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• W1993587850 abstract "α-l-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human α-l-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant α-l-iduronidase at each of its sixN-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-β-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides andl-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion byN-glycanase unless first dephosphorylated, but it was sensitive to endo-β-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells. α-l-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human α-l-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant α-l-iduronidase at each of its sixN-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-β-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides andl-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion byN-glycanase unless first dephosphorylated, but it was sensitive to endo-β-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells. α-l-Iduronidase (EC 3.2.1.76), a lysosomal enzyme that participates in the degradation of dermatan sulfate and heparan sulfate, is deficient in the Hurler, Hurler/Scheie, and Scheie syndromes, collectively known as mucopolysaccharidosis I (1Neufeld E.F. Muenzer J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 2465-2494Google Scholar). In the absence of α-l-iduronidase, lysosomal accumulation of partially degraded glycosaminoglycans causes characteristic clinical manifestations that include corneal clouding, skeletal abnormalities, cardiovascular disease, limited joint mobility, and organomegaly. Mental retardation and death in childhood characterize the Hurler syndrome, while intelligence is normal and life span nearly so in the Scheie syndrome. There exist canine and feline forms of α-l-iduronidase deficiency, and a murine form has recently been generated by homologous recombination (2Clarke L.A. Russell C. Warrington C. Pownall S. Borowski A. Jirik E.R. Toone J. Dimmick J. Am. J. Hum. Genet. 1996; 59: A196Google Scholar). The disorders have been extensively reviewed, as have recent studies of their molecular basis (1Neufeld E.F. Muenzer J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 2465-2494Google Scholar, 3Scott H.S. Bunge S. Gal A. Clarke L.A. Morris C.P. Hopwood J.J. Hum. Mutat. 1995; 6: 288-302Crossref PubMed Scopus (166) Google Scholar). Early work in cell culture had suggested that mucopolysaccharidosis I might be amenable to enzyme replacement therapy, since exogenous enzyme could be taken up by receptor-mediated endocytosis and delivered to lysosomes (1Neufeld E.F. Muenzer J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 2465-2494Google Scholar). To provide sufficient enzyme, we isolated a stably transfected Chinese hamster ovary (CHO) 1The abbreviations used are: CHO, Chinese hamster ovary; ConA, concanavalin A; dMM, deoxymannojirimycin; endo-H, endo-N-acetylglucosaminidase H; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; PNGase-F, peptideN-glycosidase F (N-glycanase); TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; TLCK,N-tosyl-l-lysine chloromethyl ketone; HPLC, high pressure liquid chromatography; Mes, 4-morpholineethanesulfonic acid. cell line that synthesized and secreted large amounts of recombinant human α-l-iduronidase (4Kakkis E.D. Matynia A. Jonas A.J. Neufeld E.F. Protein Exp. Purif. 1994; 5: 225-232Crossref PubMed Scopus (83) Google Scholar). The secreted enzyme had properties desirable for replacement purposes, including efficient endocytosis by cultured fibroblasts through a mannose 6-phosphate-dependent system and a 5-day half-life within the cells. When used in replacement trials for the canine model of α-l-iduronidase deficiency, the recombinant human enzyme was taken up to the largest extent by liver, in lesser amounts by lung, kidney, and spleen, and little if at all by brain, cartilage, myocardium, and cornea (5Shull R.M. Kakkis E.D. McEntee M.F. Kania S.A. Jonas A.J. Neufeld E.