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Beta-hexosaminidase A subunit alpha (HEXA)

Alex Donatelle

Section 008

Joseph Koussa

Alex Donatelle Section 008 Joseph Koussa

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Table of Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Gene Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Gene and protein function and role in disease . . . . . . . . . . . . . . . . 5

Studies in model systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

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Abstract

The HEXA gene encodes for the alpha subunit of beta-hexosaminidase A. HEXA plays an important role in the degradation of GM2 in neural cells. Throughout the past 50 years, researchers have developed information regarding the gene structure, protein structure, and function of the HEXA gene. The HEXA gene is found on chromosome 15 in humans and is flanked by CELF6 and TMEM202. The gene has 14 exons, 13 introns, and two splice variants. The HEXA gene in Mus musculus shows relatively high homology with the gene in Homo sapiens. The beta-hexosaminidase A protein is a dimer. The alpha subunit is composed of two domains. As a member of glycosyl hydrolase family 20, it has a functional domain important for the removal of D-hexosamine residues from N-acetyl-beta-D-hexosaminides. Intermolecular interactions take place between the alpha subunit and the beta subunit, three three N-Acetyl-D-Glucosamine molecules, and a sulfate ion. Beta-hexosaminidase A degrades GM2 in neural cells by cleaving a monosaccharide from a glycan chain on GM2 via hydrolysis. Mutations in the HEXA gene have been linked to Tay-Sachs Disease, an deadly autosomal recessive neurodegenerative disease linked to the accumulation of GM2 in neural cells. Myerowitz and Costigan (1998) determined the most common DNA lesion leading to the disease, a 4-base pair insertion in exon 11 of the HEXA gene. This results in a deficiency of the HEXA mRNA and an accumulation of GM2 causing early cell death in neurons. Several studies in the model organism Mus musculus have increased our understanding of HEXA’s function in humans. Platt et al. (1997) tested a treatment on HEXA knockout mice that may be useful in humans with Tay-Sachs Disease.

Data

Gene Structure

The HEXA gene is located on chromosome 15 in Homo sapiens. It is flanked by CELF6, HEXA-AS1, RPL12P35, and TMEM202. The gene starts at the 4,707th base pair and ends at the 37,745th base pair, spanning 33,039 base pairs total. The gene is composed of 14 exons, located at positions 4,707-5,460 (Exon 1), 24,563-24,655, (Exon 2), 25,556-25,621 (Exon 3), 27,443-27,489 (Exon 4), 28,002-28,112 (Exon 5), 29,946-30,047 (Exon 6), 30,530-30,662 (Exon 7), 31,921-32,101 (Exon 8), 33,046-33,132 (Exon 9), 33,422-33,494 (Exon 10), 34,470-34,653 (Exon 11), 34,855-34,945 (Exon 12), 35,630-35,734 (Exon 13), and 37,040-37,745 (Exon 14). The gene is composed of 13 introns. The total exonic length of the gene is 7,792 base pairs, and the total intronic length of the gene is 25,247 base pairs (NCBI).

Figure 1.1 – Location of HEXA and flanking genes on chromosome 15 in Homo sapiens (NCBI).

The coding region of the gene starts at the 5,208th base pair and ends at the 37,103rd base pair, spanning 31,896 base pairs total. The 5’ UTR starts at the 4,707th base pair and ends at the 5,208th base pair, spanning 502 base pairs total. The 3’ UTR starts at the 37,103rd base pair and

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ends at the 37,745th base pair, spanning 642 base pairs total. The total UTR length of the gene is thus 1,144 base pairs. HEXA in humans has 2 splice variants. One contains an augmented exon two, and one contains all exons excluding Exon 10, Exon 11, and Exon 14 (see Figure 1.2). The transcription start site is located at the 4,707th base pair (NCBI).

(Intron 1) Exon 2 Exons 10, 11 Exon 14

Figure 1.2 – Transcript variants of the HEXA gene in Homo sapiens (NCBI).

A homologous gene is located on chromosome 9 of Mus musculus, the house mouse. The two genes display rather high homology, sharing 84.3% protein identity and 84.4% DNA identity. Like its counterpart in Homo sapiens, HEXA in Mus musculus is flanked by CELF6 and TMEM202 (see Figure 1.3). HEXA in Mus musculus is also composed of 14 exons and 13 introns, and is marked by a similarly long intron 1. HEXA in mice also has 2 splice variants. One contains all exons excluding Exon 1, and one contains all exons excluding Exon 10 (see Figure 1.4) (NCBI).

Figure 1.3 – Location of HEXA and flanking genes on chromosome 9 in Mus musculus (NCBI).

