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 (iii) that high-uptake forms are particularly abundant in ex- glucuronosohydrolase, EC 3.2.1.31), like many other glycopro- tracts of blood platelets (9); (iv) that the high-uptake platelet tein lysosomal hydrolases, is specifically taken up from the is converted intracellularly to the less acidic low-uptake culture medium by human fibroblasts. Prior work has indicated enzyme that the enzyme exhibits charge heterogeneity and that "high- forms following pinocytosis (8). In this paper, specific com- uptake" forms, i.e., those rapidly internalized by human fibro- petitive inhibition of enzyme pinocytosis by certain yeast blasts, are more acidic than, slowly internalized forms. Here we , phosphates, and is demonstrated, as is present two lines of evidence that the acidic group required for the destruction of the uptake activity of high-uptake enzyme the high-uptake property of certain forms of the enzyme is a by treatment with alkaline phosphatase [orthophosphoric- phosphate on, or in proximity to, a D--type carbohy- optimum), EC 3.1.3.1]. drate. The first line of evidence was obtained from analysis of monoester phosphohydrolase (alkaline inhibition of enzyme pinocytosis by yeast mannans, phospho- These results suggest that the "recognition component" on rylated sugars, and sugars. Mannans that contained phosphate human platelet f-glucuronidase contains phosphohexose that were more potent inhibitors than those that did not contain is essential to its high uptake and contributes to its acidic phosphate. iD-Mannose 6-phosphate was a more potent inhibitor properties. than either D-mannose 1-phosphate or 2-deoxy-D- 6- phosphate. D-Mannose and certain related sugars were weak MATERIALS AND METHODS pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and Most reagents were purchased from Sigma Chemical Co., St. the apparent K is estimated for the following compounds: Sac- Louis, MO. Mannans from mutant Saccharomyces cerevtsiae charomyces cerevisiae mannan from mutant X2180-mnnl, 3 were generously supplied by Clinton Ballou. Homogeneous 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-, 4 X 10-2 M; and Escherichia coli alkaline phosphatase was a gift of Milton D-mannose, 6 X 10-2 M. The second line of evidence comes from Schlesinger. This enzyme contained no detectable hydrolytic the observation that alkaline phosphatase [orthophosphoric- activity toward the following substrates: p-nitrocatechol sulfate, monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] bis(p-nitrophenyl)phosphate, and 4-methylumbelliferyl de- treatment of human platelet fl-glucuronidase abolished its rivatives of f3-D-, f3-N-acetyl-D-glucosamine, and "high-ptake" activity, without diminishing its catalytic activity, a-D-mannose. Blood platelets were gifts of the American Red and converted some forms of the heterogeneous enzyme to less Cross Blood Bank, St. Louis, MO. Orosomucoid was the gift of acidic forms. the American Red Cross Laboratory in Bethesda, MD. Asialo- Pinocytosis of lysosomal enzymes, identified by Neufeld and and agalacto-orosomucoid were prepared chemically by the coworkers (1) as uptake of corrective factors by enzyme-defi- methods of Spiro (10). cient fibroblasts, displays the selectivity and saturability ex- Chromatography of Human Platelet ,BGlucuronidase. pected for a receptor-mediated process (2). Several investigators f3-Glucuronidase was partially purified by antibody affinity (3-5) have studied kinetic aspects of this process with enzymes chromatography as previously described (9). Typically, a 2 X from different sources. Hickman and Neufeld (6) suggested that 107 unit (nmol/hr) batch of this preparation in 3 M urea and many lysosomal hydrolases have similar components that are 0.01 M Tris-HCI at pH 8 was loaded at 40 on a 2.5 X 22 cm