencing identified a deletion of a cytosine (C) at base pair 96,140,631 (NCBI v38) on chromosome 9, within exon 5 (of 16 total exons) of Gk5 (Figure 3), which corresponds to position 519 of the Gk5 cDNA transcript (ENSMUST00000085217).
The deleted C is in red in the C57BL/6J sequence above. The toku transcript codes a premature stop codon after amino acid 174.
TALEN-mediated targeting of Gk5 confirmed that the causative mutation for the alopecia phenotype was in Gk5 (Figure 4).
Gk5 gene encodes the 534 amino acid (aa) glycerol kinase 5 (GK5) protein. Although the protein domains of GK5 have not been documented, SMART predicts that GK5 contains two domains that are found in the FGGY family of carbohydrate kinases (Figure 5), which also includes glycerol kinase (Gyk; alternatively, GK (in humans); it will be referred to as GK for clarity), glucokinase (Gck), xylulokinase (Xylb), and Hsc70 (or Hspa8). The FGGY kinases contain conserved motifs at both the N- and C-termini (amino acids 25-287 and aa 396-416 in mouse GK5, respectively; SMART). The FGGY_N and FGGY_C termini are structurally similar and adopt a ribonuclease H-like fold (2;3). Between the FGGY_N and FGGY_C domains is a catalytic cleft where a sugar substrate and ATP bind (4). The substrate interacts mainly with the N-terminus; ATP contacts both the N- and C-terminal domains (4). Upon binding of the substrate (e.g., glycerol), a conformational change occurs whereby the N- and C-termini close to prevent any solvents from entering the catalytic cleft [(5-8); reviewed in (2;4)]. A highly conserved 29-aa extension at the C-terminus of GK is proposed to mediate the binding of GK to the voltage-dependent anion channel (VDAC) at the mitochondrial outer membrane (9); the function of the C-terminus of GK5 has not been elucidated.
The FGGY proteins have five specificity-determining positions (SDPs) within the catalytic cleft (4). The side chains of the amino acids at the SDPs point toward the center of the substrate binding site, interacting with the substrates (4). In addition to the SDPs, other amino acids have been identified to be essential for the function of GK. For example, Asp10 and Asp245 (both in in E. coli) are proposed to contribute to a Mg2+-binding site that may function in GK-mediated ATP hydrolysis based on the structures of hexokinase, HSC70, and actin [(10); reviewed in (2)]. Hurley et al. also determined that Asp245 (in E. coli) forms a hydrogen bond with the 3-hydroxyl of glycerol, indicating that it is the catalytic base in the phosphorylation reaction (2).
The crystal structure of E. coli GK in complex with IIAGlc (or IIIGlc) has been solved [Figure 6; PDB: 1GLB; (2)]. IIAGlc is a member of the phosphoenolpyruvate:glucose phosphotransferase system in bacteria that is responsible for the uptake and phosphorylation of glucose. GK forms functional dimers and tetramers [(11-13); reviewed in (2)]. The FGGY domains (denoted here as domains I and II) are subdivided: IA (aa 1-35, 49-83, 165-173, and 221-253), IIA (aa 254-306 and 373-472), IB (aa 36-48 and 82-164), IC (aa 174-220), IIB (aa 307-372), and IIC (aa 456-501)). Domains IB, IC, IIB, and IIC are insertions into, or extensions of, the ATPase core in hexokinase (14), HSC70 (10;15), and actin (16); subdomains IC and IIC are unique to GK (2). Also unique from other FGGY family members is that the IB subdomain of GK contains two insertions, making it larger than other IB sub-domains; it contains a central five-stranded antiparallel β-sheet (2). Sub-domain IIB is topologically identical to those found in HSC70 and actin (2).
Gk5 has three isoforms, the canonical 2,823 bp transcript (ENSMUST00000085217) that encodes the 534 amino acid protein, a 4,474 bp transcript (ENSMUST00000122383) that encodes a 516 amino acid protein product, and a third 2,506 bp isoform (ENSMUST00000129774) that encodes a 59 amino acid polypeptide; the 59 aa product is expected to undergo nonsense-mediated decay (Ensembl). A fourth transcript, ENSMUST00000136496, does not encode a protein product. The 516 amino acid protein product of isoform two differs from the canonical 534 amino acid sequence at the C-terminus (Uniprot: Q8BX05).
Gk5 is expressed in several mouse tissues; however, protein expression was observed solely in the skin (Figure 7) (1). GK5 is localized to the sebaceous glands adjacent to the hair follicles and it is cytoplasmic (Figure 8).
