Multiple isoelectric and recognition forms of human β-glucuronidase activity

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

doi:10.1016/0003-9861(75)90417-8

Cited by 79 publications

(30 citation statements)

References 14 publications

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“…5). Furthermore, "mature" lysosomal enzymes in organ extracts have little or no M6P residues (33). Hex A and Hex B in our preparation are not significantly taken up by MPRC' rich cultured skin fibroblasts (data not shown).…”

Section: Discussionmentioning

confidence: 69%

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Rattazzi¹

1991

J. Clin. Invest.

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Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen-or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental jl-hexosaminidase and partially purified bovine liver ,j-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. colifl-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle. (J. Clin. Invest.

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Section: Discussionmentioning

confidence: 69%

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Rattazzi¹

1991

J. Clin. Invest.

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“…We cannot exclude the possibility that the low-uptake form consists of a small amount of high-uptake enzyme in combination with a "no-uptake" form. High-and low-uptake forms of human a-N-acetylglucosaminidase (6), amannosidase A (6), a-L-iduronidase (5), and fl-glucuronidase (22) have been reported by others. We cannot exclude the existence of a ,B-hexosaminidase form with greater affinity for the uptake mechanism than that of the high-uptake form we observed.…”

Section: Discussionmentioning

confidence: 80%

Excretion-Reuptake Route of β-Hexosaminidase in Normal and I-Cell Disease Cultured Fibroblasts

Vladutiu¹,

Rattazzi²

1979

J. Clin. Invest.

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A B S T R A C T It has been proposed that in cultured fibroblasts the final packaging of enzymes in lysosomes re(luires excretion followed by pinocytosis by neighboring cells via a carbohydrate-specific receptor mechanism. It has also been proposed that the abnormally high activity of lysosomal enzymiies in the medium of cultured fibroblasts from patients with I-cell disease (mucolipidosis II) results from an altered carbohydrate recognition residue on the enzymes which prevents reuptake into the cells. With 8-hexosaminidase as a marker, and competitive inhibition of uptake by 2 mM mannose-6-phosphate, we have determined that only 12% of the total (intra-and extracellular) B-hexosaminidase in normal fibroblasts is channeled through the excretion-reuptake route. After 9 d of exposure to mannose-6-phosphate, normal fibroblast cultures accumulated in the medium only a fraction of the enzyme excreted by I-cell disease fibroblasts in the same period. Furthermore, this minimal loss of enzyme to the medium did not result in a decrease of intracellular enzyme activity. Finally, if the defect in I-cell disease were only because of an impairment of a reuptake mechanism that involves only 12% of the total enzyme, then 88% of the newly synthesized enzyme should be retained by I-cell fibroblasts, resulting in intracellular activity three to nine times higher than that which is observed. These data are consistent with our previous proposal that excessive lysosomal enzyme activity in the medium of I-cell disease fibroblasts results from preferential exocytosis.

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“…The effect of alkaline phosphatase treatment on the isoelectric focusing pattern of high-uptake human platelet f-glucuronidase was studied in polyacrylamide gels. Samples were focused for 3 hr in gels (3.4% acrylamide) containing 6 M urea and 1.25% Ampholine (pH 6-8) as described by Owens et al (13) and stained for /3-glucuronidase activity as described previously (8). Inhibition of enzyme pinocytosis by sugar phosphates The inhibition of enzyme pinocytosis by several phosphorylated sugars was much more dramatic than the inhibition by sugars.…”

Section: Mostmentioning

confidence: 99%

“…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 pinocytosed, forms of the enzyme (8);…”

mentioning

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].…”

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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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Multiple isoelectric and recognition forms of human β-glucuronidase activity

Cited by 79 publications

References 14 publications

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Excretion-Reuptake Route of β-Hexosaminidase in Normal and I-Cell Disease Cultured Fibroblasts

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

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