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12937-12941Crossref PubMed Scopus (150) Google Scholar, 6Kakkis E.D. McEntee M.F. Schmidtchen A. Neufeld E.F. Ward D.A. Gompf R. Kania S. Bedolla C. Chien S.L. Shull R.M. Biochem. Mol. Med. 1996; 58: 156-167Crossref PubMed Scopus (159) Google Scholar). Similar results were found in replacement trials for the feline model of the disease (7Haskins M.E. Kakkis E.D. Wan O. Weil M.A. Aguirre G.D. Schuchman E.H. Am. J. Hum. Genet. 1995; 57: A39Google Scholar). It is not known whether this distribution is the result of accessibility of the circulating enzyme to tissues, of uptake of the enzyme by specific receptor systems, or of some combination of these factors. Overexpressing CHO cell lines have been engineered for production of a number of other soluble lysosomal enzymes (8Ioannou Y.A. Bishop D.F. Desnick R.J. J. Cell Biol. 1992; 119: 1137-1150Crossref PubMed Scopus (128) Google Scholar, 9Anson D.S. Taylor J.A. Bielicki J. Harper G.S. Peters C. Gibson G.J. Hopwood J.J. Biochem. J. 1992; 284: 789-794Crossref PubMed Scopus (61) Google Scholar, 10Bielicki J. Hopwood J.J. Wilson P.J. Anson D.S. Biochem. J. 1993; 289: 241-246Crossref PubMed Scopus (58) Google Scholar, 11Ling P. Roberts M. Biol. Reprod. 1993; 49: 1317-1327Crossref PubMed Scopus (12) Google Scholar, 12Unger E.G. Durrant J. Anson D.S. Hopwood J.J. Biochem. J. 1994; 304: 43-49Crossref PubMed Scopus (52) Google Scholar, 13Van Hove J.L.K. Yang H.W. Wu J.Y. Brady R.O. Chen Y.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 65-70Crossref PubMed Scopus (105) Google Scholar). In contrast to normal cultured cells, which secrete very little lysosomal enzyme except in the presence of NH3 or lysosomotropic amines, engineered CHO lines secrete a substantial fraction of the newly synthesized recombinant enzymes even in the absence of these weak bases (4Kakkis E.D. Matynia A. Jonas A.J. Neufeld E.F. Protein Exp. Purif. 1994; 5: 225-232Crossref PubMed Scopus (83) Google Scholar, 8Ioannou Y.A. Bishop D.F. Desnick R.J. J. Cell Biol. 1992; 119: 1137-1150Crossref PubMed Scopus (128) Google Scholar, 11Ling P. Roberts M. Biol. Reprod. 1993; 49: 1317-1327Crossref PubMed Scopus (12) Google Scholar, 13Van Hove J.L.K. Yang H.W. Wu J.Y. Brady R.O. Chen Y.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 65-70Crossref PubMed Scopus (105) Google Scholar). Such secretion occurs although the enzymes appear to have the mannose 6-phosphate signal for targeting to lysosomes, as evidenced by mannose 6-phosphate inhibition of their uptake. There is no detailed information on the structures of the carbohydrate constituents of recombinant lysosomal enzymes. We undertook an analysis of the N-linked carbohydrates of secreted α-l-iduronidase to shed light on processing by overexpressing CHO cells as well as to facilitate interpretation of enzyme uptake in vivo. Since the structure of carbohydrates at each of the six glycosylation sites (Asn residues 110, 190, 336, 372, 415, and 451 (14Scott H.S. Anson D.S. Orsborn A.M. Nelson P.V. Clements P.R. Morris C.P. Hopwood J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9695-9699Crossref PubMed Scopus (133) Google Scholar)) would be more useful for both purposes than the structure of pooled carbohydrates, we used MALDI-TOF mass spectrometry of isolated glycopeptides before and after treatment with glycosidases and phosphatase (15Huberty M.C. Vath J.E. Yu W. Martin S.A. Anal. Chem. 1993; 65: 2791-2800Crossref PubMed Scopus (176) Google Scholar, 16Sutton C.W. O'Neill J.A. Cottrell J.S. Anal. Biochem. 