Human {alpha}-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Masaya Ohta, Tomomi Ohnishi, Yiannis A. Ioannou2, Mark E. Hodgson2, Fumito Matsuura and Robert J. Desnick1,2

Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729–0292, Japan, 2Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, NY 10029–6574

Received on June 24, 1999; revised on August 30, 1999; accepted on September 9, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human {alpha}-N-acetylgalactosaminidase ({alpha}-GalNAc; also known as {alpha}-galactosidase B) is the lysosomal exoglyco­hydrolase that cleaves {alpha}-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (~53%), hybrid (~12%), and high mannose-type (~33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (~53%). Approximately 80% of the complex oligo­saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with {alpha}-2,3-linked sialic acid residues. The majority of hybrid type oligo­saccharides were GalGlcNAcMan6GlcNAc­­Fuc0–­­1Glc­NAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man5-7GlcNAc2 were the predominant species (~90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of {alpha}-GalNAc and {alpha}-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that {alpha}-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.

Key words: recombinant human {alpha}-N-acetylgalactosaminidase/N-glycosylation/Chinese hamster ovary cells/human {alpha}-galactosidase A


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human {alpha}-N-acetylgalactosaminidase ({alpha}-GalNAc: EC. 3.3.1.49) is the lysosomal hydrolase that cleaves {alpha}-N-acetylgalactosaminyl residues from various glycolipids, O- and N-glyco­proteins and keratan sulfate type II (Dean et al., 1977Go; Schram et al., 1977Go; Desnick and Wang, 1995Go). In the early 1970s, this activity was originally designated {alpha}-galactosidase B ({alpha}-Gal B) based on its ability to cleave the artificial water soluble substrate, 4-methylumbelliberyl {alpha}-D-galactopyranoside (Kint, 1971Go; Beutler and Kuhl, 1972aGo; Wood and Nadler, 1972Go; Desnick et al., 1973Go). About 80–90% of total {alpha}-galacto­sidase activity in human tissue extracts was due to {alpha}-galacto­sidase A ({alpha}-Gal A), the lysosomal hydrolase deficient in Fabry disease (Beutler and Kuhl, 1972aGo,b; Ho et al., 1972Go; Wood and Nadler, 1972Go; Desnick et al., 1973Go), while the remaining 10–20% of activity was designated {alpha}-Gal B. Treatment with neuraminidase resulted in the electrophoretic comigration of both {alpha}-Gal A and {alpha}-Gal B, suggesting that both enzymes were the result of differential glycosylation (Kint, 1971Go; Ho et al., 1972Go; Wood and Nadler, 1972Go; Romeo et al., 1975Go). However, subsequent kinetic, inhibitor, immunologic, and gene mapping studies demonstrated that {alpha}-Gal A and B were genetically distinct lysosomal enzymes with different substrate specificities. {alpha}-Gal A, encoded by an X-linked gene, was a true {alpha}-galactosidase, whereas {alpha}-Gal B, encoded by a gene localized to chromosome 22q13-qter, was actually an {alpha}-N-acetyl­galactosaminidase (Dean et al., 1977Go; Schram et al., 1977Go; Dean and Sweeley, 1979Go).

In 1987, the deficient activity of {alpha}-GalNAc was shown to be the metabolic defect in Schindler disease (van Diggelen et al., 1987Go), an autosomal recessive disorder with severe infantile and milder adult forms that are characterized by the increased excretion of oligosaccharides and glycopeptides containing {alpha}-N-acetylgalactosaminyl moieties (van Diggelen et al., 1987Go; Linden et al., 1989Go; Schindler et al., 1989Go; Hirabayashi et al., 1990Go; Schindler et al., 1990Go; Kanzaki et al., 1991Go; Desnick and Wang, 1995Go).

The 3.6 kb full-length human {alpha}-GalNAc cDNA sequence was isolated, found to encode a precursor peptide of 411 amino acids including a 17-residue signal peptide (Tsuji et al., 1989Go; Wang et al., 1990Go). Interestingly, the predicted mature {alpha}-GalNAc amino acid sequence had remarkable identity with that encoded by human {alpha}-Gal A (46.9%) gene. Moreover, the structural organization of the human and mouse {alpha}-GalNAc and {alpha}-Gal A genes, with introns interrupting the exons at identical positions in both species indicated that these genes evolved from a common ancestral gene (Wang and Desnick, 1991Go; Herrmann et al., 1998Go; Wang et al., 1998Go). Of note, six putative N-glycosylation consensus sites (N124, 177, 201, 359, 385, and 391) were present in the human {alpha}-GalNAc amino acid sequence, four of which were conserved in identical positions in human and murine {alpha}-Gal A (Tsuji et al., 1989Go; Wang et al., 1990Go). When compared with the mouse and chicken {alpha}-GalNAc cDNA sequences (Zhu and Goldstein, 1993Go; Herrmann et al., 1998Go; Wang et al., 1998Go), only three N-glycosylation sites were conserved among the these species, suggesting that human residues N177, N201 and N385 were site occupied and important for enzyme synthesis, function, and/or stability. Recent site-specific mutagenesis studies of the four homologous human {alpha}-Gal A N-glycosylation sites revealed that the first three (N139, N192, and N215) were glycosylated (Ioannou et al., 1998Go), indicating that the human {alpha}-GalNAc N124 site may be occupied. However, the site occupancy of the six potential N-glycosylation sites has not been reported.

