Purification and some properties of liver and brain β-N-Acetyl-hexosaminidase S

Potier, Michel; Teitelbaum, Jeremy; Mélançon, S. B.; Dallaire, Louis

doi:10.1016/0005-2744(79)90251-1

Cited by 14 publications

(12 citation statements)

References 19 publications

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“…The sugar chain is elaborated with repeating N-acetylglucosamine and N-acetylmuramic acid units, and N-acetylmuramic acid is cross-linked by peptides to produce a dense three-dimensional network (47,48). b-Hexosaminidase affects the N-acetylglucosamine or N-acetylgalactosamine residues in the termini of glycoproteins or glycolipids (49)(50)(51). Lysozyme, which is also known as muramidase, is found in considerable quantities in the granules of sentinel cells such as neutrophils (52), and this enzyme cleaves bacterial PGN by hydrolyzing the 1,4-b bond between N-acetylmuramic acid and N-acetylglucosamine (53).…”

Section: Discussionmentioning

confidence: 99%

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

Fukuishi

Murakami

Ohno

et al. 2014

The Journal of Immunology

101

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β-Hexosaminidase, which is generally present in the lysosome, is essential for glycoprotein metabolism in the maintenance of cell homeostasis. In mast cells (MCs), large amounts of β-hexosaminidase are present in the granules as opposed to the lysosome, and the biological role of MC β-hexosaminidase has yet to be fully elucidated. Therefore, we investigated the biological role of β-hexosaminidase in MC granules. Bone marrow-derived MCs from C57BL/6 (BL/6-BMMC) or β-hexosaminidase gene–deficient (hexb−/−-BMMC) mice were transplanted into MC-deficient (WBB6F1/J-KitW/KitW-v [W/Wv]) mice to generate MC-reconstituted models. In asthma model experiments, no differences were observed in the symptoms of BL/6, W/Wv, BL/6-BMMC–reconstituted W/Wv, or hexb−/−-BMMC–reconstituted W/Wv mice. In Staphylococcus epidermidis experimental infection model experiments, the severity of symptoms and frequency of death were markedly higher in W/Wv and hexb−/−-BMMC–reconstituted W/Wv mice than in BL/6 and BL/6-BMMC–reconstituted W/Wv mice. The growth of S. epidermidis in an in vitro study was clearly inhibited by addition of BL/6-BMMC lysate, but not by addition of hexb−/−-BMMC lysate. Moreover, suppression of bacterial proliferation was completely recovered when bacteria were incubated with hexb−/−-BMMC lysate plus β-hexosaminidase. Transmission electron microscopy indicated that the cell wall of S. epidermidis was heavily degraded following coincubation of bacteria with BL/6-BMMC lysate, but not following coincubation with hexb−/−-BMMC lysate. These findings strongly suggest that MC granule β-hexosaminidase is crucial for defense against bacterial invasion, but is not involved in the allergic response. Our results also suggest that the bactericidal mechanism of β-hexosaminidase involves degradation of bacterial cell wall peptidoglycan.

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Section: Discussionmentioning

confidence: 99%

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

Fukuishi

Murakami

Ohno

et al. 2014

The Journal of Immunology

101

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“…Human native β-hexosaminidases have been purified from liver [7][8][9][10][11], kidney [12], brain [10], and placenta [13][14][15]. On the other hand, human β-hexosaminidases have been recombinantly produced in CHO [5,16], HeLa [17], and NG141 and NG125 cell lines [18], as well as in insect cells [17,19,20] and yeast (O. minuta and Pichia pastoris KM71H) [21][22][23].…”

Section: Characterization Of Recombinant Human Lysosomal Beta-hexosammentioning

confidence: 99%

“…Human β-hexosaminidases extracted from human liver have a molecular weight between 100 and 103 kDa [7]. However, Hex-S extracted from brain and liver displayed a molecular weight of 152 and 154 kDa, respectively [10]. The estimated molecular weight of Hex-A and Hex-B produced in CHO and HeLa cells was between 63 and 69 kDa [5,17].…”

Section: Characterization Of Recombinant Human Lysosomal Beta-hexosammentioning

confidence: 99%

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris

et al. 2016

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β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant β-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14.606 U.mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.

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“…When 47 nanomoles/min of fibroblast hexosaminidase B were rechromatographed under the same conditions less than 0.02% of this enzyme activity is recovered in column fractions eluting after the application of the salt gradient. b) Hexosaminidase S has been identified in normal fibroblasts and in Sandhoff disease fibroblasts (Beutler et al 1975b) in Sandhoff disease brain and liver (Potier et al 1979, Ikonne et al 1975, Geiger et al 1977, and as a breakdown product of merthiolate treated human placental hexosaminidase A (Mahuran & Lowden 1980). However, this species is absent in Tay Sach Disease tissues (Beutler et al 1975b) and its thermolability and ionic properties are unlike those of TSD anionic hexosaminidase.…”

Section: Discussionmentioning

confidence: 99%

A new from of residual hexosaminidase activity in infantile Tay Sachs disease fibroblasts

Hechtman

Khoo

Isaacs³

1983

Clinical Genetics

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Fibrolast cells lines obtained from five patients with the early onset form of Tay Sachs disease (TSD) possess a species of β‐N‐acetylhexosaminidase (Hex) which is more anionic than Hex B but which is stable to heating under conditions which completely inactivate Hex A. This speices, which comprised between 3 and 20% of th total hexosaminidase acitivity in homozygous TSD fibroblasts, appeared to be unstable and upon isoelectric focussing produced a mixture of Hex B (pI=7.2) and an isozayme with a pI of 6.2. This intermediate form of hexosaminidase was not seen in two normal fibroblast cell lines but was observed following anion exchange chromatography of extracts of fibroblast cell lines obtained from two obligate heterozygotes. A species of hexosaminidase with the same chromatographic properties, thermostability and isoelectric point as the intermediate form found in fibroblasts with the TSD genotypes can be recovered after anion exchange chromatography of a partially purified preparation of human liver Hex A that had been treated with merthiolate. We hypothesize that in TSD cells a form of the β subunit which is usually incorporated into Hex A accumulates due to the absence of a subunits. This form of the β subunit is more anionic than the β subunit found in Hex B. In the absence of α subunits these anionic β subunits form tetramers with a pI = 6.2. This form of the enzyme is unstable in the presence of cellular proteases and may be modified to Hexosaminidase B.

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Purification and some properties of liver and brain β-N-Acetyl-hexosaminidase S

Cited by 14 publications

References 19 publications

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris

A new from of residual hexosaminidase activity in infantile Tay Sachs disease fibroblasts

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