Evidence that the mutant enzyme in fibroblasts of a patient with mannosidosis does not crossreact with antiserum raised against normal acidic α‐D‐mannosidase

Burditt, Lynda J.; Chotai, K; G, Winchester Bryan

doi:10.1016/0014-5793(78)81168-5

Cited by 12 publications

(9 citation statements)

References 17 publications

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“…This activity could be a mutant form of the a-D-mannosidase affected in genetic mannosidosis or another form of lysosomal a-D-mannosidase that is unaffected in genetic mannosidosis. The fact that the residual acidic a-D-mannosidase in this mannosidosis cell line does not cross-react with antiserum raised against normal human a-D-mannosidase favours the latter suggestion (Burditt et al, 1978). Reversibility of swainsonine-induced accumulation ofoligosaccharides It has been shown previously that the rapid uptake of swainsonine into fibroblasts in culture results in inhibition of the intracellular lysosomal a-D-mannosidase (Chotai et al, 1983).…”

Section: Chemicalsmentioning

confidence: 63%

“…A. Lee by the method of Colegate et al (1979) Normal human fibroblasts and fibroblasts from a child with mannosidosis were grown as described previously (Burditt et al, 1978). To investigate the effect of swainsonine on the metabolism of glycoproteins, the cells were incubated in culture medium containing 100 pM-swainsonine for the required length of time.…”

Section: Chemicalsmentioning

confidence: 99%

“…a-D-Mannosidase assays a-D-Mannosidase was assayed in cell extracts as described previously (Burditt et al, 1978).…”

Section: Chemicalsmentioning

confidence: 99%

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The storage products in genetic and swainsonine-induced human mannosidosis

Bello¹,

Dorling²,

Winchester³

1983

Biochemical Journal

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Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal a-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi a-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal a-D-mannosidase that is unaffected in genetic mannosidosis.The 1,2,8-trihydroxylated indolizidine alkaloid swainsonine occurs in plants of the genus Swainsona (Colegate et al., 1979), in certain species of locoweeds (Astragalus and Oxytropis spp.) (Molyneux & James, 1982) and in the fungus Rhizoctonia leguminicola (Schneider et al., 1982). It is a potent inhibitor of a-D-mannosidase activity (Dorling et al., 1980). Prolonged ingestion of swainsonine by animals leads to a neurological disorder that is a phenocopy of the genetically induced lysosomal storage disease mannosidosis (Dorling et al., 1978).Mannosidosis is characterized by accumulation in cells, and excretion in the urine, of mannose-rich oligosaccharides resulting from a deficiency of lysosomal a-D-mannosidase (Jolly et al., 1981). Swainsonine is a lysosomotropic compound and accumulates rapidly in lysosomes of normal human fibroblasts in culture to produce inhibition of intracellular lysosomal a-D-mannosidase (Chotai et al., 1983). It is assumed that this is its mode of action in vivo. Swainsonine also affects one (Elbein et al., 1981(Elbein et al., , 1982Gross et al., 1983). In the present paper we show that swainsonine induces the storage of mannose-rich oligosaccharides and alters the processing of glycoproteins in human fibroblasts in culture. The storage products in swainsonineinduced and genetic human mannosidosis are compared. Methods ChemicalsAll reagents were of analytical grade and were obtained from BDH or Sigma, both of Poole, Dorset, U.K. Swainsonine was isolated from the plant Swainsona canescens (Benth.) A. Lee by the method of Colegate et al. (1979) Normal human fibroblasts and fibroblasts from a child with mannosidosis were grown as described previously (Burditt et al., 1978). To investigate the effect of swainsonine on the metabolism of glycoproteins, the cells were incubated in culture medium containing 100 pM-swainsonine for the required length of time. The reversal of swainsonine-induced effects was followed by replacing the swainsoninecontaining medium after the required length of time with normal medium. The monolayers of cells were

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Section: Chemicalsmentioning

confidence: 63%

Section: Chemicalsmentioning

confidence: 99%

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The storage products in genetic and swainsonine-induced human mannosidosis

Bello¹,

Dorling²,

Winchester³

1983

Biochemical Journal

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“…The last possibility was eliminated by a series of mixing experiments between normal and Chediak-Higashi cultured cell extracts (results not shown). Evidence does exist in other autosomal recessive disorders that a structural gene mutation can result in an alteration of post-translational modifications leading to subsequent activity changes (Burditt et al, 1978). Our current studies do not yet allow us to distinguish between the first two possibilities.…”

Section: Discussionmentioning

confidence: 66%

Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts

Miller¹,

Stein²,

Sundsmo³

et al. 1986

Biochemical Journal

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Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.

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“…a-D-mannosidase was assayed routinely at 37 "C by using the synthetic fluorigenic substrate, 4-methylumbelliferyl a-D-mannopyranoside (Koch-Light Laboratories, Colnbrook, Bucks, United Kingdom) with a substrate concentration of 5 mM in the reaction mixture [5]. Activity measured at pH 4.0 and pH 5.5 was defined as acidic (lysosomal) and intermediate a-D-mannosidase, respectively [6].…”

Section: Assay For A-d-mannosidasementioning

confidence: 99%

The uptake of swainsonine, a specific inhibitor of α‐D‐mannosidase, into normal human fibroblasts in culture

Chotai

Jennings

Winchester

et al. 1983

J of Cellular Biochemistry

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Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.

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Evidence that the mutant enzyme in fibroblasts of a patient with mannosidosis does not crossreact with antiserum raised against normal acidic α‐D‐mannosidase

Cited by 12 publications

References 17 publications

The storage products in genetic and swainsonine-induced human mannosidosis

The storage products in genetic and swainsonine-induced human mannosidosis

Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts

The uptake of swainsonine, a specific inhibitor of α‐D‐mannosidase, into normal human fibroblasts in culture

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