Exon 1 (Intron 1) Exon 10

Figure 1.4 – Transcript variants of the HEXA gene in Mus musculus (NCBI).

Protein Structure

The HEXA gene encodes for the alpha subunit of the beta-hexosaminidase A protein (see Figure 2.1). The alpha subunit of this protein has an alpha helix to beta sheet ratio of approximately 60:40. It is composed of two domains. The smaller of the two domains is composed of antiparallel beta sheets linked to alpha helices. The larger of the two domains is composed of a tunnel of parallel beta sheets linked to alpha helices. The alpha subunit is one of two polypeptide subunits that compose the beta-hexosaminidase A protein dimer (RCSB).

As a member of the glycosyl hydrolase family 20 (GH20), the alpha subunit of beta-hexosaminidase A has a functional domain that catalyzes the removal of beta-1,4-linked N-acetyl-D-hexosamine residues from the non-reducing ends of N-acetyl-beta-D-hexosaminides. Proteins of this family are thought to achieve catalytic activity by reacting with a nucleophilic substrate. This domain is found in a wide variety of eukaryotic enzymes. It is responsible for many cellular processes including structure, signaling, and lysosomal catabolism (NCBI).

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Figure 2.1 – Crystallographic structure of beta-hexosaminidase A alpha (red) and beta (blue) subunits (RCSB).

Intermolecular interactions include those with Chain B of Human Beta-Hexosaminidase A along the plane of the polypeptide, three N-Acetyl-D-Glucosamine molecules located on the poles of the polypeptide, and one sulfate ion located near the beta sheet tunnel (see Figure 2.2) (NCBI). N-Acetyl-D-Glucosamines have been linked to the lysosomal catabolism of glycolipids. Deficiencies in proteins containing N-Acetyl-D-Glucosamines have been linked to several lysosomal storage diseases (PubChem). The sulfate ion located at the beta sheet tunnel functions to add sulfate groups to subtrates (Kolter).

Beta-hexosaminidase A is responsible for the lysosomal degradation of GM2 in neural cells. GM2 is a ganglioside, a sialic-acid containing glycosphingolipid found in neural cells. GM2 is made in high amounts and quickly degraded during neural development. After a glycosidase cleaves monosaccharide units from GM2 glycan chains, beta-hexosaminidase A cleaves the N-acetylgalactosaminyl residue via hydrolysis. The resulting GA2 molecule is further degraded to produce ceramides and sphingosines (Kolter).

Gene and protein function and role in disease

Tay-Sachs Disease is an autosomal recessive neurodegenerative disease linked to the HEXA gene. Tay-Sachs Disease is caused by a mutation in the HEXA gene that hinders the degradation of GM2 in neurons (OMIM).

Myerowitz and Costigan (1988) determined that a 4-base pair insertion in exon 11 of the HEXA gene is the most common mutation leading to Tay-Sachs disease in Ashkenazi Jews. Using the dot blot method, it was found that 70% of all Tay-Sachs Disease carriers tested carried this insertion. This insertion causes a frameshift and introduces a premature termination codon 9 bases downstream of the insertion (See Figure 3.1).

Figure 2.2 Figure 2.2 Figure 2.2 Figure 2.2 Figure 2.2 – Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of Schematic representation of intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions intermolecular interactions – A, alpha A, alpha A, alpha A, alpha A, alpha subunit; B, beta 1, Nsubunit; B, beta subunit; 1, N subunit; B, beta 1, Nsubunit; B, beta subunit; 1, Nsubunit; B, beta subunit; 1, N subunit; B, beta 1, N subunit; B, beta 1, Nsubunit; B, beta subunit; 1, N subunit; B, beta 1, N subunit; B, beta 1, Nsubunit; B, beta subunit; 1, Nsubunit; B, beta subunit; 1, N subunit; B, beta 1, N -AcetylAcetyl AcetylAcetylAcetyl-D-Glucosamine; 2Glucosamine; 2Glucosamine; 2 Glucosamine; 2Glucosamine; 2 Glucosamine; 2 Glucosamine; 2 , Sulfate Ion; 3, 2 , Sulfate Ion; 3, 2 , Sulfate Ion; 3, 2, Sulfate Ion; 3, 2, Sulfate Ion; 3, 2, Sulfate Ion; 3, 2 , Sulfate Ion; 3, 2 , Sulfate Ion; 3, 2 , Sulfate Ion; 3, 2 -(Acetylamino) (Acetylamino) (Acetylamino)(Acetylamino)(Acetylamino)(Acetylamino)(Acetylamino) (Acetylamino) -2-DeoxyDeoxy Deoxy-A-D-GlucopyranoseGlucopyranoseGlucopyranose Glucopyranose Glucopyranose Glucopyranose (NCBI). (NCBI).(NCBI). (NCBI).