essential for their recognition and uptake by human fibroblasts. DEAE-Sephadex column, which had been pre-equilibrated This suggestion was based on the observation that fibroblasts with this buffer. After the ion-exchanger was washed with three from patients homozygous for a single gene mutation (I-cell column volumes of 0.01 M Tris at pH 8, a linear gradient (0-0.3 disease) secrete several hydrolases which are not specifically M NaCl, in 0.01 M Tris at pH 8, 850 ml) was applied. Fractions pinocytosed (6). The finding that periodate treatment of fi- were eluted at 0.4 ml/min and assayed for (3-glucuronidase (11), hexosaminidase secreted by normal fibroblasts destroyed its A280, conductivity, and "uptake ratio" (defined below). The uptake activity led to the suggestion that the "recognition fractions that eluted between 0.065 M and 0.28 M were pooled, component" on the enzyme contains (7). concentrated by ultrafiltration, and stored at 40. These ,B-glu- We have utilized fl-glucuronidase (f3-D-glucuronide glucu- curonidase preparations (purified 2000- to 4000-fold from the ronosohydrolase, EC 3.2.1.31) to study this pinocytosis process. original platelet lysate) had specific activities of approximately Previously published studies have shown (i) that human f,- 5 X 105 units/mg and uptake ratios of 0.25-0.5. glucuronidase from all tissue sources investigated exhibits Pinocytosis Measurements. Cultured fibroblasts were es- charge heterogeneity (8); (ii) that high-uptake forms, which tablished from skin biopsies obtained from patient J.E. with are specifically pinocytosed by fibroblasts, are more acidic than f3-glucuronidase deficiency mucopolysaccharidosis (available the low-uptake, or poorly pinocytosed, forms of the enzyme (8); as cell strain GM151 from the Human Mutant Cell Repository, Camden, NJ). Fibroblasts were grown in Eagle's minimal es- Abbreviation: HPG, human platelet glycoproteins. sential medium with Earle's salts (Gibco) supplemented with t To whom reprint requests should be addressed. 15% heat-inactivated fetal calf serum and 3 mM glutamine. To 2026 Downloaded by guest on September 30, 2021 Cell Biology: Kaplan et al. Proc. Natl. Acad. Sci. USA 74 (1977) 2027 Table 1. Effect of sugars on pinocytosis of human platelet O 0 D B-glucuronidase by fibroblasts ol 6 Pinocytosis of z x g-glucuronidase z 0 u 6 _ 03 _ Rate* Inhi- L x >- a (units/ bition C E Added z :2 compound (100 mM) mg hr) (%) o c u < 03 cr None Lu - O 1 w - 0 2- 117+ 7 J:, Y O ° u < 1 D-Mannose 80 3 32 D Z) L-Fucose 72 + 1 38 20 40 60 80 100 120 ce-Methyl-D-mannoside 78 + 3 33 FRACTION NUMBER (5 ml) D- 80 + 4 32 FIG. 1. DEAE-Sephadex chromatography of partially purified D- 71 + 1 39 human platelet f-glucuronidase. Uptake activity is the product of the D-Mannoheptulose 78 + 2 33 3-glucuronidase concentration and the uptake ratio. The uptake ratio D- 66 + 1 44 was determined by measuring, as described in Materials and Meth- 2-Amino-2-deoxy-D-mannose 39 + 2 67 ods, the fraction of 0-glucuronidase that was cell-associated after a D- 92 + 1 21 20-hour at incubation 370 with 400 units of added f-glucuronidase. D-Galactose 116 + 3 1 N-Acetylneuraminic acid 121 ± 5 0 determine pinocytosis rates for f-glucuronidase, duplicate 35 N-Acetyl-2-amino-2-deoxy-D-glucose 126 + 10 0 mm petri dishes containing cells at confluence (approximately N-Acetyl-2-amino-2-deoxy-D-galactose 114 + 2 3 0.2 mg of protein per plate) were exposed to indicated con- N-Acetyl-2-amino-2-deoxy-D-mannose 108 + 17 8 centrations of enzyme in 1 ml of this medium. After a 2 hr in- D-Glucoset 120 + 3 0 cubation at 370, dishes were chilled in ice and washed six times * Rate measurements are described in Materials and Methods. 