Recently, human {alpha}-GalNAc and {alpha}-Gal A were individually overexpressed in Chinese hamster ovary (CHO) cells in which the respective human cDNA had been stably amplified (Ioannou et al., 1992Go). This provided milligram quantities of secreted recombinant human {alpha}-GalNAc, permitting isolation and structural analysis of its N-linked oligosaccharides. In a previous paper, the structures of the N-linked oligosaccharides of the recombinant cellular and secreted forms of human {alpha}-Gal A were characterized (Matsuura et al., 1998Go). Mutagenesis studies of the human {alpha}-GalNAc cDNA demonstrated glycosylation at the first five sites, N124, N177, N201, N359, and N385. Site occupancy at site 3 (N201) was important for enzyme function and stability. In this communication, the site occupancy and N-linked oligosaccharide structures of secreted recombinant human {alpha}-GalNAc were determined and compared with those of secreted recombinant human {alpha}-Gal A. All of the potential N-glycosylation sites were occupied in both enzymes with the exception of each C-terminal site. Glycosylation of site 3 in {alpha}-GalNAc (N201) and {alpha}-Gal A (N215) was critical for the activity and stability of each enzyme. Both enzymes had complex, high mannose, and hybrid-type oligosaccharide structures that were similarly phosphorylated. However, human {alpha}-GalNAc had more complete sialyl oligosaccharide structures whereas the complex oligosaccharides of {alpha}-Gal A were incompletely galactosylated and sialylated. These studies provide the detailed characterization of a fully processed overexpressed recombinant secreted lysosomal enzyme.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human {alpha}-GalNAc subunits contain five N-linked oligosaccharides
Site-directed mutagenesis of the {alpha}-GalNAc cDNA was used to eliminate each of the six putative N-glycosylation sites by altering the asparagine residues to glutamine. Each mutagenized {alpha}-GalNAc cDNA construct was expressed in COS-1 cells which were metabolically radiolabeled with [35S]methionine, and the expressed {alpha}-GalNAc glycoforms were immunoprecipitated, resolved by SDS-PAGE, and visualized by radiography as shown in Figure 1. Of the six single site mutant constructs, N124({Delta}GS-1), N177({Delta}GS-2), N201({Delta}GS-3), N359({Delta}GS-4), and N385({Delta}GS-5), each expressed enzyme subunits with increased mobility compared to the wild type enzyme, consistent with the loss of a single N-linked oligosaccharide chain. In contrast, the N391({Delta}GS-6) cDNA construct expressed a glycoform whose subunit had the same mobility as the wild type, indicating that N391 was not occupied (Figure 1).



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Fig. 1. Cos-1 cells transfected with various mutant constructs were metabolically labeled with [35S]methionine. At 48 h after transfection, {alpha}-GalNAc was immunoprecipitated from cell lysates. The immunoprecipitates were analyzed by SDS/PAGE and autoradiography as previously described (Ioannou et al., 1998Go). Lanes 1–6, immunoprecipitates from COS-1 cells expressing constructs {Delta}GS-1 through {Delta}GS-6 (N124Q, N177Q, N201Q, N359Q, N385Q, and N391Q, respectively); wt, the wild type construct; and mock, COS-1 cells transfected with the vector only.

 
The intracellular enzyme activities expressed by each mutant construct were determined at 72 h following transfection (Table I). The {Delta}GS-2, {Delta}GS-4, and {Delta}GS-6 glycoforms had intracellular activities that ranged from 48 to 73% of the intracellular wild type activity. Thus, individual elimination of the oligosaccharide at sites 2, 4, or 6 did not markedly impair enzyme activity. Although the {Delta}GS-6 glycoform was fully glycosylated, it had slightly decreased intracellular activity (~73 % of wild type), presumably due to the asparagine to glutamine substitution which made the enzyme slightly less active and less stable at lysosomal pH. In contrast, the {Delta}GS-1, {Delta}GS-3, and {Delta}GS-5 glycoforms had only 35, 28, and 44% of the wild type intracellular activity levels, respectively.


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Table I . Specific activity and stability of the {alpha}-GalNAc glycosylation mutants
 
The {Delta}GS-2 and {Delta}GS-3 mutant glycoforms were markedly unstable at 42°C and pH 4.4 for 1 hr, retaining only 25 and 14% of initial activity, respectively. In contrast, the fully glyco­sylated {Delta}GS-6 glycoform retained ~86% of initial activity after terminal inactivation. Notably, the {alpha}-GalNAc {Delta}GS-3 mutant protein had the lowest activity and stability, analogous to the findings for the homologous {Delta}GS-3 (N215) in human {alpha}-Gal A which resulted in an insoluble and inactive enzyme protein that was localized to the endoplasmic reticulum where it was presumably degraded (Ioannou et al., 1998Go).

Fractionation of the ABEE-oligosaccharides from recombinant {alpha}-GalNAc by charge
Human recombinant {alpha}-GalNAc was overexpressed in CHO cells as described previously (Ioannou et al., 1992Go) and the N-linked oligosaccharides from the purified, secreted glycoforms were analyzed. The N-linked oligosaccharide mixture was released from the secreted recombinant human {alpha}-GalNAc by hydrazinolysis, followed by re-N-acetylation, and then was subjected to HPLC on a TSKgel DEAE-5PW column after derivatization with p-aminobenzoic ethyl ester (ABEE). As shown in Figure 2, the sample was separated into a neutral (denoted N, 46.1% of the total) and four acidic fractions A (21.3%), B (17.1%), C (8.4%), and D (7.1%). Fraction A was eluted in the position identical to monosialyl ABEE-oligosaccharides (Ohta et al., 1990Go) and completely converted into neutral components by the treatment with Salmonella typhimurium {alpha}2,3-sialidase, indicating that the acidic nature was due to the presence of a single {alpha}2,3-linked sialic acid residue. This fraction was designated SI. Fraction B was eluted in the position identical to monophosphoryl ABEE-oligosaccharides obtained from human recombinant {alpha}-Gal A (Matsuura et al., 1998Go), and was completely converted to neutral components upon alkaline phosphatase digestion, indicating the presence of a single phosphate moiety in the monoester form. This fraction was designated PI. Fraction C was eluted at the position corresponding to the disialyl ABEE-oligosaccharide (Ohta et al., 1990Go). When digested with {alpha}2,3-sialidase, fraction C gave two peaks with the same positions as neutral- (75%) and monophosphoryl ABEE-oligosaccharides (25%). The latter was converted into neutral components by incubation with alkaline phosphatase. These results indicated that this fraction was composed of the oligosaccharides carrying two {alpha}2,3-linked sialic acid residues (designated SII) and oligosaccharides carrying one {alpha}2,3-linked sialic acid and one phosphate residue in monoester form (designated SP). When fraction D was digested with {alpha}2,3-sialidase, ~20% of the fraction was converted to neutral components but the rest of the fraction was unchanged and converted to neutral components by alkaline phosphatase treatment. On the basis of these results, together with the elution position on the DEAE column (Matsuura et al., 1998Go), fraction D was found to contain oligosaccharides having three {alpha}2,3-linked sialic acid residues (designated SIII) and oligosaccharides having two phosphate residues in the monoester form (designated PII).



