Metabolic correction in microglia derived from Sandhoff disease model mice

Tsuji, Daisuke; Kuroki, Aya; Ishibashi, Yasuhiro; Itakura, Tomohiro; Itoh, Kohji

doi:10.1111/j.1471-4159.2005.03317.x

Cited by 28 publications

(24 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cells were grown in Eagle's medium (Dulbecco's modified Eagle's medium) or Ham's F-10 medium supplemented with 10% fetal calf serum at 37°C in a humidified incubator flushed continuously with a 5% CO 2 -95% air mixture. Experiments for enzyme uptake by TS and SD fibroblasts and the observation of intracellular GM2 ganglioside by immunostaining against anti-GM2 antibody were performed as described previously (33,44). Briefly, for enzyme incorporation, TS and SD fibroblasts were cultured for 3 days in medium containing HexA with a particular amount of MUGS-hydrolyzing activity (0, 200, 600, or 1,800 nmol/h/well).…”

Section: Methodsmentioning

confidence: 99%

“…DEAE chromatography was performed to separate isozymes by a NaCl gradient (0 to 300 mM) as previously described by Tsuji et al (44), with a slight modification in the program of elution of isozymes. Three fractions with MUG-hydrolyzing activity were separately collected as recombinant HexB (␤␤), HexA (␣␤), and HexS (␣␣); these fractions were eluted at NaCl concentrations of 50 mM, 90 to 150 mM, and 190 to 250 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The replacement of recombinant ␤-hexosaminidases in CHO cells applied to SD mouse microglia, Schwann cells, and SD human fibroblasts has been reported (33,44). To further investigate the use of ERT for GM2 gangliosidosis, in this report we have produced yeast recombinant HexA that can be incorporated into TS and SD cells and hydrolyzes accumulating intracellular GM2 gangliosides.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Akeboshi

Chiba

Kasahara

et al. 2007

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Human ␤-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of ␣-and ␤-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using ␣-1,6-mannosyltransferase-deficient (och1⌬) yeast as the host. Genes encoding the ␣-and ␤-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (␣␣) and HexB (␤␤). A total of 57 mg of ␤-hexosaminidase isozymes, of which 13 mg was HexA (␣␤), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the ␤-subunit. The purified HexA was treated with ␣-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ؎ 0.1 and 1.7 ؎ 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the ␤-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Akeboshi

Chiba

Kasahara

et al. 2007

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Enzyme replacement using recombinant human b-hexosaminidase (mixture of all isozymes) from CHO cells has been examined in SD mouse microglia cells, 100) SD mouse Schwann cells, and fibroblast cells from SD patients. 101) Since further ERT experiments in mouse models and molecular studies on recombinant HexA are required, the b-hexosaminidase was expressed in a yeast expression system in order to obtain the required large amounts of enzyme (Fig.…”

Section: Treatment Of Lysosomal Storage Diseasesmentioning

confidence: 99%

“…As in other mammalian expression systems, co-expression of the two HexA subunits produces not only HexA but also the homodimeric isozymes HexS (aa) and HexB (bb). 100,104) The O. minuta strain produced 14.3 mg of b-hexosaminidase isozymes from 1 l of culture broth; HexA constituted 23% of the total isozymes, based on the MUG hydrolyzing activities of the isolated isozymes. 105) The host O. minuta strain was manipulated to increase the amount of phosphorylation on recombinant HexA N-glycans.…”

Section: Treatment Of Lysosomal Storage Diseasesmentioning

confidence: 99%

Glycan Engineering and Production of 'Humanized' Glycoprotein in Yeast Cells

Chiba

Akeboshi

2009

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Protein therapeutics, such as antibodies and cytokines, is the largest class of new drug candidates being developed by pharmaceutical companies. Although most of these glycoproteins are produced in mammalian cells, there is concern that their large-scale production could be affected by an inadequate supply of bovine serum. There is also the risk of viral infection spreading through the use of contaminated protein therapeutics. Consequently, protein expression systems in yeast have been established because protein manufacturing costs are cheaper than in mammalian cells, and yeast systems are virus-free. However, yeasts cannot generate human-type glycans, and thus cannot produce therapeutic glycoproteins for human use. There has therefore been considerable interest in glycan remodeling, from yeast-type to human-type. 'Humanized' glycoproteins can now be generated in yeast by disrupting yeast-specific glycosyltransferases and introducing genes responsible for sugar-nucleotide synthesis, its transported from the cytosol to Golgi lumen, as well as their transfer and hydrolysis. A compound that inhibits yeast O-mannosyltransferase suppresses yeast-specific O-mannosyl modification, and can produce mucin-type glycoproteins. These systems are just being developed to the stage where the production in glycoengineered yeast of biopharmaceutical glycoproteins such as cytokines, antibodies for therapeutics, and enzymes for replacement therapy for lysosomal diseases are being evaluated for clinical applications. Yeast glycoprotein expression systems are expected to become the dominant approach for the production of human glycoproteins in the near future.

show abstract

The Blood – Brain Barrier and its Effect on Absorption and Distribution

Boer

Gaillard²

2010

Pharmaceutical Sciences Encyclopedia

View full text Add to dashboard Cite

Potential drugs for the treatment of most brain diseases are often not able to cross barriers protecting the central nervous system (CNS). As a result, various drug delivery and targeting strategies are currently being developed to enhance the absorption and distribution of drugs into the brain. This article discusses the biology and physiology of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier with respect to drug transport (absorption, distribution), the in vitro and in vivo methods to measure BBB transport, and the possibilities to deliver large molecular drugs, by viral and receptor‐mediated nonviral drug delivery, to the (human) brain.

show abstract

Metabolic correction in microglia derived from Sandhoff disease model mice

Cited by 28 publications

References 30 publications

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Glycan Engineering and Production of 'Humanized' Glycoprotein in Yeast Cells

The Blood – Brain Barrier and its Effect on Absorption and Distribution

Contact Info

Product

Resources

About