1994; 218: 34-46Crossref PubMed Scopus (157) Google Scholar, 17Zhao K.W. Stevens R.L. Faull K.F. Kakkis E.D. Neufeld E.F. FASEB J. 1996; 10: A1108Google Scholar). α-l-Iduronidase was collected from secretions of the overexpressing CHO cell line 2.131 and purified to apparent homogeneity as described previously (4Kakkis E.D. Matynia A. Jonas A.J. Neufeld E.F. Protein Exp. Purif. 1994; 5: 225-232Crossref PubMed Scopus (83) Google Scholar). Antiserum to this enzyme was raised in rabbits. Reagents were purchased from the following vendors: 33Pi from Amersham Corp.;l-[5,6-3H]fucose from ICN; Expre35s35s 35S protein labeling mix from NEN Life Science Products; fetal bovine serum and other tissue culture reagents from Life Technologies, Inc., except for methionine-free Dulbecco's modified Eagle's medium, which was from Biofluids; TPCK-treated trypsin and TLCK-treated chymotrypsin, bradykinin, heparin-acrylic beads, and agarose-linked lectins from Sigma; Pansorbin, Escherichia coli alkaline phosphatase, andVibrio cholerae neuraminidase from Calbiochem; recombinant endo-N-acetylglucosaminidase H (endo-H) from New England Biolabs; deoxymannojirimycin (dMM) from Genzyme; N-glycanase (PNGase-F), V. cholerae sialidase, and α-mannosidase from Oxford Glycosystems; glycopeptidase A from Seikagaku; α-cyano-4-hydroxycinnamic acid from Aldrich; sequencing grade carboxypeptidase Y and pyroglutamate aminopeptidase from Boehringer Mannheim. α-Glucosidase II was a gift from Drs. Alan Elbein and Y. Zeng of the University of Arkansas for Medical Sciences. Centricon-30 microconcentrators were from Amicon, and Sep-Pak C-18 cartridges were from Waters. The CHO cell line 2.131 was maintained in minimal essential medium-α (with nucleosides) supplemented with 10% fetal bovine serum, nonessential amino acids, 20 mm Hepes, 0.4 mg/ml Geneticin, and 1% penicillin/streptomycin, with a final pH of 6.8, at 37 °C and 5% CO2. Cells were transplanted to 6-well plates (35-mm well diameter) and used after reaching confluence. They were preincubated under similar conditions in methionine- or phosphate-free Dulbecco's modified Eagle's Medium supplemented as above except for the use of dialyzed fetal bovine serum and of 2.5 mm Hepes (starvation medium). After 30 min at 37 °C, the starvation medium in each well was replaced with 0.5 ml of medium of similar composition containing 25 μCi of 35S protein labeling mix or 25 μCi of33Pi. After 1 h of labeling, a chase was initiated by the addition of unlabeled methionine to a final concentration of 0.1 mg/ml or of unlabeled phosphate to 1 mm. Where deoxymannojirimycin was used, it was added to the medium 6 h before labeling and kept through the labeling and chase period. No change was made in medium components when labeling withl-[5,6-3H]fucose. Immunoprecipitation of α-l-iduronidase with rabbit antiserum and Pansorbin was carried out essentially as described for β-hexosaminidase (18Proia R.L. D'Azzo A. Neufeld E.F. J. Biol. Chem. 1984; 259: 3350-3354Abstract Full Text PDF PubMed Google Scholar), except that 10 mm sodium phosphate, pH 5.8, was substituted for Tris-HCl. Because of the high level of expression, the enzyme could be immunoprecipitated from the medium without prior concentration. The immunoprecipitates were dissolved in 0.1% SDS, 0.01% NaN3 and heated at 98 °C for 3 min; after centrifugation, 0.1 volume of 7.5% Nonidet P-40 and 1.5% β-mercaptoethanol were added to the supernatant fluids. The mixtures were heated again at 98 °C for 3 min and centrifuged prior to endoglycosidase digestion. Immunoprecipitates were divided into aliquots for treatment at 37 °C with one of the following: 50 milliunits of PNGase-F in 0.1 m Tris-HCl, pH 7.8; 100 units of endo-H in 0.1 m citrate buffer, pH 5.5 (the amount of enzyme was reduced to 50 units to observe glycosylation intermediates); 5 milliunits of neuraminidase in 0.1 m sodium acetate buffer, pH 4.6; or 250 milliunits of alkaline phosphatase in 0.1m Tris-HCl, pH 8.2, for the indicated period of time. The immunoprecipitates, with or without enzymatic treatment, were subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% gel (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar), and radiolabeled α-l-iduronidase was visualized by fluorography. Enzyme purified to apparent homogeneity (4Kakkis E.D. Matynia A. Jonas A.J. Neufeld E.F. Protein Exp. Purif. 