Glycobiology Advance Access originally published online on January 31, 2006
Glycobiology 2006 16(5):440-461; doi:10.1093/glycob/cwj081
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Site-specific glycosylation analysis of the bovine lysosomal 
-mannosidase
2 Unité Mixte de Recherche CNRS/USTL 8576, «Glycobiologie Structurale et Fonctionnelle», IFR 118, Bâtiment C9, Université des Sciences et Technologies de Lille 1, 59655 Villeneuve d’Ascq Cedex, France; and 3 Department of Medical Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
1 To whom correspondence should be addressed; e-mail: willy.morelle{at}univ-lille1.fr
Received on December 7, 2005; revised on January 23, 2006; accepted on January 23, 2006
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Abstract |
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Top
Abstract
IntroductionLysosomal
-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder -mannosidosis. This enzyme of about 1000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N497 glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc0–1Glc0–1Man4–9GlcNAc2), hybrid-type (Gal0–1Man4–5GlcNAc4), and complex-type (Fuc0–1Gal0–2Man3GlcNAc3–5) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS–PAGE and in-gel deglycosylation. These experiments revealed that the N497 and N930 sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc0–1Man5–9GlcNAc2, including the evolutionary conserved Glc1Man9GlcNAc2 glycan, and Fuc0–1Man3–5GlcNAc2, respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N692 and N766 sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc0–1Man3–7GlcNAc2), hybrid (Fuc0–1 Gal0–1Man4–5GlcNAc4), and complex (Fuc0–1Gal0–2Man3GlcNAc4–5) structures; and (2) the N367 site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc0–1Man3–5GlcNAc2). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N367 site, the N133 site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc0–1Man4–5GlcNAc2), complex-type (Fuc0–1Gal0–1Man3GlcNAc5), and hybrid-type (Fuc0–1Gal0–1Man5GlcNAc4) glycans.
Key words:
-mannosidosis
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glycosylation sites
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lysosomal storage disorder
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mass spectrometry
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structure analysis
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Introduction |
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Lysosomal
-mannosidase (LAMAN; EC 3.2.1.24
[EC]
) belonging to class II -mannosidases is an exoglycosidase that cleaves all -mannosyl linkages known during the degradation of N-linked oligosaccharides (Aronson and Kuranda, 1989
). LAMAN is expressed in all tissues and in many species. This enzyme is characterized by broad natural substrate specificity, its sensitivity to the swainsonine inhibitor, its capacity to retain the anomeric configuration of the related mannae residues (Howard-et-al-1997Howard et al., 1997), and its capability to hydrolyze p-nitrophenylmannopyranoside (for review, see Daniel et al., 1994; Moremen et al., 1994). Contrary to the other members of the sequence-based glycoside–hydrolase family 38 (GH 38) (Henrissat and Bairoch, 1993), LAMAN is characterized by low-pH activation (Heikinheimo et al., 2003), depending on the intracellular location on the lysosome and lack of activity at neutral pH.
Numerous metabolic studies, particularly on human LAMAN (Pohlmann et al., 1983; Cheng et al., 1986; Tsuji and Suzuki, 1987; Nilssen et al., 1997), have shown that the enzyme is synthesized as a single-chain precursor close to 110 kDa, which is further proteolytically processed into several peptides (two to 10) during its intracellular traffic from the endoplasmic reticulum (ER) to the lysosome. These evolutionary preserved cleavages play no physiological role, since the enzyme exists as a functionally single-chain polypeptide (Berg et al., 1999). Human LAMAN consists of two immunologically identical isoforms A and B, which are selectively separated by ion-exchange chromatography and differ in their isoelectric point, arising from different states of sialylation and/or phosphorylation of mannose residues (Cheng et al., 1986). The human enzyme is first synthesized as three glycopeptides of 70, 42, and 15 kDa, generated from the proteolysis of 110 kDa chain precursor which is partly secreted into the extracellular medium. The 70-kDa glycopeptide will further undergo a second proteolytic process on its way to the lysosome to produce three more glycopeptides, joined by two disulfide bridges. Moreover, another post-translational modification appears to be evolutionary conserved involving one N-glycosylation consensus site (497N-I/V-S/T499) and two proximate cysteines (C493 and C501) organized as a loop. This structural modification appears to be essential for the maintenance of lysosomal stability.
Lack of this enzyme causes a lysosomal storage disorder, named -mannosidosis (OMIM 248500
[OMIM]
). This genetic defect is responsible for a massive accumulation of unprocessed mannose-containing oligosaccharides within lysosomes in most cell types of patients, resulting in varying neural, immune, and skeletal abnormalities. Patients are characterized by varying clinic presentations including mental retardation, recurrent infections, hearing loss, hepatosplenomegaly, and dysostosis multiplex as the most known (Thomas and Beaudet, 1995; Michalski, 1996; Michalski and Klein, 1999). -Mannosidosis is known to occur in human (Nilssen et al., 1997), cattle (Berg, Healy, et al., 1997; Tollersrud et al., 1997), Persian cat (Berg, Tollersrud, et al., 1997), and guinea pig (Berg and Hopwood, 2001), and a gene knockout model in mice has been established. Several groups have cloned the LAMAN gene (MANB), comprising 24 exons, spanning 21.5 kb and located in chromosome 19p13.2-q12 (Liao et al., 1996; Nilssen et al., 1997; Riise et al., 1997). More than 50 -mannosidosis-causing mutations have been reported (Nilssen et al., 1997; Gotoda et al., 1998; Berg et al., 1999). These disease-causing mutations are responsible for the total or partial inactivation of LAMAN by disturbing its intracellular traffic (Hansen et al., 2004), structure, and activity (Heikinheimo et al., 2003).
