Evidence for two different active sites on human beta-hexosaminidase A. Interaction of GM2 activator protein with beta-hexosaminidase A.

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

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An α-subunit loop structure is required for GM2 activator protein binding by β-hexosaminidase A

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.09.159 ◽

2004 ◽

Vol 324 (3) ◽

pp. 1048-1052 ◽

Cited By ~ 10

Author(s):

Maryam Zarghooni ◽

Scott Bukovac ◽

Michael Tropak ◽

John Callahan ◽

Don Mahuran

Keyword(s):

Protein Binding ◽

Loop Structure ◽

Α Subunit ◽

Activator Protein ◽

Hexosaminidase A ◽

Gm2 Activator ◽

Gm2 Activator Protein

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Ganglioside GM2-Activator Protein and Vesicular Transport in Collecting Duct Intercalated Cells

Journal of the American Society of Nephrology ◽

10.1681/asn.v103435 ◽

1999 ◽

Vol 10 (3) ◽

pp. 435-443

Author(s):

THOMAS M. MUNDEL ◽

HANS W. HEID ◽

DON J. MAHURAN ◽

WILHELM KRIZ ◽

PETER MUNDEL

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

The Body ◽

Kidney Cortex ◽

Intercalated Cells ◽

Rat Kidney Cortex ◽

Activator Protein ◽

Hexosaminidase A ◽

Gm2 Activator ◽

Gm2 Activator Protein

Abstract. This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with “studs,” which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.

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In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

PLoS ONE ◽

10.1371/journal.pone.0057908 ◽

2013 ◽

Vol 8 (3) ◽

pp. e57908 ◽

Cited By ~ 8

Author(s):

Incilay Sinici ◽

Sayuri Yonekawa ◽

Ilona Tkachyova ◽

Steven J. Gray ◽

R. Jude Samulski ◽

...

Keyword(s):

Gm2 Ganglioside ◽

Activator Protein ◽

Hexosaminidase A ◽

Hybrid Construct ◽

Gm2 Activator ◽

Gm2 Activator Protein

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In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

10.32920/14639667.v1 ◽

2021 ◽

Author(s):

Warren W. Wakarchuk ◽

Incilay Sinici ◽

Sayuri Yonekawa ◽

Ilona Tkachyova ◽

Steven J. Gray ◽

...

Keyword(s):

In Vitro Assays ◽

Variant Form ◽

Gm2 Ganglioside ◽

Activator Protein ◽

Hexosaminidase A ◽

Hybrid Construct ◽

Gm2 Activator ◽

Gm2 Activator Protein

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

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Formation of a ternary complex between GM2 activator protein, GM2 ganglioside and hexosaminidase A

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/s0167-4838(97)00027-7 ◽

1997 ◽

Vol 1340 (1) ◽

pp. 45-52 ◽

Cited By ~ 12

Author(s):

Franeli Yadao ◽

Peter Hechtman ◽

Feige Kaplan

Keyword(s):

Ternary Complex ◽

Gm2 Ganglioside ◽

Activator Protein ◽

Hexosaminidase A ◽

Gm2 Activator ◽

Gm2 Activator Protein

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The human GM2 activator protein. A substrate specific cofactor of beta-hexosaminidase A

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)52375-9 ◽

1991 ◽

Vol 266 (3) ◽

pp. 1879-1887 ◽

Cited By ~ 1

Author(s):

E M Meier ◽

G Schwarzmann ◽

W Fürst ◽

K Sandhoff

Keyword(s):

Protein A ◽

Activator Protein ◽

Hexosaminidase A ◽

Gm2 Activator ◽

Gm2 Activator Protein

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Two patients from Turkey with a novel variant in the GM2A gene and review of the literature

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2020-0655 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Aslı İnci ◽

Filiz Başak Cengiz Ergin ◽

Gürsel Biberoğlu ◽

İlyas Okur ◽

Fatih Süheyl Ezgü ◽

...

Keyword(s):

Sandhoff Disease ◽

Pathogenic Variant ◽

Protein Deficiency ◽

Gm2 Gangliosidosis ◽

Infantile Form ◽

Rare Form ◽

Inborn Errors ◽

Activator Protein ◽

Gm2 Activator ◽

Gm2 Activator Protein

Abstract Objectives GM2 gangliosidosis is a rare form of inborn errors of metabolism including Tay-Sachs disease, Sandhoff disease, and GM2 activator deficiency. GM2 activator protein deficiency is an ultra-rare form of GM2 gangliosidosis. To date, 16 cases of GM2 activator protein deficiency have been reported in the literature, and among them, 11 cases were the infantile form of the disease. Here we report the first two patients from Turkey with the infantile form of the disease with a novel likely pathogenic variant. Case presentation A boy of eight months old presented to the metabolic department with very mild neurological deterioration, although he had achieved early developmental milestones at the appropriate time. The parents also had a daughter who had lost skills progressively before one year of age. The boy was evaluated and bilateral cherry-red spots were found with no abnormality in either metabolic screening including β-hexosaminidase or cranial magnetic resonance imaging. A novel homozygous likely pathogenic variant in GM2A was detected in a next-generation sequence panel revealing GM2 activator protein deficiency. His sister was investigated after he was diagnosed with GM2 activator deficiency and it was found that she had the same variant as her brother. Conclusions This case report emphasizes that in the event of normal β-hexosaminidase activity, GM2 activator protein deficiency could be underdiagnosed, and further molecular analysis should be performed. To the best of our knowledge, this boy is one of the youngest patient diagnosed with very mild symptoms. With this novel pathogenic variant, these patients have expanded the mutation spectrum of GM2 activator protein deficiency.

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