1994; 5: 225-232Crossref PubMed Scopus (83) Google Scholar) was stored in 0.15 m NaCl buffered to pH 5.8 with 10 mm sodium phosphate (buffer A). To change the pH for proteolysis, 2 nmol of enzyme (140 μg of protein, excluding the weight of the carbohydrate chains) was buffer-exchanged in a Centricon-30 microconcentrator that had been pretreated overnight with 5% ethylene glycol to block nonspecific binding to the plastic wall. The exchange buffer was 100 mm Tris-HCl containing 10 mm sodium phosphate, 2 mm EDTA, and 0.02% NaN3, pH 8.2 (buffer B). After three cycles of dilution and concentration, the final protein solution in the reservoir (∼100 μl) was recovered by reverse centrifugation for 5 min. To prepare [33P]α-l-iduronidase for protease digestion, biosynthetic labeling of CHO 2.131 cells was carried out in a 100-mm plate in phosphate-free medium as described above, except for absence of fetal bovine serum during both the starvation and labeling periods. After 30 min preincubation, the medium was changed to 2 ml of the same serum-free starvation medium containing 200 μCi of 33Pi. At the end of a 6-h labeling period, the medium was collected and centrifuged, and the supernatant fluid was applied to a small column (0.8 ml) of heparin-acrylic beads prewashed with buffer A containing 0.02% NaN3. α-l-Iduronidase was eluted in fractions of 0.5 ml with 0.6 m NaCl, 10 mm sodium phosphate, pH 5.8, 0.02% NaN3. The purified radiolabeled α-l-iduronidase was concentrated and combined with 2–3 nmol of unlabeled α-l-iduronidase for further processing. The concentrated samples of α-l-iduronidase were denatured under reducing conditions by the addition of 4 μmol of dithiothreitol, 1.2 mmol of solid guanidinium HCl, and buffer B to a total volume of 200 μl. After 2 h at ambient temperature, solid iodoacetamide (18 μmol) was added, and the volume was adjusted to 0.4 ml with buffer B. After further incubation for 3 h in the dark, the samples were dialyzed overnight at 4 °C against 2 liters of 50 mm NH4HCO3, 10 mmsodium phosphate, pH 8.2. The samples were then incubated with TPCK-treated trypsin or TLCK-treated chymotrypsin for 12–16 h at 37 °C (protease to substrate ratio ∼1:50, w/w). Phenylmethylsulfonyl fluoride was added to the tryptic or chymotryptic digests to a final concentration of 1 mm; the mixture was adjusted to pH 5.8 by the addition of 1 m HCl. After the addition of CaCl2 and MgCl2 to a final concentration of 1 mm each, the digest was passed over a series of lectin columns, each having a 0.5-ml bed volume, beginning with ConA-agarose. The column was extensively washed with buffer A untilA 280 was negligible. Glycopeptides were eluted from the ConA-agarose column with 5 ml of 10 mmα-methylglucoside and 5 ml of 0.5 m α-methylmannoside in buffer A, in fractions of 0.5 ml. Unbound fractions were pooled, adjusted to pH 7.4, and passed over a column of wheat germ agglutinin-agarose joined to a column of castor bean agglutinin-agarose. After washing with buffer A, the columns were disconnected and eluted with 0.1 m N-acetylglucosamine or 0.1 m galactose in buffer A, respectively. Lectin column eluates were pooled, concentrated to about 0.5 ml in a Speedvac concentrator, and applied to a reverse phase HPLC column (Keystone Scientific betasil, 2 × 250 mm, particle size 5 μm, and pore size 100 Å). The column was developed with 0.1% trifluoroacetic