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Fig. 2. Ion-exchange HPLC of ABEE-oligosaccharides from recombinant secreted human {alpha}-GalNAc. N-linked oligosaccharides were released from {alpha}-GalNAc by hydrazinolysis and then tagged with ABEE. The ABEE-oligosaccharides were applied to a column (0.75 x 7.5 cm) of TSKgel DEAE-5PW and were eluted isocratically with 10 mM NaH2PO4 for 10 min and then with a linear gradient of the same buffer to 170 mM NaH2PO4 over 40 min at a flow rate 0.5 ml/min. The indicated fractions were pooled. Arrows indicate the elution positions of authentic ABEE-derivatives of (1) neutral, (2) monosialyl, (3) monophosphoryl, (4) disialyl, (5) trisialyl, and (6) diphosphoryl oligosaccharides.

 
Subfractionation of the neutral ABEE-oligosaccharides by affinity chromatography
To isolate individual ABEE-oligosaccharides, fraction N was first subjected to HPLC on a Concanavalin A (Con A) column. The neutral oligosaccharides were separated into flow-through (denoted NF), weakly-bound (NW), and bound (NB) fractions in a percent ratio of 13.5:40.1:46.4.

Structural analysis of the oligosaccharides in fraction NF
As shown in Table II, methylation analysis of the fraction NF was shown to contain C-2,4-, C-2,6-, and C-3,6-disubstituted mannose residues at the branching points, consistent with the fraction containing mainly tri- and tetraantennary complex-type oligosaccharides. The abundance of terminal galactose and terminal fucose residues suggested that these complex-type oligosaccharides had mature outer chains, and had a fucose residue linked to the reducing terminal N-acetyl­glucosamine (core-region fucose). Subsequent fractionation of fraction NF resulted in 10 new peaks, denoted NFa-NFj (Figure 3). The elution positions of peaks NFe, NFf, NFg, NFh, and NFj on the 2D-map coincided with those of authentic ABEE-oligosaccharides with, 2,4-branched tri-, 2,6-branched tri-, fucosylated 2,4-branched tri-, fucosylated 2,6-branched tri-, and fucosylated tetra-antennary structures, respectively (Matsuura et al., 1992Go). Serial enzymatic digestion with {alpha}-L-fucosidase, ß-galactosidase, ß-N-acetylhexosaminidase followed by jack bean {alpha}-mannosidase gave results supporting the structures proposed above by their chromatographic behaviors (for example, the results for the most abundant oligosaccharide, NFh, are shown in Figure 4). The elution positions of other minor peaks suggested that they were fucosylated complex-type oligosaccharide with outer chains having N-acetylgluco­samine at the nonreducing termini (Ohta et al., 1991Go; Matsuura et al., 1992Go). 2D-mapping analysis of fractions NFa, NFb, NFc, NFd, and NFi before and after digestion with {alpha}-L-fucosidase followed by ß-galactosidase treatment indicated that NFa was a fucosyl 2,4-branched triantennary type having one galactose residue at the nonreducing end, NFb was fucosyl 2,6-branched triantennary type having one galactose, and NFc, NFd, and NFi were fucosyl tetraantennary types having one, one, and three galactose residues, respectively. NFc and NFd, which had the same backbone structure and one galactose residue, presumably were resolved by the different locations of their respective galactose residue which remains to be determined. These results, together with methylation analysis, established the structure of NFa-j shown in Table III.


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Table II. Methylation analysis of ABEE-oligosaccharides from recombinant human {alpha}-GalNAc
 


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Fig. 3. HPLC of the neutral ABEE-oligosaccharides from recombinant secreted human {alpha}-GalNAc. Fraction N shown in Figure 2 was further characterized by HPLC on Con A-agarose. The three resulting fractions were designated flow-through (NF), weakly bound (NW) and strongly bound (NB). Fractions NF (A), NW (B), and NB (C) were analyzed by HPLC using a TSKgel Amide-80 column (0.46 cm x 25 cm) equilibrated with an 80:20 mixture of solvent A (acetonitrile:water, 9:1 by vol) and solvent B (acetonitrile:water, 1:9 by vol). After injection, the sample was eluted using a linear gradient from an 80:20 to a 50:50 ratio of solvents A to B over 60 min at a flow rate of 0.8 ml/min at 40°C. Each fraction was collected for subsequent structural characterization. Arrows indicate the elution positions of glucose oligomers (numbers indicate the glucose units). The smaller peaks in chromatograms A and B between 0 to 10 min and the larger peak in chromatogram C were non-sugar materials.

 