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One would expect this premature termination codon to result in the translation of a truncated alpha subunit protein. However, a complete deficiency HEXA mRNA was observed. It is possible that the early termination of transcription results in reduced mRNA stability, increased mRNA degradation, or compromised mRNA processing (Myerowitz and Costigan). Frisch et al. (2004) identified a conserved haplotype in chromosomes from 55 unrelated Ashkenazi Jews carrying this 4-base pair insertion. This suggested the spread of this insertion resulted from the founder effect in a rapidly expanding population that emerged from a bottleneck.

The lack of alpha subunit protein in the neural cells of patients with Tay-Sachs Disease leads to impaired degradation of the GM2 ganglioside. The alpha subunit active site that is necessary for the cleaving of N-acetylgalactosaminyl acid is not present in Tay-Sachs patients. Thus, GM2 accumulates in the neurons. Structures called zebra bodies, masses of indigestible lysosomal membranes, fill the neuron. This, combined with the toxicity of GM2, causes the early death of neural cells (OMIM).

Early death of neural cells causes the shortening of the thalamus, prolongation of the basal ganglia, and widespread changes to white matter. Development begins to slow at 6 months of age. Symptoms of Tay-Sachs Disease include intellectual disability, paralysis, dementia, blindness. The startle reaction, an early, persistent, increased response to sound caused by the increased excitability of brainstem neurons, is often the first sign of Tay-Sachs Disease. Other signs include the presence of a cherry-red spot on the optic fundus and ballooned neurons, both caused by increased storage of GM2 in neural ganglia (See Figure 3.2). Tay-Sachs patients usually die before the age four (OMIM).

Studies in model systems

Several studies in the model organism Mus musculus have increased the understanding of the function of the HEXA gene and its role in disease. Taniike et al. (1995) performed a targeted disruption of the HEXA gene in mice. They found that knockout mice displayed depleted beta-

Figure 3.1 Figure 3.1 Figure 3.1 Figure 3.1 Figure 3.1 Figure 3.1 Figure 3.1 – Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal Nucleotide sequence analysis of normal and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 1 and mutant HEXA genes in region of exon 11 containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988). containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).containing the insertion (Myerowitz et al. 1988).

Figure 3.2 – Signs of Tay-Sachs Disease.

Top - Cherry-red spot (Retina & Macula).

Bottom - Ballooned neurons (Utah).

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hexosaminidase A activity and increased GM2 accumulation. This suggests that HEXA has similar biochemical function in mice and humans, and that model studies with mice may be useful in studying the function of HEXA in humans. Cohen-Tannoudji et al. (1995) performed a similar targeted disruption of the HEXA gene in mice. They found that no apparent motor or behavioral disorder resulted. This suggests that while human and mouse HEXA share similar biochemical function, HEXA is not an absolute requirement for ganglioside degradation in mice. It was stated, however, that animal models such as mice should be useful for the testing of new forms of therapy in humans.

Platt et al. (1997) tested a treatment on HEXA knockout mice. Mutant mice were treated with N-butyldeoxynojirimycin, a glycosphingolipid synthesis inhibitor. Treated mice displayed lower GM2 levels than untreated mice (See Figures 4.1 and 4.2). It was concluded that the decreased synthesis of GM2 prevented the accumulation of the ganglioside. It is possible that a similar treatment in humans could similarly prevent GM2 ganglioside accumulation. However, inhibition of glycosphingolipid synthesis must be controlled, as these lipids are necessary for development and function in many tissues and organ systems throughout the human body.

Discussion

Through several studies, researchers have formed a vast body of knowledge of the HEXA gene, the structure of the beta-hexosaminidase A protein, and the degradation of GM2. The HEXA genes in Homo sapiens and Mus musculus have shown rather high homology throughout studies of the genes. The genes are flanked by CELF6 and TMEM202 in both species. HEXA in both humans and mice contains 14 exons and 13 introns. Both genes have a long Intron 2 accounting for over half the length of the gene. The high homology between the two genes suggests that the function of the gene is evolutionarily important and that the gene has been conserved over time. In mice, the beta-hexosaminidase A alpha subunit contains the same conserved functional group, namely the glycosyl hydrolase family 20 domain, as its counterpart in humans. This similarly suggests that the function of the alpha subunits in both species is evolutionarily important and

Figure 4.2 – GM2 storage in ventromedial hypothalamus of untreated (A) and NB-DNJ–treated (B) mice (Platt et al.).

Figure 4.1 – Mean GM2 levels in untreated and NB-DNJ treated mice expressed in arbitrary units and determined via densitometry (Platt et al.).