acid for 10 min and eluted with a gradient of 0–50% acetonitrile, 0.1% trifluoroacetic acid for 100 min in fractions of 0.2 ml/min. All fractions were stored at 4 °C. Immediately before use, endo-H was diluted to 20 units/μl with water; PNGase-F to 10 milliunits/μl and alkaline phosphatase to 2 milliunits/μl with 50 mmNH4HCO3; α-mannosidase and sialidase to 0.2 milliunits/μl with buffers provided by the supplier; and glycopeptidase A to 0.2 milliunits/μl with 50 mm(NH4)2 H citrate. α-Glucosidase II, provided in 100 mm Mes buffer, pH 6.8, containing 0.1% Triton X-100 (20Kaushal G.P. Pastuszak I. Hatanaka K. Elbein A.D. J. Biol. Chem. 1990; 265: 16271-16279Abstract Full Text PDF PubMed Google Scholar) was diluted 300-fold with 50 mm sodium citrate buffer, pH 7.2. In each case, 1 μl of enzyme was incubated with 1 μl of HPLC fraction overnight at ambient temperature. HPLC eluates or their deglycosylation products, 1 μl, were co-spotted on a stainless steel plate with 1 μl of matrix (α-cyano-4-hydroxycinnamic acid (5 mg/ml) in water/acetonitrile/trifluoroacetic acid (50:50:0.1) containing 0.5 nmol/ml bradykinin). The bradykinin served as an internal standard for calibration. After the spots had been air-dried, each was washed for 30 s with 5 μl of water and dried again. The wash step improved the detection efficiency and resolution of the resulting signals. The dried samples were examined by laser desorption mass spectrometry using a reflection time of flight instrument in the linear mode. Mass signals were analyzed without smoothing. The measured mass of deglycosylated peptides was compared with that calculated from the deduced amino acid sequence of α-l-iduronidase cDNA (4Kakkis E.D. Matynia A. Jonas A.J. Neufeld E.F. Protein Exp. Purif. 1994; 5: 225-232Crossref PubMed Scopus (83) Google Scholar, 14Scott H.S. Anson D.S. Orsborn A.M. Nelson P.V. Clements P.R. Morris C.P. Hopwood J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9695-9699Crossref PubMed Scopus (133) Google Scholar) using the MacBiospec computer program (PE Sciex Instruments, Ontario, Canada). Amino-terminal sequence analysis was carried out on a Porton 2090E Sequencer at the UCLA Microsequencing Facility. Cleavage of amino-terminal pyroglutamate was carried out by incubating 1 μl of pyroglutamate aminopeptidase (20 ng/μl in 50 mm NH4HCO3, 5 mm EDTA, 5 mm dithiothreitol) with 1 μl of PNGase-F-treated HPLC fraction overnight at ambient temperature. Carboxyl-terminal sequence analysis was carried out by graded cleavage of the peptide with carboxypeptidase Y and determination of the mass of the resulting peptides (21Anal. Chem. 67, 369–384Patterson, D. H., Tarr, G. E., Regnier, F. E., and Martin, S. A. Anal. Chem. , 67, 369–384.Google Scholar). Equal volumes (0.5 μl) of carboxypeptidase Y (serially diluted in (NH4)2H citrate to give 150–0.3 μg/μl) and PNGase-F-treated HPLC fractions were mixed on the steel plate. After incubation for 2 h at 37 °C in a sealed Petri dish humidified with wet cotton wads, the reaction was stopped, and the samples were crystallized by the addition of 1 μl of 10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid. After the wash step as above, the peptides were analyzed by MALDI-TOF mass spectrometry. Susceptibility of radiolabeled α-l-iduronidase to cleavage by endo-H and PNGase-F provided information on the ratio of high mannose to complex oligosaccharides. Newly made intracellular enzyme differed from secreted enzyme in that the former was completely cleaved by endo-H (Fig. 1 A, lanes 1and 3), whereas secreted enzyme was partially resistant (Fig. 1 A, lanes 7 and 9). The carbohydrate chains not removed from the secreted enzyme by endo-H could be cleaved, although not completely, by PNGase-F (Fig.1 A, lanes 11 and 12); a possible explanation for the incomplete hydrolysis by