The members of the FGGY family phosphorylate sugar substrates in an ATP-dependent manner (4). GK mediates an ATP-dependent glycerol phosphorylation to G3P, an important precursor protein in glucose and lipid metabolism (17). G3P is essential for the production of glycerides, glucose, glycogen, and dihydroxyacetone phosphate (DHAP), an intermediate in several metabolic pathways (e.g. glycolysis and glycogenesis) (18-20). G3P is also involved in cancer development and is a substrate in acylglycerol synthesis, a process involved in the energy shuttle system of the mitochondria that provides energy to the cell during cell proliferation and development (21). After GK-mediated phosphorylation of glycerol to G3P, approximately 70-90% of the G3P is oxidized via G3P dehydrogensase to dihydroxyacetone phosphate, which can enter the Embden-Meyerhof pathway (i.e. glycolysis) [reviewed in (22)]. The remainder combines with free fatty acids to form triglycerides [(23); reviewed in (22)]. In non-hepatic tissues that lack significant GK activity, glycerol returns to the liver to be phosphorylated (24). In the liver, the glycerol can then be reesterified to triglyceride; the remainder will enter the glycolytic/gluconeogenic metabolite pool (24). In the glycolytic pathway, the glycerol will result in the conversion of pyruvate to lactate or acetyl CoA; in the gluconeogenic pathway, there is an eventual release of glucose or a storage of glycogen (19;24).
GK5 exhibits glycerol kinase activity albeit at significantly lower rates than GK (1). GK5 functions specifically in the skin to regulate SREBP-1/-2-mediated free cholesterol, cholesteryl esters, triglycerides, and ceramides production. In the skin, GK5 specifically functions in regulating the processing of the SREBPs, which promote cholesterol biosynthesis and homeostasis by stimulating the transcription of sterol-regulated genes (e.g., Hmgcr, Hmgcs1, Hmgcs2, Acaca, Fasn, Scd1, Ldlr, and Fdps) (1).
GK5 is one of several transcripts that are upregulated in patients that have received intestinal transplant grafts and that experience early acute cellular rejection (25).
The levels of the transcriptionally active forms of SREBP-1 and SREBP-2 were increased in the skin of the toku mice (Figure 10). The expression level of HMGCR, a SREBP-1/-2 target, was increased in the toku skin compared to that in wild-type skin (Figure 10). Treatment of the toku mice with simvastatin, a statin inhibitor of HMGCR, the rate-determining cholesterol biosynthesis enzyme, showed that blockade of the levels of sterol precursors or cholesterol in the skin could rescue the hair loss phenotype (Figure 11). Together, these findings indicate that increased sterol precursors and/or cholesterol in the skin caused the hair loss in Gk5-deficient mice (1).
Toku genotyping is performed by amplifying the region containing the mutation using PCR followed by sequencing of the amplified region to detect the nucleotide change. The following primers were used for PCR amplification:
Primers for PCR amplification
Toku (F): 5'- TTCAGAAGGTTGAGAGCCACCACG -3'
Toku (R): 5'- CATGCCTGAGTCACCCAAAATGTTG -3'
Primers for Sequencing
Toku_seq (F): 5'- GGTGCTTTAACTGAACCCAGG -3'
Toku_seq (R): 5'- CTCATCGTTAGACATCGTTAGAGC -3'
1) 94° C 2:00
2) 94° C 0:30
3) 60° C 0:30
4) 72° C 1:00
5) repeat steps (2-4) 29x
6) 72° C 7:00
7) 4° C ∞
The following sequence of 751 nucleotides (from Genbank genomic region: NC_000075.6 of the linear genomic sequence of Gk5) is amplified:
20790 t tcagaaggtt gagagccacc acgtgggtgc 20821 tttaactgaa cccaggggct ctgcaagagc agcaagtgtt cttaatcact gagatgcctc 20881 tctaacctct gtttttgttc tgtaagaaac ggattaagct ggacatagtg gtgcacatca 20941 ttaatcctag tgtgggggaa gcagaaacag gtggacctga actccataag ttcaatgcca 21001 gccaggacta catagttata ccctgtcttg aaaaggggag ggggtcgaga aagaaaaccc 21061 aagtgccctt aaatgtattt ccttaaagat ctcttttgtt tttaaaacag ctattgcatg 21121 gggccacccg agtccttcat ttcttcagta gaagtaaagt aatgctaacg gtcagccgct 21181 tcaatttcag cacccagcat gccaccttaa gattgacctg gattttacaa aacctatctg 21241 aggtaagaga agattgtgtg tgtggagagg ggatcatgac tgtggggaag aaaaatttaa 21301 agagtaagaa caataatcca gccttttggg gaagtgacca gtactcctgg gctagaagac 21361 agagttttca attggttcct gtgtttaaag tctttgtgtt atgtcataac acaaaaaaga 21421 ttgacagtaa gtttgaagct aacctgggct acatccagga gctctaacga tgtctaacga 21481 tgagatgaca ttcttatgta tgctagatag gtacccaaca ttttgggtga ctcaggcatg
PCR primer binding sites are underlined; Sequencing primer binding sites are highlighted; the deleted C is highlighted in red.
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2. Hurley, J. H., Faber, H. R., Worthylake, D., Meadow, N. D., Roseman, S., Pettigrew, D. W., and Remington, S. J. (1993) Structure of the Regulatory Complex of Escherichia Coli IIIGlc with Glycerol Kinase. Science. 259, 673-677.
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|Science Writers||Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||Duanwu Zhang, Wataru Tomisato, Bruce Beutler|