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Fig. 4. Two dimensional map of the major ABEE-oligosaccharides from recombinant secreted human {alpha}-GalNAc and their digestion products with various glycosidases. Each ABEE-oligosaccharide was analyzed by HPLC on TSKgel Amide-80 and on Wakosil 5C18-200 columns. Elution positions of ABEE-oligosaccharides from the Amide-80 column are expressed as the number of glucose units, and from the ODS column as the relative retention time to glucose-ABEE. Amide/HPLC was performed as in Figure 3. The Wakosil 5C18-200 (0.4 cm x 25 cm) column was eluted with a linear gradient from 9% acetonitrile in 100 mM acetic acid to 11% acetonitrile in 100 mM acetic acid over 60 min at a flow rate of 0.8 ml/min at 40°C. Arrows (dashed arrow), (solid arrow), (dotted arrow), (dash-dot arrow), and (dash-dot-dot arrow) indicate digestions with bovine kidney {alpha}-L-fucosidase, jack bean ß-galactosidase, jack bean ß-N-acetylhexosaminidase, A.saitoi {alpha}1,2-mannosidase and jack bean {alpha}-mannosidase, respectively. (1) Indicates the elution position of authentic ManGlcNAc2-ABEE, (2) Man3GlcNAc2-ABEE, (3) GlcNAc2Man3GlcNAc2-ABEE, (4) 2,6-branched GlcNAc3Man3GlcNAc2-ABEE, (5) Gal2GlcNAc2Man3GlcNAc2-ABEE, (6) 2,6-branched Gal3GlcNAc3Man3GlcNAc2-ABEE, (7) Gal2GlcNAc2Man3GlcNAcFucGlcNAc-ABEE, (8) 2,6-branched Gal3GlcNAc3Man3GlcNAcFucGlcNAc-ABEE, (9) Man5GlcNAc2-I-ABEE, (10) Man6GlcNAc2-I-ABEE, (11) Man7GlcNAc2-ABEE, (12) GlcNAcMan5GlcNAc2-ABEE, (13) GalGlcNAcMan5GlcNAc2-ABEE and (14) GalGlcNAcMan6GlcNAc2-ABEE.

 
Structural analysis of the oligosaccharides in fraction NW
Methylation analysis of fraction NW indicated that the predominant residues were terminal galactose, terminal fucose, C-2 monosubstituted mannose, C-4 monosubstituted N-acetyl­glucosamine, and C-3,6 disubstituted mannose (Table II). These results suggested that the structures of the oligosaccharides in this fraction were biantennary oligosaccharides, some with a core-region fucose. Amide HPLC fractionated this fraction into four subfractions, denoted NWa-NWd (Figure 3). NWc and NWd, the most abundant components in fraction NW comigrated with biantennary ABEE-oligosaccharides with the structures of Gal2GlcNAc2Man3GlcNAc2-ABEE and Gal2GlcNAc2Man3GlcNAcFucGlcNAc-ABEE, respectively, on 2D-mapping analysis. Sequential enzymatic analysis with {alpha}-L-fucosidase, ß-galactosidase, ß-N-acetylhexosaminidase, and {alpha}-mannosidase gave results supporting the structures initially proposed by their chromatographic behaviors, as shown in Figure 4. A minor component, NWa, mapped with authentic Man{alpha}1-3Man{alpha}1-6(Man{alpha}1-2Man{alpha}1-3)Manß1-4GlcNAcß1-4GlcNAc-ABEE (Man5GlcNAc2-II-ABEE) (Matsuura et al., 1998Go). The elution position of NWa shifted to that of authentic Man{alpha}1-3Man{alpha}1-6(Man{alpha}1-3)Manß1-4GlcNAcß1-4GlcNAc-ABEE (Man4GlcNAc2-ABEE) on digestion with Aspergillus saitoi {alpha}-mannosidase I ({alpha}1,2-mannosidase), and then to that of ManGlcNAc2-ABEE on subsequent {alpha}-mannosidase digestion (data not shown). Another minor component, NWb, comigrated with the monoantennary ABEE-oligosaccharide, Galß1-4Glc­NAcß1-2Man{alpha}1-6(Man{alpha}1-3)Manß1-4GlcNAcß1-4(Fuc{alpha}1-6)GlcNAc-ABEE (Matsuura et al., 1992Go). The results of enzymatic sequencing analysis by using {alpha}-L-fucosidase, ß-galactosidase, ß-N-acetylhexosaminidase, and jack bean {alpha}-mannosidase supported the proposed structure (see Matsuura et al., 1992Go).

Structural analysis of the oligosaccharides in fraction NB
Methylation analysis of fraction NB indicated that the predominant oligosaccharides were high mannose-type with terminal C-2 monosubstituted and C-3,6 disubstituted mannose residues; minor components had terminal galactose and N-acetylglucosamine consistent with hybrid-type structures. The detection of small amounts of terminal fucose and C-4,6 disubstituted N-acetylglucosamine residues suggested that some oligosaccharides had a core-region fucose (Table II). When analyzed by Amide HPLC, eight peaks denoted NBa-NBh were obtained as shown in Figure 3. NBa mapped with authentic Man3GlcNAc2-ABEE and NBb with authentic Man3GlcNAcFucGlcNAc-ABEE found in {alpha}-Gal A (Matsuura et al., 1998Go). The results of the enzymatic sequencing analysis with {alpha}-L-fucosidase and {alpha}-mannosidase (data not shown) and of methylation studies supported the proposed structures (Table III).





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Table III. Proposed structures of N-linked oligosaccharides of recombinant human {alpha}-GalNAc

* Indicates the mannose-6-phosphate residue in monophosphoryl oligosaccharides (PI). # Indicates the mannose-6-phosphate residue in diphosphoryl oligosaccharides (PIT).

aAbbreviated structural formula.

 
Fractions NBc, NBd, and NBf mapped with authentic high mannose-type ABEE-oligosaccharides with the structures Man{alpha}1-6(Man{alpha}1-3)Man{alpha}1-6(Man{alpha}1-3)Manß1-4GlcNAcß1-4GlcNAc-ABEE (Man5GlcNAc2-I-ABEE), Man{alpha}1-6(Man{alpha}1-3)Man{alpha}1-6(Man{alpha}1-2Man{alpha}1-3)Manß1-4GlcNAcß1-4Glc­NAc-ABEE (Man6GlcNAc2-I-ABEE) and Man{alpha}1-2Man{alpha}1-6(Man{alpha}1-3)Man{alpha}1-6(Man{alpha}1-2Man{alpha}1-3)Manß1-4GlcNAcß1-4GlcNAc-ABEE (Man7GlcNAc2-ABEE), respectively. The results of sequential glycosidase digestions (Figure 3), acetolysis, and 2D-mapping analysis of the endo-ß-N-acetylglucosaminidase H digest were the same as those for corresponding ABEE-oligo­saccharides in {alpha}-Gal A (Matsuura et al., 1998Go).