PNGase-F will be provided below. Only after the labeling had been performed in the presence of dMM, an inhibitor of the Golgi α-mannosidase I and hence of the mannose trimming required for complex sugar formation (22Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (348) Google Scholar), were the migration and endo-H susceptibility of the secreted enzyme equal to those of newly made intracellular enzyme (Fig. 1 A,lanes 2, 4, 8, and 10). Thus, the difference is due to complex oligosaccharides on the secreted enzyme. Intermediate deglycosylation steps were made visible by partial endo-H digestion. Treatment of newly made intracellular α-l-iduronidase with a reduced amount of endo-H gave a ladder of six distinct bands, not including the starting material (Fig.1 B, left panel), showing that all six sites were of the high mannose type. Similar treatment of the secreted enzyme with endo-H resulted in only 3 or 4 bands (Fig. 1 B, right panel), indicating that two or three sites had been processed to the complex form. The bands derived from the secreted enzyme were diffuse and poorly resolved, although the sample had been pretreated with neuraminidase to reduce heterogeneity due to different degrees of sialylation. The diffuse appearance and poor resolution of the bands are attributed to incomplete desialylation and/or glycoform heterogeneity of secreted α-l-iduronidase. When the secreted α-l-iduronidase was biosynthetically labeled with 33P, all of the phosphate label could be removed by treatment with endo-H (Fig.2 A, lanes 1–4), indicating that it was associated with the carbohydrate and not with the protein. The deglycosylated protein itself could be visualized with [35S]methionine labeling (Fig. 2 A, lanes 7 and 8). The secreted enzyme became strongly labeled after continuous exposure of the cell to 33Pifor 6 and 10 h (Fig. 2 B, lanes 3 and4), but the intracellular enzyme contained a surprisingly low level of radioisotope at all times (Fig. 2 B, lanes 1 and 2), suggesting that it was dephosphorylated after delivery to lysosomes. On the other hand, l-[3H]fucose was incorporated only into endo-H-resistant, PNGase-F-sensitive oligosaccharides of secreted α-l-iduronidase (Fig.2 C), showing that it was present solely onN-linked complex oligosaccharides. Fig.3, A and C, shows the HPLC profile of α-l-iduronidase glycopeptides produced by tryptic and chymotryptic digestion and isolated by α-methylmannoside elution from ConA-agarose; Fig. 3,C and D, show the corresponding radioactivity profile of 33P-labeled α-l-iduronidase glycopeptides similarly treated with trypsin and chymotrypsin, respectively. MALDI-TOF analysis of HPLC fractions 69–70 of the tryptic digest (Fig.3 A) gave a prominent signal at m/z3802.8. This signal disappeared when the fractions were treated with endo-H or PNGase-F and was replaced by signals ofm/z 2306.2 or 2103.7, respectively. This decrease in mass resulted from the loss of carbohydrate, the structure of which could be deduced from the magnitude of the decrease (TableI). The carbohydrate itself was not detected, since it does not crystallize with the matrix and/or fails to protonate under the conditions used. Treatment of fractions 69–70 with α-mannosidase produced no change in mass, whereas treatment with alkaline phosphatase resulted in a prominent signal atm/z 3644.7. The changes in mass observed upon treatment with endo-H, PNGase-F, and phosphatase were compatible with the structure P2Man7GlcNAc2 (Table I). The33P radiolabel in fractions 69–70 (Fig. 3 B) confirmed the phosphorylated structure of the oligosaccharide. Some minor signals could also be seen in the intact sample, ofm/z 3638.9, 3556.7, and 3390.1, compatible with structures P2Man6GlcNAc2, P1Man6GlcNAc2, and P1Man5GlcNAc2; these accounted for ∼6%, 6%, and 2% of the total, respectively. Amino- and