Invitro correction of deficient human fibroblasts by β-glucuronidase from different human sources

Brot, Frederick E.; Glaser, J H; Roozen, Kenneth J.; Sly, William S.; Stahl, Philip D.

doi:10.1016/s0006-291x(74)80349-9

Cited by 99 publications

(45 citation statements)

References 15 publications

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“…f3-Glucuronidase was partially purified by antibody affinity chromatography as previously described (9). Typically, a 2 X 107 unit (nmol/hr) batch of this preparation in 3 M urea and 0.01 M Tris-HCI at pH 8 was loaded at 40 on a 2.5 X 22 cm DEAE-Sephadex column, which had been pre-equilibrated with this buffer.…”

Section: Mostmentioning

confidence: 99%

“…One hundred milliliters of a platelet lysate from which over 85% of the f-glucuronidase had been removed by batch treatment with antibody-Sepharose (9) was freed of 0-glucuronidase by passage over a 10-ml antibody-Sepharose column (0.9-cm diameter) at a flow rate of 0.5 ml/min. The effluent was applied to a 10-ml concanavalin-A-Sepharose column (0.9-cm diameter) (Pharmacia) at a rate of 0.5 ml/min.…”

Section: Mostmentioning

confidence: 99%

“…(iii) that high-uptake forms are particularly abundant in extracts of blood platelets (9); (iv) that the high-uptake platelet enzyme is converted intracellularly to the less acidic low-uptake forms following pinocytosis (8). In this paper, specific competitive inhibition of enzyme pinocytosis by certain yeast mannans, sugar phosphates, and sugars is demonstrated, as is the destruction of the uptake activity of high-uptake enzyme by treatment with alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1].…”

mentioning

confidence: 99%

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Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Kaplan¹,

Achord²,

Sly³

1977

Proc. Natl. Acad. Sci. U.S.A.

538

208

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Human fl-glucuronidase (fl-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "highuptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than, slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. iD-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1-phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2-and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent K is estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10-6 M; mannan from wild-type S. cerevisiae, 3 X 10-5 M; D-mannose 6-phosphate, 6 X 10-5 M; L-fucose, 4 X 10-2 M; and D-mannose, 6 X 10-2 M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet fl-glucuronidase abolished its "high-ptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.Pinocytosis of lysosomal enzymes, identified by Neufeld and coworkers (1) as uptake of corrective factors by enzyme-deficient fibroblasts, displays the selectivity and saturability expected for a receptor-mediated process (2). Several investigators (3-5) have studied kinetic aspects of this process with enzymes from different sources. Hickman and Neufeld (6) suggested that many lysosomal hydrolases have similar components that are essential for their recognition and uptake by human fibroblasts. This suggestion was based on the observation that fibroblasts from patients homozygous for a single gene mutation (I-cell disease) secrete several hydrolases which are not specifically pinocytosed (6). The finding that periodate treatment of fihexosaminidase secreted by normal fibroblasts destroyed its uptake activity led to the suggestion that the "recognition component" on the enzyme contains carbohydrate (7).We have utilized fl-glucuronidase (f3-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) to study this pinocytosis process. Previously published studies have shown (i) that human f,-glucuronidase from all tissue sources investigated exhibits charge heterogeneity (8); (ii) that high-uptake forms, which are specifically pinocytosed by fibroblasts, are more acidic than the low-uptake, or poorly ...

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Section: Mostmentioning

confidence: 99%

Section: Mostmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Kaplan¹,

Achord²,

Sly³

1977

Proc. Natl. Acad. Sci. U.S.A.

538

208

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“…The velocities needed to separate the various organelles differ significantly from those used in liver cells [23], and indicate different apparent densities of the subcellular particles in the two tissue sources.…”

Section: C'dl Distxp T Iorimentioning

confidence: 91%

“…Human platelet /~-glucuronidase which is readily taken up by fibroblasts [23,24] was used in these studies. Incubation of 1500 U fi-glucuronidase per flask for 48 h resulted in an approximately lo",, recovery of the added enzyme within the cells.…”

Section: Up Fuke Suhcclltilui Disrrihu Lion Ciiid Lcircwc:r Of' E~mentioning

confidence: 99%

Intracellular Localization of Exogenous β‐Glucuronidase in Cultured Skin Fibroblasts

Bach

Liebmann‐Eisenberg

1979

European Journal of Biochemistry

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The intracellular localization of exogenously supplied human platelet β‐glucuronidase in cultured skin fibroblasts derived from a β‐glucuronidase‐deficient patient was studied. Four cellular fractions were obtained by differential speed centrifugation. Following two days of incubation, the exogenously supplied enzyme exhibited a distribution pattern identical to that of endogenous β‐hexosaminidase. Disruption of membranes by freezing and thawing caused a 35% increase of the enzyme activity, thus indicating a latent activity following the internalization. This indicated localization in the lysosomal fractions. Longer incubation periods led to an intracellular shift of the engulfed enzyme from the lighter lysosomal fraction to heavier particles. Once located in the heavier fraction, the enzyme was relatively stable, and participated in the catabolism of 35S‐labeled mucopolysaccharides which had accumulated in the lysosomes of these fibroblasts. A marked reduction in the accumulated mucopolysaccharides of the lysosomal fraction was observed following addition of the enzyme. This was accompanied by the formation of smaller sized molecules.

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The Phosphomannosyl Recognition System for Intracellular and Intercellular Transport of Lysosomal Enzymes

Sly

1982

J of Cellular Biochemistry

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364

152

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Invitro correction of deficient human fibroblasts by β-glucuronidase from different human sources

Cited by 99 publications

References 15 publications

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Intracellular Localization of Exogenous β‐Glucuronidase in Cultured Skin Fibroblasts

The Phosphomannosyl Recognition System for Intracellular and Intercellular Transport of Lysosomal Enzymes

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