NBg and NBh mapped with authentic hybrid-type oligo­saccharides, GalGlcNAcMan6GlcNAc2-II-ABEE without or with a core region fucose obtained from {alpha}-Gal A (Matsuura et al., 1998Go). Both NBg and the defucosylated NBh had the same sequential map positions following a series of glycosidase digestions using ß-galactosidase, ß-N-acetylhexosaminidase, and {alpha}-mannosidase (Figure 4), confirming the proposed structures (Table III). The low abundance fraction NBe was found to be a mixture of two hybrid-type oligosaccharides by the following experiments. Digestion with ß-galactosidase shifted ~75% of the fraction (designated NBe1) to a position corresponding to the release of one galactose residue (Figure 4). When the residual ß-galactosidase resistant ABEE-oligosac­charide (designated NBe2) was digested with {alpha}1,2-mannosidase, its position shifted to that of the ß-galactosidase digest of NBe1. When both digests were then incubated with ß-N-acetylhexosaminidase and coinjected on ODS/HPLC, a single peak corresponding to Man5GlcNAc2-I-ABEE was detected (Figure 4). Furthermore, the elution position of NBe2 shifted to that of Man{alpha}1-2Man{alpha}1-6(Man{alpha}1-3)Man{alpha}1-6(Man{alpha}1-3)Manß1-4GlcNAcß1-4GlcNAc-ABEE (Man6Glc­NAc2-II-ABEE) (Matsuura et al., 1998Go) upon ß-N-acetylhexosaminidase digestion. These results indicated that NBe1 and NBe2 were oligosaccharides with the structures shown in Table III.

Structures of core oligosaccharides of sialyloligosaccharides
The elution profiles of the desialylated ABEE-oligosaccharides from mono- (SIN), di- (SIIN), and trisialyl (SIIIN) oligosaccharides from {alpha}-GalNAc on an Amide-80 column are shown in Figure 5. SIN gave seven peaks denoted SINa-g. The 2D-mapping positions of SINa, b, d, e, f, and g, and their digests with various glycosidases coincided with those of NWb, NWc, NWd, NFg, NFh, and NFj described above (Table III), respectively. Fraction SINc was not carbohydrate. Fraction SIIN gave six peaks at the elution positions corresponding to SINb-SINg, and SIIIN had three peaks at the elution positions corresponding to SINe-SINg. When each isolated fraction from SIIN and SIIIN was coinjected with the corresponding fraction from SIN, they coeluted as a single peak, respectively. From these results, the structures of the ABEE-oligosaccharides in fractions SIN, SIIN, and SIIIN were proposed as in Table III.



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Fig. 5. HPLC of the neutral ABEE-oligosaccharides derived from sialic acid-containing oligosaccharides present on recombinant secreted human {alpha}-GalNAc. Monosialyl (SI), disialyl (SII), trisialyl (SIII), and monosialyl monophosphoryl (SP) fractions were exclusively digested with {alpha}2,3-sialidase and/or alkaline phosphatase and the resulting neural ABEE-oligosaccharides SIN (A), SIIN (B), SIIIN (C), and SPN (D) were then analyzed by HPLC on TSKgel Amide 80. HPLC was performed as in Figure 4. Arrows indicate the elution positions of authentic 1, Gal2GlcNAc2Man3GlcNAc2-ABEE; 2, 2,4-branched Gal3GlcNAc3Man3GlcNAc2-ABEE; 3, 2,6-branched Gal3GlcNAc3Man3GlcNAc2-ABEE; 4, Gal4GlcNAc4Man3GlcNAc2-ABEE; 5, Gal2GlcNAc2Man3GlcNAcFucGlcNAc-ABEE; 6, 2,4-branched Gal3GlcNAc3Man3GlcNAcFucGlcNAc-ABEE; 7, 2,6-branched Gal3GlcNAc3Man3GlcNAcFucGlcNAc-ABEE and 8, Gal4GlcNAc4Man3GlcNAcFucGlcNAc-ABEE.

 
Structures of the mono- and diphosphoryl oligosaccharides
As shown in Figure 6, the ABEE-derivatives of phosphoryl oligosaccharides in fractions PI and PII were separated by ODS/HPLC into five (PIa-PIe) and two (PIIa and PIIb) peaks, respectively. When each fraction was subjected to 2D-mapping analysis and sequential enzymatic digestion after exhaustive alkaline phosphatase digestion, peak PIa-PIe gave the same results as those of NBd (Man6GlcNAc2-I), NWa(Man5GlcNAc2-II), NBf(Man7GlcNAc2), NBg (GalGlc­NAcMan6GlcNAc2), and NBh(GalGlcNAcMan6GlcNAcFuc­GlcNAc), respectively. In a similar manner, the core oligosaccharide of PIIa was determined to be Man7GlcNAc2-ABEE.



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Fig. 6. HPLC of the ABEE-derivatives of the phosphoryl oligosaccharides from recombinant human secreted {alpha}-GalNAc. (A) Monophosphoryl oligosaccharides (PI); (B) diphosphoryl oligosaccharide (PII). HPLC was performed on a Wakosil 5C18-200 column eluted isocratically with 8% acetonitrile in 50 mM NaH2PO4 at a flow rate of 0.8 ml/min.

 
Determination of the location of the mannose-6-phosphate residue(s) was performed by taking advantage of the fact that {alpha}1,2-mannosidase can not hydrolyze phosphorylated {alpha}1,2-residues at the nonreducing end as described previously (Matsuura et al., 1998Go). Both PIa and PIb were resistant to digestion with {alpha}1,2-mannosidase, indicating that the phosphate residue was attached to the {alpha}1,2-linked mannose residue of the Man{alpha}1-2Man{alpha}1-3Manß1- branch (Table III). Digestion of PIc with {alpha}1,2-mannosidase followed by alkaline phosphatase yielded Man6GlcNAc2-II-ABEE, indicating that its phosphate was on the {alpha}1,2-linked mannose residue of the Man{alpha}1-2Man{alpha}1-6Man{alpha}1-6Manß1- branch (Table III). Both PId and PIe were resistant to digestion with {alpha}1,2-mannosidase, indicating that a phosphate was on the {alpha}1,2-linked mannose residue of the Man{alpha}1-2Man{alpha}1-6Man{alpha}1-6Manß1- branch (Table III).