03- with 3-ml portions of ice-cold phosphate-buffered saline. The Glucuronidase concentration was 1000 units/ml. cells were lysed with 0.5 ml of 1% sodium deoxycholate and the t No detectable inhibition by 0.1 M concentrations of the following lysates were assayed in duplicate for cell protein (12) and f- was observed: mannitol, D-fucose, L-lyxose, L- glucuronidase activity. Results are reported in the tables as the , D-, and a- or f-methyl-D-galactose. 0.1 M D- mean of duplicates plus or minus the mean deviation and in the arabinose and 0.1 M myo-inositol inhibited pinocytosis less than 20%. figures as the mean of duplicates. Uptake Ratio. The uptake ratio of f-glucuronidase samples was determined by measuring, as described above, the fraction the rate seen for low-uptake forms. When incubated with this of added f3-glucuronidase that was cell-associated after a 24-hr pooled high-uptake fraction for several days, deficient human incubation at 370 with 400 units of added ,B-glucuronidase. fibroblasts accumulated enzyme to concentrations exceeding #-Glucuronidase-Deficient Human Platelet Glycoproteins 100 times the level of enzyme in normal human fibroblasts. The ("fl-Glucuronidase-Free HPG"). One hundred milliliters of a pinocytosis rate at 40 was 7% that at 370. The rate of fluid pi- platelet lysate from which over 85% of the f-glucuronidase had nocytosis ([3H]methoxydextran uptake) was less than 0.5% the been removed by batch treatment with antibody-Sepharose (9) rate of enzyme pinocytosis. The enzyme pinocytosis rate was was freed of 0-glucuronidase by passage over a 10-ml anti- constant for at least 6 hr even at the low enzyme concentrations body-Sepharose column (0.9-cm diameter) at a flow rate of 0.5 employed here for inhibitor studies. ml/min. The effluent was applied to a 10-ml concanavalin- Inhibition of enzyme pinocytosis by A-Sepharose column (0.9-cm diameter) (Pharmacia) at a rate sugars of 0.5 ml/min. The column was washed with three volumes of Table 1 presents data comparing the inhibition of specific en- 0.01 M Tris at pH 8. Glycoproteins were eluted at room tem- zyme pinocytosis by sugars added to the medium. Conforma- perature with 20 ml of 0.75 M a-methyl-D-mannoside con- tional features common to the first nine sugars include taining 0.025 M EDTA at pH 8, dialyzed against 0.1 M NaCl, ring structures, axial 2-hydroxyl, and equatorial 4-hydroxyl and concentrated by ultrafiltration. Ten milligrams of glyco- moieties, or their equivalents. Closely related sugars that lack protein, containing less than 10 units 3-glucuronidase per mg pyranose ring structure (mannitol, myo-inositol), that lack the of protein, was obtained. axial 2-hydroxyl (D-glucose), that lack the equatorial 4-hydroxyl Polyacrylamide Gel Isoelectric Focusing. The effect of (D-talose), or that contain a substituted 2 position (N-acetyl- alkaline phosphatase treatment on the isoelectric focusing 2-amino-2-deoxy-D-mannose) show weak or insignificant in- pattern of high-uptake human platelet f-glucuronidase was hibition. Lack of inhibition by the other sugars tested may be studied in polyacrylamide gels. Samples were focused for 3 hr explained by the absence of one or more of the critical features in gels (3.4% acrylamide) containing 6 M urea and 1.25% common to the first nine sugars. Ampholine (pH 6-8) as described by Owens et al. (13) and Caution is required in interpreting the inhibitory effects of stained for /3-glucuronidase activity as described previously such high concentrations of sugars. On prolonged exposure, 0.1 (8). M D-mannose and 0.1 M L-fucose markedly inhibit growth of fibroblasts, indicating that such concentrations disturb many RESULTS metabolic processes. While these general toxic effects could Specific pinocytosis of ,B-glucuronidase explain some of the effects of sugars on enzyme pinocytosis, 0.1 M D-mannose and 0.1 M L-fucose did not detectably alter the Fig. 1 presents a chromatogram showing the separation of f- rate of fluid endocytosis ([3H]methoxydextran uptake) or iso- glucuronidase forms on DEAE-Sephadex. The pooled high- leucine transport for up to 3 hr. Similar studies have not yet uptake forms (0.065-0.28 M NaCl) are internalized at 250 times been done with the other inhibitory sugars. Downloaded by guest on September 30, 2021 2028 Cell Biology: Kaplan et al. Proc. Natl. Acad. Sci. USA 74 (1977)

