Isolation and Characterization of Phosphorylated Oligosaccharides from α‐<i>N</i>‐Acetylglucosaminidase that Are Recognized by Cell‐Surface Receptors

130

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-p-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated frm -hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to a-N-acetylglucosamine residues linked to mannose 6phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Manal,6-(Manal,3)Manal,f(Manal,3)ManBGlcNAc and either one or two al,21linked mannose residues.Lysosomal enzymes share an oligosaccharide recognition marker, which plays an important role in the transfer of these enzymes into lysosomes.(1). In 1977 Kaplan et al. (2) suggested, primarily on the basis of competition experiments, that phosphorylated mannose residues were the principal component of the recognition marker of f3-glucuronidase. The same conclusion was reached for other enzymes and in other laboratories (3)(4)(5). Later on phosphate groups were found in several lysosomal glycoproteins (6-10) and were localized on carbon 6 of mannose (7-9).To study the structure of the oligosaccharide bearing the phosphorylated group, we used procedures developed (9, 11) to isolate mannose 6-phosphate residues from lysosomal enzymes. We labeled cultured human fibroblasts in the presence of NH4Cl, collected the secretions, immunoprecipitated precursors of f,-hexosaminidase and of cathepsin D, and isolated the oligosaccharides that were cleaved by endo-f3-N-acetylglucosaminidase H. We have observed that the phosphate groups are to a large extent buried in a diester with N-acetylglucosamine and that the oligosaccharides may contain up to three such phosphodiester residues.During the preparation of this manuscript Tabas and Kornfeld reported that (3-glucuronidase from mouse lymphoma cells contains high-mannose oligosaccharides with phosphate residues blocked by N-acetylglucosamine (12).MATERIALS AND METHODS Labeling and Immunoprecipitation of Lysosomal Enzymes. Human skin fibroblasts were maintained at 370C in 5% CO2 in Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 10% fetal calf serum (Flow Laboratories, Bonn, Federal Republic of Germany) as described (13). Conditions of labeling cells with [2-3H]mannose and 32P, and immunoprecipitation of f3-hexosaminidase and cathepsin D were those described (11) with the following modifications. The labeling medium was prepared without glucose and supplemented with 10 mM NH4Cl and with 0.5% fetal calf serum that had been incubated at pH 10.4 for 30 min at 37°C to destroy acid hydrolases. NH4Cl has been included in the medium to increase the yield of secreted precursors of lysosomal enzymes (11). Labe...

supporting

confidence: 57%

Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups.

Hasilík¹,

Klein²,

Waheed³

et al. 1980

130

“…Similar results on the effects of endoglycosidase H on enzyme uptake have been seen with 3-galactosidase from bovine testes (G. W. Jourdian, personal communication), a-L-iduronidase (R. Myerowitz and E. F. Neufeld, personal communication), and a-N-acetylglucosaminidase (32) from human urine and 3-hexosaminidase from fibroblast secretions (unpublished data). Furthermore, von Figura and Klein recently showed release of acidic oligosaccharides from a-Nacetylglucosaminidase by endoglycosidase H treatment and conversion of the oligosaccharides to neutral species by phosphatase treatment (32). Thus, the Man6P in the common recognition marker for uptake of acid hydrolases by fibroblasts (5) appears to be present on endoglycosidase H-sensitive structures on all of these enzymes.…”

Section: Discussionmentioning

confidence: 95%

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Natowicz¹,

Maggie²,

Lowry³

et al. 1979

208

Human fB-glucuronidase (fl-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycopr tein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in P-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of fl-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme. Furthermore, the mannose 6-phosphate content of the enzyme varies directly with its susceptibility to pinocytosis by fi roblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enz me b treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms,.that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.