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has been conserved. Due to the high gene and protein homology in HEXA in humans and mice, many valuable studies can be conducted using Mus musculus as a model. A study by Taniike et al. (1995) showing a link between HEXA disruption and GM2 accumulation in mice suggested that HEXA has similar biochemical function in mice and humans. A study by Cohen-Tannoudji et al. (1995) showing no link between HEXA disruption and behavioral disorder in mice suggested that humans and mice evolved slightly different uses for the alpha subunit over time. A study by Platt et al. (1997) showing a correlation between treatment with N-butyldeoxynojirimycin and decreased GM2 accumulation suggests a possible method of therapy for humans with Tay-Sachs. However, more research on the drug’s effect on glycosphingolipid synthesis must be conducted to prevent negative impacts on the development of neural cells in human trials. Further studies using Mus musculus as a model organism may prove valuable in developing treatment for individuals with Tay-Sachs disease in the future.

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References

2-acetamido-2-deoxy-beta-D-glucopyranosylamine. (n.d.). PubChem Open Chemistry Database. Retrieved December 9, 2017, from https://pubchem.ncbi.nlm.nih.gov/compound/439454

Cherry-red spot (n.d.). Retina & Macula. Retrieved December 10, 2017, from http://maculaspecialists.com.au/case/july-2014/

Cohen-Tannoudji, M., Marchand, P., Akli, S., Sheardown, S. A., Puech, J.-P., Kress, C., Gressens, P., Nassogne, M.-C., Beccari, T., Muggleton-Harris, A. L., Evrard, P., Stirling, J. L., Poenaru, L., Babinet, C. (1995) Disruption of murine Hexa gene leads to enzymatic deficiency and to neuronal lysosomal storage, similar to that observed in Tay-Sachs disease. Mammalian Genome 6: 844-849.

Conserved Protein Domain Family GH20_hexosaminidase. (n.d.). NCBI National Center for Biotechnology Information. Retrieved December 9, 2017, from https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cl02948

Crystallographic Structure of Human Beta-hexosaminidase a. (n.d.) NCBI National Center for Biotechnology Information. Retrieved December 9, 2017, from https://www.ncbi.nlm.nih.gov/Structure/pdb/2GJX

Crystallographic structure of human beta-Hexosaminidase A. (n.d.). RCSB Protein Data Bank. Retrieved December 9, 2017, from https://www.rcsb.org/pdb/explore/explore.do?structureId=2GJX

Enlarged pale neurons (n.d.). University of Utah Spencer S. Eclles Health Sciences Library. Retrieved December 10, 2017, from https://library.med.utah.edu

Frisch A., Colombo R., Michaelovsky E., Karpati M., Goldman B., Peleg L. (2004) Origin and spread of the 1278insTATC mutation causing Tay-Sachs disease in Ashkenazi Jews: genetic drift as a robust and parsimonious hypothesis. Human Genetics 113(4): 366-76.

Hexa hexosaminidase A [ Mus musculus (house mouse) ]. (n.d.). NCBI National Center for Biotechnology Information. Retrieved December 8, 2017, from https://www.ncbi.nlm.nih.gov/gene/15211

HEXA hexosaminidase subunit alpha [ Homo sapiens (human) ]. (n.d.). NCBI National Center for Biotechnology Information. Retrieved December 8, 2017, from https://www.ncbi.nlm.nih.gov/gene/3073

HomoloGene:20146. Gene conserved in Eukaryota. (n.d.). NCBI National Center for Biotechnology Information. Retrieved December 9, 2017, from https://www.ncbi.nlm.nih.gov/homologene?cmd=Retrieve&dopt=AlignmentScores&list_uids=20146

Kolter, T (2012) Ganglioside Biochemistry. ISRN Biochemistry 2012(01), 36. doi: 10.5402/2012/506160

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Myerowitz M., Costigan F. C. (1988) The Major Defect in Ashkenazi Jews with Tay-Sachs Disease Is an Insertion in the Gene or the a-Chain of b-Hexosaminidase. Journal of Biological Chemistry 263(33): 18587-18589.

Platt, F. M., Neises, G. R., Reinkensmeier, G., Townsend, M. J., Perry, V. H., Proia, R. L., Winchester, B., Dwek, R. A., Butters, T. D. (1997) Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science 276: 428-431.

Taniike, M., Yamanaka, S., Proia, R. L., Langaman, C., Bone-Turrentine, T., Suzuki, K. (1995) Neuropathology of mice with targeted disruption of Hexa gene, a model of Tay-Sachs disease. Acta Neuropath. 89: 296-304.

Tay-Sachs Disease; TSD. (n.d.). OMIM Online Mendelian Inheritance in Man. Retrieved December 10, 2017, from http://omim.org/entry/272800

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