Table 2. Effect ofsugar phosphates on enzyme pinocytosis Table 3. Effect of mannans and glycoproteins on enzyme pinocytosis Pinocytosis of j3-glucuronidase Pinocytosis of ,-glucuronidase Added compound Rate* Inhibition (0.5 mM) (units/mg hr) (%) Added mannan or protein Ratet Inhibition (1 mg/ml)* (units/mg hr) (%) None 132 + 5 D-Man-6-P 25 + 1 81 None 136+ 9 D-Fru-1-P 39 + 2 70 Mannan (wild type, Sigma) 64 + 2 53 D-Gal-6-P 108 4 18 Mannan (X2180-mnnl) 33 +4 76 D-Glc-6-P 97 +10 27 Mannan (X2180-mnn2) 109 2 20 2-Deoxy-D-Glc-6-P 127 + 4 4 Mannan(X1280-mnn4) 110 10 19 D-Fru-6-P 113 +3 14 ,-Glucuronidase-free HPGt 72 + 7 47 D-Fru-1,6-P2 96 +3 27 Phosvitin, 113 3 17 D -Sedoheptulose-1, 7-P2 109+ 1 17 Thyroglobulin 131 ± 9 4 2-Deoxy-D-Rib-5-P 121 + 1 8 Human immunoglobulin G 169 ± 2 0 Orosomucoid 143 ± 1 0 * * See footnote in Table 1. Asialo-orosomucoid 142 ± 4 0 Agalacto-orosomucoid 163 ± 4 0 Inhibition of enzyme pinocytosis by sugar phosphates Ovalbumin 150 ± 3 0 The inhibition of enzyme pinocytosis by several phosphorylated * Final concentration except for j3-glucuronidase-free HPG (0.2 sugars was much more dramatic than the inhibition by sugars. mg/ml), asialo-orosomucoid (0.7 mg/ml), agalacto-orosomucoid (1.1 Table 2 presents data showing that D-Man-6-P and D-Fru-1-P mg/ml). are potent inhibitors of specific pinocytosis at concentrations t See footnote * in Table 1. f,-Glucuronidase-free HPG refers to ,B-glucuronidase-free human 200-fold lower than inhibitory concentrations of D-mannose platelet glycoproteins eluted from concanavalin-A-Sepharose as and D-fructose (Table 1). The weaker inhibition by D-Glc-6-P described in Materials and Methods. and negligible effects of 2-deoxy-D-Glc-6-P are compatible with the requirement for an axial 2-hydroxyl for maximal in- hibition. The weak inhibition by D-Fru-6-P compared to D- Competitive inhibition by D-mannose, L-fucose, and Fru-1-P again suggests the requirement for two appropriately phosphomannosyl-compounds positioned carbohydrate oxygens. Although the fructose Pinocytosis by both normal and enzyme deficient fibroblasts phosphates are predominantly in the f- forms, the is a saturable function of f,-glucuronidase concentration. Fig. relative positions of the 1-P, the anomeric oxygen, and the 4- 2 presents data showing competitive inhibition for several oxygen of D-Fru-1-P are equivalent to the positions of the 6-P, compounds. Thus, a common binding site for these inhibitors the 4-oxygen, and the 2-oxygen, respectively, of D-Man-6-P. and f3-glucuronidase seems plausible. However, the possibility This correspondence does not hold for D-Fru-6-P and is also that the inhibitors selectively interfere with internalization absent in D-Man-1-P, which was 100-fold less potent as an in- rather than binding to the pinocytosis receptor has not been hibitor than D-Man-6-P. The reasons for the weaker inhibition excluded. The apparent Kis for inhibitors were estimated to be: by D-Fru-1,6-P2 and D-sedoheptulose-1,7-P2 are not yet D-mannose, 6 X 10-2 M; L-fucose, 4 X 10-2 M; D-Man-6-P, 6 clear. X 10-5 M; mannan (wild type), 3 X 10-5 M; mannan (mnnl), The striking inhibition by 0.5 mM D-Man-6-P is not likely to be due to a general toxic effect on the cells, because cell growth was not detectably inhibited by including 1 mM D- Man-6-P in the growth medium. Inorganic phosphate was a weak inhibitor of enzyme pinocytosis (50% inhibition at 0.05 M) compared to D-Man-6-P.