carboxyl-terminal sequence analyses as well as the mass of the deglycosylated glycopeptide identified it as Val-325 to Tyr-343 (TableII). The phosphorylated high mannose oligosaccharide was therefore assigned to Asn-336.Table IExamples of structure assignment based on reduction in mass after enzyme treatmentFractionsTreatmentMass [M + H]+Residues removed−Δ massObservedCalculated69–70None3804.8α-Mannosidase3804.0None0.80Phosphatase3644.72P160.1160.0Endo-H2306.2P2Man7GlcNAc1498.61497.9PNGase-F2103.7P2Man7GlcNAc21701.11701.166None3530.7Phosphatase3530.2None0.50α-Mannosidase2557.76Man973.0972.6Endo-H1867.3Man9GlcNAc1663.41662.1PNGase-F1664.9Man9GlcNAc21865.81865.3None3693.4Phosphatase3693.2None0.20α-Glucosidase II3530.5Glc162.9162.1α-Mannosidase3368.52Man324.9324.2Endo-H1867.3GlcMan9GlcNAc1826.11824.2PNGase-F1664.9GlcMan9GlcNAc22028.52027.4Residue mass used for calculations: Glc and Man, 162.1; GlcNAc, 203.2; P, 80.0. Open table in a new tab Table IISummary of glycopeptidesPeptidesMassProteaseHPLC fractionConA elutionEndo-H cleavageComments[M + H]+ obs[M + H]+ cal110(R)GLSYDFTHLDGYLDLL(R)1842.71843.1Try84Me-Glc−a(L)SYDFTHL(D)882.6883.0Chy60 –61Me-Glc−(L)SYDFTHL(D)884.8883.0Chy57 –58Me-ManND190(W)NEPDHHDFDDVSMTM(Q)1791.61789.7Chy58Me-Glc−b(W)NEPDHHDFDDVSMTMQGF(L)2122.92123.3(W)NEPDHHDFDDVSM*TMQGF(L)2137.82139.3(W)NEPDHHDFDDVSM*TM(Q)1806.41806.9Chy54Me-Glc−(W)NEPDHHDFDDVSM*TM*(Q)1822.71822.9336(K)VIAQHQNLLLADTTSAFPY(A)2103.72103.4Try69 –70Me-Glc−(K)VIAQHQNLLLADTTSAFPY(A)2102.92103.4Try69 –70Me-Man+c(L)LLADTTSAFPY(A)1200.01199.3Chy61 –62Me-Man+372(R)FQVDNTRPPHVQLL(R)1664.81664.9Try66Me-Man+d(R)FQVDNTRPPHVQLLR(K)1819.71821.1Try61 –62Me-Man(F)pEVDNTRPPHVQ(L)1275.01274.4Chy37 –41Me-Man+(F)QVD NTRPPHVQL(L)1404.71404.6Chy50 –51Me-Man+(F)pEVDNTRPPHVQL(L)1387.31387.6+e(F)QVDNTRPPHVQLL(R)1517.41517.7Chy59 –62Me-Man+(F)pEVDNTRPPHVQLL(R)1500.61500.7+415(W)AEVSOAGTVLDSDHTVGVL(A)1897.91899.1Chy61 –62Me-Man+(T)VLDSD …Chy58Me-Glcf451(Y)ASDDTRAHPDRSVAV(T)1597.01597.7Chy39 –41Me-Man+(Y)ASDDTRAHPDRSVAVT(L)1699.31698.8Chy37 –38Me-Man+(Y)ASDDTRAHP DRSVAVTL(R)1810.81812.0Chy50 –51Me-Man+g(Y)ASDDTRAHPDRSVAVTL(R)1811.91812.0Chy51 –52Me-Glc−/+hDeglycosylated peptides were prepared by PNGase-F digestion, which converts asparagine residues at the carbohydrate-protein junction to aspartic, with a net gain of 1 Da. The junction Asp residues are numbered. Experimentally determined amino acid sequences are underlined (amino terminus) or italicized (carboxyl terminus). Adjacent amino acids in the intact α-l-iduronidase are shown in parentheses. pE, pyroglutamic; M*, oxidized methionine. ND, not determined.2-aGlycopeptide data in Fig. 7.2-b Found in mixture with Asn-415 peptide.2-c Glycopeptide data in Table I.2-d Glycopeptide data in Fig. 4 and Table I.2-e Glycopeptide data in Fig. 5; mixture with Asn-451 glycopeptide.2-fPeptide seen in mixture with Asn-190 peptide by amino-terminal sequence analysis; not seen in mass spectrometry.2-gGlycopeptide data in Fig. 5; mixture with Asn-372 glycopeptide.2-hEndo-H-cleavable oligosaccharide same as that of methylmannoside-eluted glycopeptide. Open table in a new tab Residue mass used for calculations: Glc and Man, 162.1; GlcNAc, 203.2; P, 80.0. Deglycosylated peptides were prepared by PNGase-F digestion, which converts asparagine residues at the carbohydrate-protein junction to aspartic, with a net gain of 1 Da. The junction Asp residues are numbered. Experimentally determined amino acid sequences are underlined (amino terminus) or italicized (carboxyl terminus). Adjacent amino acids in the intact α-l-iduronidase are shown in parentheses. pE, pyroglutamic; M*, oxidized methionine. ND, not determined. 2-aGlycopeptide data in Fig. 7. 2-b Found in mixture with Asn-415 peptide. 2-c Glycopeptide data in Table" @default.
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