Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 2026-2030, May 1977

Cell Biology

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts (jB-glucuronidase/glycoproteins/phosphoproteins/phosphohexoses/storage disease)

ARNOLD KAPLAN*, DANIEL T. ACHORDt, AND WILLIAM S. SLYt t * Department of Microbiology, St. Louis University, St. Louis, Missouri 63104; and t Division of Medical Genetics, Department of Pediatrics, Washington University, St. Louis, Missouri 63110

Communicated by Oliver H. Lowry, February 22, 1977

ABSTRACT 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 6phosphate. 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 pinocytosed, forms of the enzyme (8);

(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]. These results suggest that the "recognition component" on human platelet f-glucuronidase contains phosphohexose that is essential to its high uptake and contributes to its acidic properties. MATERIALS AND METHODS Most reagents were purchased from Sigma Chemical Co., St. Louis, MO. Mannans from mutant Saccharomyces cerevtsiae were generously supplied by Clinton Ballou. Homogeneous Escherichia coli alkaline phosphatase was a gift of Milton Schlesinger. This enzyme contained no detectable hydrolytic activity toward the following substrates: p-nitrocatechol sulfate, bis(p-nitrophenyl)phosphate, and 4-methylumbelliferyl derivatives of f3-D-galactose, f3-N-acetyl-D-glucosamine, and a-D-mannose. Blood platelets were gifts of the American Red Cross Blood Bank, St. Louis, MO. Orosomucoid was the gift of the American Red Cross Laboratory in Bethesda, MD. Asialoand agalacto-orosomucoid were prepared chemically by the methods of Spiro (10). Chromatography of Human Platelet ,BGlucuronidase. 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. After the ion-exchanger was washed with three column volumes of 0.01 M Tris at pH 8, a linear gradient (0-0.3 M NaCl, in 0.01 M Tris at pH 8, 850 ml) was applied. Fractions were eluted at 0.4 ml/min and assayed for (3-glucuronidase (11), A280, conductivity, and "uptake ratio" (defined below). The fractions that eluted between 0.065 M and 0.28 M were pooled, concentrated by ultrafiltration, and stored at 40. These ,B-glucuronidase preparations (purified 2000- to 4000-fold from the original platelet lysate) had specific activities of approximately 5 X 105 units/mg and uptake ratios of 0.25-0.5. Pinocytosis Measurements. Cultured fibroblasts were established from skin biopsies obtained from patient J.E. with f3-glucuronidase deficiency mucopolysaccharidosis (available as cell strain GM151 from the Human Mutant Cell Repository, Camden, NJ). Fibroblasts were grown in Eagle's minimal essential medium with Earle's salts (Gibco) supplemented with 15% heat-inactivated fetal calf serum and 3 mM glutamine. To

Abbreviation: HPG, human platelet glycoproteins. t To whom reprint requests should be addressed.

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Cell Biology:

Kaplan et al.

Proc. Natl. Acad. Sci. USA 74 (1977) D

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Table 1. Effect of sugars on pinocytosis of human platelet B-glucuronidase by fibroblasts

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Pinocytosis of g-glucuronidase

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

Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 2026-2030, May 1977 Cell Biology Phosphohexosyl components of a lysosomal enzyme are recognized by pi...
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