scholarly journals 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

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.

scholarly journals 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

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.

scholarly journals 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 ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e57908 ◽  
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

Degradation of GM1 and GM2 by mammalian sialidases

2001 ◽  
Vol 360 (1) ◽  
pp. 233-237 ◽  
Author(s):  
Su-Chen LI ◽  
Yu-Teh LI ◽  
Setsuko MORIYA ◽  
Taeko MIYAGI
Keyword(s):  
Clinical Symptoms ◽  
Activator Protein ◽  
Tay Sachs Disease ◽  
In Vitro Hydrolysis ◽  
Mammalian Tissues ◽  
Gm2 Activator ◽  
Gm2 Activator Protein ◽  
Cytosolic Sialidase

In mammalian tissues, the pathway known for the catabolism ofGM1[Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer;where Cer is ceramide] is the conversion of this ganglioside into GM2 [GalNAcβ4(Neu5Acα3)Galβ4GlcβCer] by β-galactosidase followed by the conversion of GM2 into GM3 (Neu5Acα3Galβ4GlcβCer) by β-N-acetylhexosaminidase A (Hex A). However, the question of whether or not GM1 and GM2 can also be respectively converted into asialo-GM1 (Galβ3GalNAcβ4Galβ4GlcCer; GA1) and asialo-GM2 (GalNAcβ4Galβ4GlcβCer, GA2) by mammalian sialidases has not been resolved. This is due to the fact that sialidases purified from mammalian tissues always contained detergents that interfered with the in vitro hydrolysis of GM1 and GM2 in the presence of an activator protein. The mouse model of human type B Tay–Sachs disease created by the disruption of the Hexa gene showed no neurological abnormalities, with milder clinical symptoms than the human counterpart, and the accumulation of GM2 in the brains of affected mice was only limited to certain regions [Sango, Yamanaka, Hoffmann, Okuda, Grinberg, Westphal, McDonald, Crawley, Sandhoff, Suzuki and Proia (1995) Nat. Genet. 11, 170–176]. These results suggest the possible presence of an alternative catabolic pathway (the GA2 pathway) in mouse to convert GM2 into GA2 by sialidase. To show the existence of this pathway, we have used recombinant mammalian cytosolic sialidase and membrane-associated sialidase to study the desialylation of GM1 and GM2. We found that the mouse membrane-bound sialidase was able to convert GM1 and GM2 into their respective asialo-derivatives in the presence of human or mouse GM2 activator protein. The cytosolic sialidase did not exhibit this activity. Our results suggest that, in vivo, the stable NeuAc of GM1 and GM2 may be removed by the mammalian membrane-associated sialidase in the presence of GM2 activator protein. They also support the presence of the GA2 pathway for the catabolism of GM2 in mouse.

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

2004 ◽  
Vol 324 (3) ◽  
pp. 1048-1052 ◽  
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

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

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.

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

1991 ◽  
Vol 266 (3) ◽  
pp. 1879-1887 ◽  
Author(s):  
E M Meier ◽  
G Schwarzmann ◽  
W Fürst ◽  
K Sandhoff
Keyword(s):  
Protein A ◽  
Activator Protein ◽  
Hexosaminidase A ◽  
Gm2 Activator ◽  
Gm2 Activator Protein

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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