When PIIa was treated with {alpha}1,2-mannosidase followed by alkaline phosphatase, it yielded an ABEE-oligosaccharide that cochromatographed with Man7GlcNAc2-ABEE. On the basis of these results, the structure of PIIa was proposed as in Table III. PIIb was resistant to various kinds of glycosidases and alkaline phosphatase, suggesting that it was not carbohydrate.

Core structures of monosialyl monophosphoryl oligosaccharides
The neutral ABEE-oligosaccharides (SPN) obtained by the digestions with sialidase followed by alkaline phosphatase gave two peaks, denoted SPNa and SPNb, as shown in Figure 5D. The 2D-mapping positions of SPNa and SPNb, and their digests with various glycosidases coincided with those of hybrid-type oligosaccharides, NBg and NBh, respectively (Table III). The location of the phosphate and sialic acid residues of the oligosaccharides could not be determined due to the limited amounts of the samples available.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this communication, the first five of the six potential N-glyco­sylation sites in human {alpha}-GalNAc were shown to be site-occupied and site 3 occupancy to be important for enzyme function and stability. Site 2 also was shown to be important for enzyme stability. In addition, the oligosaccharide structures of the secreted glycoforms of recombinant human exoglycosidase overexpressed by CHO cells were extensively characterized. Previously, only limited studies of the carbohydrate content and type of oligosaccharide structures of the human placental and fibroblast intracellular {alpha}-GalNAc were investigated (Kusiak et al., 1978Go; Sweeley et al., 1983Go). The intra­cellular lysosomal {alpha}-GalNAc glycoforms were found to contain predominantly high mannose oligosaccharides (Man8-9Glc­NAc) and little, if any, sialic acid residues, consistent with the findings for other lysosomal hydrolases isolated from mammalian tissue lysosomes (Howard et al., 1982Go; Nakao et al., 1984Go; Mutsaers et al., 1987Go). In contrast, the secreted glycoforms of recombinant human {alpha}-GalNAc were highly heterogeneous with complex (53%), hybrid (~12%), and high mannose (33%) type oligosaccharides. It should be noted that the structure and composition of oligo­saccharides vary with the expression host and culture conditions (Goochee and Monica, 1990Go; Jenkins et al., 1996Go). The complex oligosaccharides were mono-, bi-, 2,4-tri, 2,6-tri and tetraantennary structures, in which the biantennary chains were predominant (~60% of total complex species). Of note, most of the complex oligosaccharides had complete outer chains terminating in lactosamine and sialic acids linked to galactose via {alpha}2,3-linkages (~50%) and ~80% had a core region fucose. Of the high mannose oligosaccharides, the predominant species (~57%) was Man6GlcNAc2-I and about 50% of the high mannose oligosaccharides were phosphorylated with one or two phosphomonoesters, PIa (Table III) being the predominant (>50%) phosphorylated species. Unusual high mannose oligosaccharides, Man5GlcNAc2-II (NWa), and its phosphorylated species (PIb) were present at low concentrations. Interestingly, among oligosaccharides having 2–3 mannose residues, which are typically abundant in other intracellular glycosidases (Takahashi et al., 1984Go; Taniguchi et al., 1985Go; Mutsaers et al., 1987Go), Man3GlcNAc(Fuc)0–1GlcNAc were present at low levels, but Man2GlcNAc(Fuc)0–1GlcNAc was absent.

The hybrid-type oligosaccharides were highly phosphorylated (~54% of total hybrid species), and about 30% of the phosphorylated hybrid structures also were sialylated. The latter finding of phosphorylated and sialylated oligosaccharide chains is unique, having been identified previously only in a mixture of mouse cellular glycoproteins (Varki and Kornfeld, 1983Go). Of note, none of the hybrid-type oligosaccharides from {alpha}-GalNAc had bisecting N-acetylglucosamine.

Since {alpha}-GalNAc is evolutionarily related to the highly homologous {alpha}-Gal A (Wang and Desnick, 1991Go; Zhu and Goldstein, 1993Go; Herrmann et al., 1998Go; Wang et al., 1998Go), it was of interest to compare their oligosaccharide structures. Table IV compares the oligosaccharide profiles of recombinant human {alpha}-GalNAc and {alpha}-Gal A secreted by stably over­expressing CHO DG44 cells. Compared to {alpha}-Gal A, {alpha}-GalNAc had more complex (30% vs. 53%) and hybrid-type (5% vs. 12%) oligosaccharides, while {alpha}-Gal A had predominantly high mannose-type oligosaccharides (60%). Of the high mannose-type structures, Man5-7GlcNAc2 was the predominant species in both enzymes. It should be noted that {alpha}-Gal A had a 6-fold greater amount of short oligosaccharide chains, Man2-4GlcNAc(Fuc)0–1GlcNAc. Although not the major oligosaccharide type, {alpha}-GalNAc had a two-fold greater amount of hybrid-type structures (12% vs. 5%) and the major hybrid species (~60% of total hybrid species) in {alpha}-GalNAc (NBh) did not occur in {alpha}-Gal A. In addition, the sialyl phosphoryl hybrid-type oligosaccharides in {alpha}-GalNAc did not occur in {alpha}-Gal A. Whereas the major complex oligosaccharides in {alpha}-GalNAc were biantennary (~60% of total complex species) and triantennary (>20%) species, in {alpha}-Gal A the monoantennary and biantennary structures predominated (~26% each). These findings were consistent with the {alpha}-GalNAc complex oligosaccharides being more completely processed than those in {alpha}-Gal A. Notably, {alpha}-GalNAc had markedly more sialylated residues than {alpha}-Gal A (28.9% vs. 8.5% of total oligosaccharides), although both secreted enzymes had similar amounts of phosphorylated residues (23% vs. 24%), respectively.