Inhibition by glycoproteins Table 3 presents data on the effects of various glycoproteins on f3-glucuronidase pinocytosis. Two yeast mannans that contain phosphate (14), S. cerevissze wild-type mannan, and mannan from a mutant lacking terminal 1 -- 3-mannosyl moieties (mnnl), were potent inhibitors, as was ,B-glucuronidase-free HGP. Mannans that lack dimannosylphosphate (mnn4 and - mnn2) or lack both dimannosylphosphate and the 1 2- -2 -1 0 1 2 3 4 5 mannosyl branches from the 1 -> 6 linear polymannose outer /J3-GLUCURONIDASE CONCENTRATION chain (mnn2) were weakly inhibitory. Several other glyco- (ml /unit x 104) proteins that do not contain phosphate, but have FIG. 2. Double reciprocal plots of the effects offl-glucuronidase components terminating in sialic concentration on enzyme pinocytosis by human fibroblasts in the acid, D-galactose, N-acetyl- absence and presence of inhibitors. The pinocytosis measurements 2-amino-2-deoxy-D-glucose, or D-mannose, did not inhibit. were as described for those in Table 1 except that the indicated con- Phosvitin, which contains many phosphates as phosphoserine centrations of enzyme and inhibitor were used. 0, No additions; *, moieties, and also has an oligosaccharide side chain (15), was 0.1 M D-Man; A, 0.1 M L-Fuc; O, 2.5 MM mnnl mannan; *, 25 IAM a poor inhibitor. wild-type mannan (Sigma Chemical Co.); 0, 300MM D-Man-6-P. Downloaded by guest on September 30, 2021 Cell Biology: Kaplan et al. Proc. Natl. Acad. Sci. USA 74 (1977) 2029

x 4

E "I IM 3

Zn-S V) 2\ 0

01

CL

0 0.1 0.2 0.3 ALKALINE PHOSPHATASE CONCENTRATION (pmol/min ml) FIG. 3. Effect of alkaline phosphatase concentration on the de- gree of inactivation of uptake activity of human platelet f3-glucu- ronidase. To 0.1 ml aliquots of human platelet ,3-glucuronidase (3 X i0-5 units/ml) in 0.15 M NaCl and 0.01 M Tris.HCl at pH 8, 0.01 ml of alkaline phosphatase in the same buffer was added to give the in- dicated phosphatase concentrations. The mixtures were incubated for 1 hr at 370, then diluted to 5 ml with ice-cold minimal essential medium with Earle's salts supplemented with 15% heat-inactivated fetal calf serum and 3 mM glutamine. Duplicate 1 ml aliquots of the diluted mixture were assayed for fl-glucuronidase and pinocytosis rates were determined as described in Table 1 and Materials and Methods except that the fl-glucuronidase concentration in the me- A B C dium added to cells was 6000 units/ml. D

3 X 10-6 M. The Km for 3-glucuronidase was 4000 units/ml (approximately 6 X 10-9 M) and Vmax, 600 units/mg per hr. Effect of alkaline phosphatase treatment on high- FIG. 4. Effect of alkaline phosphatase treatment on polyacryl- amide gel isoelectric focusing (pH 6-8) of ,B-glucuronidase. High- uptake platelet fl-glucuronidase uptake platelet f-glucuronidase (3 X 105 units/ml) was treated with As indicated above, the sensitivity of the uptake activity of alkaline phosphatase at 1 unit (jmol per min)/ml for 4 hr at 370 at pH hexosaminidase to periodate treatment led others to suggest 8, after which 8 M urea was added to bring samples to 6 M urea. The samples were chilled to 40, and applied to gels. Gels A and C contained carbohydrate as a portion of the recognition component of the 2830 and 8490 units of alkaline-phosphatase-treated 0-glucuronidase. enzyme. The studies presented here suggested phosphate in- Gel B received 2830 units of ,B-glucuronidase from a mixture similar volvement also. To test this hypothesis, we studied the effect to that from which A and C were prepared except that urea was added of alkaline phosphatase on the uptake activity and electro- before the alkaline phosphatase and the sample was chilled imme- phoretic properties of human (3-glucuronidase. Treatment with diately. Gel D received 8490 units of ,B-glucuronidase from an incu- 1 unit (,umol/min)/ml at 370 for 1 hr at pH 8 destroyed over bation exactly like that from which gels A and C were prepared except 85% of