“…The column was washed successively with 3 ml ofwater, 6 ml of0.1 M HCl, 6 ml of 0.2 M HCI, and 2 ml of 0.5 M HCL. The mannose 6-phosphate released from the oligosaccharides by hydrolysis was eluted in the 0.2 M HClfraction.…”

Section: Methodsmentioning

confidence: 99%

“…This variant form of pseudo Hurler polydystrophy demonstrates the biological importance of this recognition mechanism in the generation of the phosphomannosyl marker. Acid hydrolases are a heterogeneous group of proteins that acquire a common phosphomannosyl recognition marker which is responsible for targeting them to their common destination, the lysosomes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). This posttranslational modification of the high-mannose-type oligosaccharides of lysosomal enzymes is generated by the sequential action of two enzymes: UDP-Nacetylglucosamine:lysosomal enzyme N-acetylglucosamine-lphosphotransferase [referred to herein as N-acetylglucosaminylphosphotransferase (GlcNAcPlase)] and a-N-acetylglucosaminyl phosphodiesterase.…”

mentioning

confidence: 99%

Identification of a variant of mucolipidosis III (pseudo-Hurler polydystrophy): a catalytically active N-acetylglucosaminylphosphotransferase that fails to phosphorylate lysosomal enzymes.

Varki¹,

Reitman²,

Kornfeld³

1981

Fibroblasts from patients with I-cell disease (mucolipidosis H) or with pseudo Hurler polydystrophy (mucolipidosis EII) are markedly deficient in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-l-phosphotransferase. As a consequence, the common phosphomannosyl recognition marker ofacid hydrolases is not generated, and these enzymes are not targeted to lysosomes. We have developed a sensitive assay for the transferase that uses a-methyl mannoside as an acceptor, and this has allowed us to distinguish between fibroblasts from these two types of patients. The enzyme activity is less in the former than in the latter (<0.4-2.0 pmol/mg per hr vs 2.9-39.4). This may provide an explanation for the difference in clinical severity, between the two syndromes. However, in two siblings with pseudo Hurler polydystrophy (GM 3391 and GM 3392), the enzyme activity was normal when assayed by using a-methyl mannoside as acceptor whereas it was low when assayed with endogenous glycoprotein acceptors or with human placental &-hexosaminidase A. The apparent Km values of the mutant enzyme toward a-methyl mannoside, high-mannose oligosaccharides, and UDP-GlcNAc were not different from those of the normal enzyme. Mixing experiments demonstrated that the mutant fibroblasts contained endogenous acceptors and were free ofinhibitors. We conclude that the N-acetylglucosaminylphosphotransferase in the mutant fibroblasts has normal catalytic activity but is defective in the ability to recognize lysosomal enzymes as specific substrates for phosphorylation. This variant form of pseudo Hurler polydystrophy demonstrates the biological importance of this recognition mechanism in the generation of the phosphomannosyl marker. Acid hydrolases are a heterogeneous group of proteins that acquire a common phosphomannosyl recognition marker which is responsible for targeting them to their common destination, the lysosomes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). This posttranslational modification of the high-mannose-type oligosaccharides of lysosomal enzymes is generated by the sequential action of two enzymes: UDP-Nacetylglucosamine:lysosomal enzyme N-acetylglucosamine-lphosphotransferase [referred to herein as N-acetylglucosaminylphosphotransferase (GlcNAcPlase)] and a-N-acetylglucosaminyl phosphodiesterase. The first enzyme catalyzes the transfer ofGlcNAc 1-phosphate to the 6 position ofmannose residues on high-mannose-type oligosaccharides; the second enzyme removes the outer GlcNAc residues to generate a phosphomonoester (12-18). We have recently shown that fibroblasts from patients with I-cell disease (mucolipidosis TI) or pseudo Hurler polydystrophy (mucolipidosis III) are markedly deficient in the first enzyme of this sequence (19). This explains why both diseases are characterized by decreased intracellular activities of many lysosomal enzymes and markedly increased levels of the same enzymes in the body fluids. Similar findings have been reported by Hasilik et al (20).In this report, we describe a variant form of pseudo Hurler ...