View this table:
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Table IV. The summary of oligosaccharides obtained from recombinant human {alpha}-N-acetylgalactosaminidase and {alpha}-galactosidase A
 
Certain findings in the oligosaccharide structures of {alpha}-GalNAc and {alpha}-Gal A suggest differences in enzyme folding or conformation which limit the accessibility of the oligosaccharides to various glycosidic modifying enzymes in the biosynthesis of the respective enzymes. For example, it was noted that {alpha}-GalNAc had only the Man6GlcNAc2-I isomer, while {alpha}-Gal A had both Man6GlcNAc2-I and II in a 2:5 ratio. {alpha}-GalNAc had 2,4- and 2,6-branched triantennary oligosaccharides in a 1:2 ratio, while {alpha}-Gal A had mostly 2,4-branched triantennary structures. In {alpha}-GalNAc, Man6GlcNAc2-II and hybrid oligosaccharide NBh were the predominant phosphorylated species, while in {alpha}-Gal A Man7GlcNAc2 was the predominant phosphorylated structure. Similarly, {alpha}-GalNAc had only one diphosphorylated oligosaccharide, while {alpha}-Gal A had several diphosphorylated structures. Presumably, these differences reflect the accessibility or steric hindrance of the respective enzymes to various modifying enzymes during their biosynthesis, as well as the relative rate of overexpression by CHO cells.

Although over 80% of the complex chains were fucosylated in both secreted enzymes, none of the {alpha}-Gal A hybrid-type oligosaccharides were fucosylated, while over 50% of the {alpha}-GalNAc hybrid-type chains had fucose residues, the predominant (>60% hybrid chains) being NBh. Interestingly, the major hybrid type oligosaccharide of {alpha}-Gal A, NBg, representing almost 80% of the hybrid chains, had the same defucosylated structure as NBh from {alpha}-GalNAc. This finding indicates that the fucosyltransferase was unable to fucosylate {alpha}-Gal A NBg, presumably due to inaccessibility to a sterically hindered domain or aggregation of the enzyme.

Even though human {alpha}-GalNAc and {alpha}-Gal A are highly homologous, their secreted recombinant glycoforms differed markedly. Was this due to differences in the number or relative position of the glycosylation sites? Recent mutagenesis studies of human {alpha}-Gal A (Ioannou et al., 1998Go) indicate that the first three of the four human {alpha}-Gal A N-glycosylation sites were occupied, the putative fourth site (N408) presumably not glyco­sylated, since it contains a proline residue (Shakin-Eshleman et al., 1996Go). For comparison, three chicken {alpha}-GalNAc N-glycosylation sites (corresponding to human site 2 (N177), site 3 (N201) and site 5 (N385)) were occupied, while the corresponding human site 1 (N124) and site 4 (N359) were obliterated (Zhu et al., 1998Go). The mutagenesis studies of human {alpha}-GalNAc described here indicated that the first five sites were occupied, the sixth site containing a proline residue was not, analogous to the proline-containing C-terminal N408 site in {alpha}-Gal A. Interestingly, the elimination of site 3 in {alpha}-GalNAc (N201) and {alpha}-Gal A (N215) resulted in the most inactive and unstable of the mutant glycoforms in the respective enzymes, indicating the importance of the oligosaccharides for proper folding of these related enzymes. Of note, {alpha}-GalNAc C-terminal site 4(N359) and site 5 (N385) may be easily accessed by glycosylation modifying enzymes, consistent with the presence of over 50% complex structures. Alternatively, secreted human {alpha}-GalNAc may have more completely fucosylated and sialylated complex structures since it remained soluble when overexpressed in CHO cells. In contrast, the secreted {alpha}-Gal A oligosaccharides may have been incompletely processed because a significant percentage of the enzyme was aggregated in the trans Golgi apparatus (Ioannou et al., 1992Go), thereby preventing modification by enzymes in the terminal steps of glycosylation (i.e., fucosylation and sialylation). To determine if {alpha}-Gal A is underglycosylated due to inaccessibility of certain normally folded domains or due to aggregation of the highly overexpressed enzyme, future studies should analyze the oligosaccharide structures of recombinant secreted {alpha}-Gal A that was stably overexpressed at a lower stage of amplification, such that all the synthesized enzyme was soluble.

In summary, determination of the complete oligosaccharide structures of {alpha}-GalNAc not only provided structural information about the secreted enzyme’s glycoforms, but even more interestingly, provided the opportunity to compare its structures with those of an evolutionarily related and highly homo­logous member of the {alpha}-galactosidase family, {alpha}-Gal A. Comparison of their structures identified certain differences that suggested that the glycosylation of {alpha}-Gal A differed primarily due to steric hindrance to certain folded domains. Support for this notion may be provided by crystallization and solution of the respective three-dimensional structures of the enzymes as well as from analysis of the oligosaccharides on overexpressed, but soluble forms of {alpha}-Gal A.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Restriction endonucleases, Vent DNA polymerase, and T4 ligase were obtained from New England Biolabs (Beverly, MA). COS-1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MS). Oligonucleotides were synthesized using an Applied Biosystems DNA synthesizer model 380B. p-Aminobenzoic ethyl ester was purchased from Wako Pure Chemical Industries (Osaka, Japan). Aspergillus saitoi {alpha}-mannosidase I ({alpha}1,2-mannosidase) was purified by the method of Amano and Kobata (Amano and Kobata, 1986Go). Jack bean ß-galactosidase, ß-N-acetylhexo­saminidase, and {alpha}-mannosidase were prepared by modifications of the published methods (Li and Li, 1972Go). Bovine kidney {alpha}-L-fucosidase and E.coli alkaline phosphatase were obtained from Sigma Chemical Co. (St. Louis, MO). Arthrobacter ureafaciens sialidase was purchased from Nacalai Tesque Inc. and cloned Salmonella typhimurium {alpha}2,3-sialidase was from Takara Shuzo Co. Ltd. (Otsu, Japan). The standard oligosaccharides tagged with ABEE were prepared from various glycoproteins as described previously (Ohta et al., 1996Go; Matsuura et al., 1998Go).