the that alkaline phosphatase was omitted. The bottoms of the gels are uptake activity of ,B-glucuronidase without affecting anodal. For conditions of gel preparation, isoelectric focusing, and its catalytic activity. The alkaline phosphatase effect was in- ,B-glucuronidase staining, see Materials and Methods. hibited by phosphate. When 1 mM Na2HPO4 was present during the alkaline phosphatase treatment, no loss in uptake implicated the carbohydrate portion of lysosomal activity was observed. Fig. 3 demonstrates the effect of different enzymes in alkaline their specific pinocytosis by fibroblasts. The competitive inhi- phosphatase concentrations on the uptake activity of bition by phosphates and by phosphoglycoproteins, to- f-glucuronidase. The pinocytosis of untreated enzyme was not gether with the effects of alkaline phosphatase, support the detectably altered by addition of comparable concentrations argument for a critical phosphomonoester at the of alkaline phosphatase to the uptake medium (which contains recognition 1 site. The simplest interpretation of these data is that the phos- mM P0). Prolonged treatment of fl-glucuronidase with alkaline phate monoester is present as a phosphohexosyl residue on the phosphatase [20 hr with 1 unit (,umol/min)/ml at pH 8] com- glycoprotein hydrolase. One may infer certain other structural pletely destroyed its uptake activity, again without diminishing features of the presumed phosphohexosyl moiety from the its catalytic activity. Fig. 4 shows that after treatment of the relative potency of inhibitors. Evidence for the proximity of electrophoretically heterogeneous fl-glucuronidase with alkaline the phosphate and the phosphatase, the isoelectric focusing pattern was changed, in- equatorial 4-oxygen or its equivalent is dicating conversion of several forms of the enzyme to less acidic provided by the potency of the inhibition of D-Man-6-P relative forms. to D-Man-l-P (100 to 1) and of D-Fru-1-P relative to D-Fru-6-P, and by the lack of inhibition by the 4-epimer of D-mannose. The requirement for the 2-axial oxygen or its equivalent is DISCUSSION suggested by weak inhibition by D-Glc-6-P and 2-deoxy-D- Human f3-glucuronidase is a glycoprotein, like most mamma- Glc-6-P compared to D-Man-6-P and by lack of inhibition by lian lysosomal enzymes (16). Work by others had indirectly D-glucose in contrast to D-mannose. Downloaded by guest on September 30, 2021 2030 Cell Biology: Kaplan et al. Proc. Natl. Acad. Sci. USA 74 (1977) Relatively few examples of glycoproteins containing phos- research which is the subject of the article. This article must therefore phohexose have been reported. The possibility of such glyco- be hereby marked "advertisement" in accordance with 18 U. S. C. proteins was raised by the discovery of UDP-GlcNAc-P-Gal §1734 solely to indicate this fact. in hen oviduct (17). Yeast mannan-proteins provide a well- 1. Neufeld, E. F. & Cantz, M. J. (1971) Ann. N.Y. Acad. Sci. 179, documented example. The phosphate linkage in yeast mannans 580-587. was shown by Ballou and coworkers (14) to be a phosphodiester 2. Neufeld, E. F., Lim, T. W. & Shapiro, L. J. (1975) Annu. Rev. in a mannosyl-l-phospho-6-mannosyl linkage. Some forms of Biochem. 44, 357-375. hemoglobin were recently reported to contain D-Glc-6-P di- 3. von Figura, K. & Kresse, H. (1973) J. Clin. Invest. 53,85-90. rectly linked to NH2-terminal amino acids (18). Human platelet 4. Wiesmann, U. N. & Herschkowitz, N. N. (1974) Pediat. Res. 8, f3-glucuronidase adds a lysosomal enzyme to the short list of 865-870. glycoproteins containing phosphohexose. 5. Lagunoff, D., Nicol, D. M. & Pritzl, P. (1973) Lab. Invest. 29, There is a possibility that phosphohexose is present in the 449-453. 6. Hickman, S. & Neufeld, E. F. (1972) Biochem. Biophys. Res. recognition site of high-uptake forms of 3-glucuronidase from Commun. 49, 992-999. other sources, as well as in the recognition site of other corrective 7. Hickman, S., Shapiro, L. J. & Neufeld, E. F. (1974) Biochem. hydrolases. Preliminary studies with hexosaminidase from Biophys. Res. Commun. 57,55-61. platelets and from fibroblast secretions suggest that the pino- 8. Glaser, J. H., Roozen, K. J., Brot, F. E. & Sly, W. S. (1975) Arch. cytosis of human hexosaminidase by fibroblasts is mediated by Biochem. Biophys. 166,536-542. the phosphohexose recognition reported for 3-glucuronidase. 9. Brot, F. E., Glaser, J. H., Roozen, K. J., Sly, W. S. & Stahl, P. D. Studies of other hydrolases should indicate whether this is the (1974) Biochem. Biophys. Res. Commun. 57, 1-8. postulated recognition component on most high-uptake forms 10. Spiro, R. G. (1966) in Methods in Enzymology, eds. Neufeld, E. of lysosomal hydrolases that is thought to be defective or masked F. & Ginsberg, V. (Academic Press, New York), Vol. 8, in pp. 26-52. hydrolases from I-cell disease patients (6). 11. Glaser, J. H. & Sly, W. S. (1973) J. Lab. Clin. Invest. 82, 969- Hieber et al. (19) recently suggested that the internalization 977. of high-uptake f-galactosidase from bovine testes by human 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. fibroblasts is mediated by a different receptor than that which (1951) J. Biol. Chem. 193,265-275. mediates uptake of f3-glucuronidase. Studies of the effects of 13. Owens, J. W., Gammon, K. L. & Stahl, P. D. (1975) Arch. Bio- D-Man-6-P on this pinocytosis system, and of the sensitivity of chem. Biophys. 166,258-272. the high-uptake enzyme to alkaline phosphatase, may clarify 14. Ballou, C. E. & Raschke, W. C. (1974) Science 184, 127-134. the relationship of that pinocytosis system to the one described 15. Shainkin, R. & Perlmann, G. E. (1971) J. Biol. Chem. 246, here. Several other systems for uptake and clearance of mam- 2278-2284. malian have been described or 16. Bouma, J. M. W. (1974) in Enzyme Therapy in Lysosomal glycoproteins suggested. These Storage Diseases, eds. Tager, J. M., Hooghwinkel, G. J. M. & include the well-characterized hepatocyte receptor for asialo- Daems, W. Th. (North-Holland/American Elsevier), pp. 197- glycoproteins (20), a less-well-characterized system in avian (21) 206. and mammalian liver (22) for clearance of agalacto-glyco- 17. Suzuki, S. (1962) J. Biol. Chem. 237, 1393-1399. proteins, and a putative system for clearance of mannosyl- 18. Haney, D. N. & Bunn, H. F. (1976) Proc. Natl. Acad. Sci. USA terminal glycoproteins (23, 24). The proposed pinocytosis re- 73,3534-3538. ceptor on fibroblasts that recognizes phosphohexosyl moieties 19. Hieber, V., Distler, J., Myerowitz, R., Schmickel, R. D. & on high-uptake forms of lysosomal hydrolases represents a Jourdian, G. W. (1976) Biochem. Biophys. Res. Commun. 73, recognition system different from any previously reported. 710-717. 20. Ashwell, G. & Morell, A. G. (1974) in Advances in Enzymology, The authors gratefully acknowledge the able technical assistance eed. Meister, A. (John Wiley and Sons, New York), Vol. 41, of David Fischer and the suggestions of Dr. Frederick Brot and Marvin pp. 99-128. Natowicz. Dr. C. J. Bellone kindly assisted us with isoelectric focusing 21. Lunney, J. & Ashwell, G. (1976) Proc. Natl. AMad. Sci. USA 73, in slab gels, and Dr. Philip Stahl and Ms. Jane Rodman assisted us with 341-343. the polyacrylamide disc gel isoelectric focusing. The research was 22. Stockert, R. J., Morell, A. G. & Scheinberg, I. H. (1976) Biochem. supported by grants from the U.S. Public Health Service (GM 20196 Biophys. Res. Commun. 68,988-993. and GM 1511), and the Ranken Jordan Trust for Crippling Diseases 23. Winkelhake, J. L. & Nicolson, G. L. (1976) J. Biol. Chem. 251, of Children. 1074-1080. The costs of publication of this article were defrayed in part by the 24. Baynes, J. W. & Wold, F. (1976) J. Biol. Chem. 251, 6016- payment of page charges from funds made available to support the 6024. Downloaded by guest on September 30, 2021