Cell culture and DNA transfections
For site occupancy studies, COS-1 cell culture, and DNA transfections were carried out as described previously (Ioannou et al., 1992Go, 1998). The pRLDN expression vector was a generous gift of Dr. John Tricc (SmithKline, PA).

Site-directed mutagenesis and site occupancy studies
For site-directed mutagenesis of the six putative N-glycosylation consensus sites, mutant cDNA constructs were synthesized using the overlap polymerase chain reaction (PCR) as previously described (Ioannou et al., 1998Go). Briefly, the full-length {alpha}-GalNAc cDNA was amplified with primers designed to replace the asparagine codon of the N-glycosylation consensus sequence with glutamine codons. Six constructs were designed to eliminate a single consensus sequence at {Delta}GS-1, {Delta}GS-2, {Delta}GS-3, {Delta}GS-4, {Delta}GS-5, and {Delta}GS-6. Each of the eight constructs was sequence confirmed, and subcloned into the eukaryotic expression vector pRLDN for expression studies.

Enzyme and protein assays
For enzyme assays, the cells in a 100 mm culture dish were washed twice with 5 ml of phosphate-buffered saline (PBS) and incubated with 1 ml of lysis buffer (50 mM sodium phosphate buffer, pH 6.5, containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.2 mM PMSF) at 4°C for 10 min. The lysates were collected and transferred to a 1.5 ml test tubes and clarified by centrifugation at 16,000 x g. The {alpha}-GalNAc activities in the cell lysates and media were determined using 50 mM 4-methylumbelliferyl-{alpha}-D-2-aceto-2-aminogalactopyranoside. The liberated fluorescence 4-methylumbelliferone was determined using a Ratio-2 System Fluorometer (Optical Technologies Devices Inc., Elmsford, NY).

Labeling of cells with [35S]-methionine and immunoprecipitation
For [35S]-methionine labeling, confluent cultures in 100 mm dishes were washed once with 5 ml of methionine-free DMEM and incubated at 37°C for 30 min with 5 ml of methionine-free DMEM. The media was removed and a fresh aliquot (1 ml) of methionine-free DMEM, supplemented with 10% dialyzed fetal calf serum and 200 µCi of [35S]methionine, was added. Cells were incubated at 37°C for 16 h, washed, and lysed as above. Polyclonal anti-human {alpha}-GalNAc antibodies were used to immunoprecipitate {alpha}-GalNAc as described previously (Wang et al., 1994Go).

Release of N-linked oligosaccharide chains from {alpha}-GalNAc
Secreted recombinant human {alpha}-GalNAc (2.5 mg) was over­expressed in DG44dhfr- CHO cells grown in CHO-SFM media (Gibco BRL, Rockville, MD) using an Endotonics hollow fiber bioreactor (Endotronics Inc., Minneapolis, MN), and the recombinant enzyme was purified as described (Ioannou et al., 1992Go). The enzyme was subjected to hydrazinolysis followed by re-N-acetylation as described previously (Ohta et al., 1991Go). The released oligosaccharides were reductively aminated with ABEE and then purified by serial chromatography on a PRE-SEP C18 cartridge and a Bio-Gel P-4 column (Matsuura and Imaoka, 1988Go).

Fractionation of ABEE-oligosaccharides by HPLC
Anion exchange HPLC of ABEE-oligosaccharides was carried out on a TSKgel DEAE-5PW column (0.75 x 7.5 cm, TOSOH) as described previously (Matsuura et al., 1993Go). Neutral ABEE-oligosaccharides were fractionated either on a TSKgel Amide-80 column (0.46 x 25 cm, TOSOH) or a Wakosil 5C18-200 column (0.4 x 25 cm, Wako Pure Chemical Industries) under the conditions described previously (Matsuura et al., 1992Go). HPLC of ABEE-derivatized, phosphoryl oligosaccharides was carried out on a Wakosil 5C18-200 column as described (Matsuura et al., 1998Go). Affinity chromatography of the ABEE-oligosaccharides was performed by HPLC on a Con A column (0.46 x 15 cm, HONEN Corp., Tokyo, Japan). The column was eluted stepwise with 0.05 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl and 10 mM and 200 mM methyl mannoside.

Two-dimensional mapping analysis of ABEE-oligosaccharides
Structures of the individual neutral oligosaccharides were analyzed by the two-dimensional (2D) mapping technique as described previously (Matsuura et al., 1992Go). Briefly, the elution positions of ABEE-oligosaccharides on the Amide-80 column (expressed as glucose units) and the ODS column (expressed as relative retention time to glucose-ABEE) were plotted on a 2D-sugar map and compared with the data for respective reference ABEE-oligosaccharides.

Other analytical procedures
Polyacrylamide gel electrophoresis was carried out under reducing conditions in a 1.5 mm thick slab containing 10% acrylamide as described previously (Ioannou et al., 1998Go). The gel was fixed in 10% acetic acid and 20% methanol for 30min and then soaked in Amplify (Amersham, Arlington Heights, IL) for 30 min with agitation. Gels were vacuum dried for 90 min and exposed to Kodak X-Omat AR film for 4–72 h. Sequential exoglycosidase digestions followed by 2D-mapping analysis of the digests was carried out as described previously (Matsuura et al., 1993Go). Methylation analysis was performed as described previously (Matsuura et al., 1993Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Kenneth Zeidner for purification of the recombinant human {alpha}-GalNAc. This work was supported by grants from the National Institutes of Health including a MERIT award (5 R01 DK34045), a grant (M01 RR00071) for the Mount Sinai General Clinical Research Center from the National Center for Research Resources, and a grant (P30 HD28822) for the Mount Sinai Child Health Research Center.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}-GalNAc, {alpha}-N-acetylgalactosaminidase; {alpha}-Gal A, {alpha}-galactosidase A; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate buffered saline; HPLC, high performance liquid chromatography; ABEE, p-amino benzoic ethyl ester; Con A, Concanavalin A; ODS, octadecyl silica.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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