The bovine enzyme is an important molecular model for understanding the human enzyme and pathophysiology of -mannosidosis. Heikinheimo et al. (2003) have recently solved its three-dimensional structure, the first structure of a mammalian enzyme in GH 38 family, providing a basis for understanding the human disease at the atomic level. The active site, formed by the a- and b-peptides, is located to the N-terminal side and is formed on the top of a distorted seven-stranded /ß-barrel. Following the barrel domain, the structure consists of a three-helix bundle, joining the b- and c-peptides, and three subsequent ß-sheet domains, formed by c-, d-, and e-peptides, with unknown function. In addition, the lysosomal enzyme has a large evolutionary conserved carbohydrate chain at the N497 glycosylation site, resting ordered against the three-helix bundle. The structure suggests signal areas for mannose phosphorylation and a low-pH activation mechanism. The bovine LAMAN is about 250 kDa homodimeric glycoprotein. The matching monomer is synthesized as a 110 kDa precursor of about 1000 amino acids, which is further post-translationally modified by N-glycosylation, disulfide bridge formation, and proteolysis into five glycopeptides named a, b, c, d, and e of 35/38, 11/13, 22, 38, and 13/15 kDa, respectively (Tollersrud et al., 1997). The a-, b-, and c-peptides are linked by two disulfide bridges, d- and e-peptides being linked via salt bridge networks (Tollersrud et al., 1997; Heikinheimo et al., 2003). Since the carbohydrate moiety plays many key roles in the biology of most cellular glycoproteins, such as resistance against proteolysis, folding, or intracellular trafficking (for reviews see Varki, 1993; Parodi, 2000; Helenius and Aebi, 2001, 2004; Spiro, 2002; Trombetta, 2003), bovine LAMAN glycosylation has been investigated. Indeed, eight putative N-glycosylation sites (N133, N367, N497, N645, N651, N692, N766, and N930) were present within the polypeptide backbone. Molecular shift analysis of the bovine enzyme by SDS–PAGE after endo-ß-N-acetylglucosaminidase H (endoH) and/or peptidyl-N-glycosidase F (PNGase F) treatments suggested that all potential sites are glycosylated (Tollersrud et al., 1997). Then, depending on their endoH resistance, it was possible to conclude that a-peptide (N133) and most sites of d-peptide (N645, N651, N692, and N766) are occupied by complex-type N-glycans. b- (N367) and c- (N497) peptides are exclusively occupied by oligomannose-type and/or hybrid-type N-glycans. With respect to e-peptide (N930), complex-type with oligomannose-type and/or hybrid-type N-glycans are present. N-glycosylation site N497 is conserved in all LAMANs from plants to mammals (Berg T, Hansen GM, et al., in preparation). In addition, an unusual monoglucosylated oligomannose glycan (Glc1Man9GlcNAc2) carried by N-glycosylation site N497 has also been described. This atypical structure is a retention signal for the calnexin/calreticulin quality-control pathway in the ER (for reviews see Rudd and Dwek, 1997; Parodi, 2000; Spiro, 2002; Trombetta, 2003; Helenius and Aebi, 2004; Trombetta and Parodi, 2005). The presence of this monoglucosylated oligomannose-type glycan in a mature and secreted glycoprotein indicates that the oligosaccharide is located in a region which is not folded by the calnexin/calreticulin system and prevents the removal of the terminal glucose residue by ER--glucosidase II (Crispin et al., 2004).
So far, no data have been reported about the detailed structure of N-glycans or about the site occupancy of the eight N-glycosylation sites. To understand the functions of N-glycans in the biology of LAMANs, we have elucidated the detailed structures of all N-glycans in the bovine LAMAN on one hand and their distribution within each site of N-glycosylation on the other hand.
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Results |
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LAMAN isolation
Bovine LAMAN (primary access number Swiss-Prot: Q29451 [GenBank] ) was isolated from homogenized Bos Taurus kidney, as published previously (Tollersrud et al., 1997
).
Strategy for determining the structure of the major glycans released from the bovine LAMAN
The general strategy employed for investigating glycosylation pattern of the bovine LAMAN is outlined in Scheme 1.
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The detailed structures of glycans of the bovine LAMAN have been determined after digestion by trypsin of the reduced carboxamidomethylated glycoprotein. Glycans were then released from the resulting peptide/glycopeptide mixture by digestion with PNGase F. The PNGase F-released N-glycans were separated from peptides using a C18 Sep-Pak cartridge and desalted on nonporous graphitized carbon solid-phase extraction cartridge. Because of the availability of only limited amounts of material, the oligosaccharides were analyzed as mixtures. PNGase F-released N-glycans were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) before and after on-plate sequential exoglycosidase digestions. Their methylated derivatives were also characterized by MALDI-MS before and after sequential exoglycosidase digestions and by linkage analysis. Structural assignments were based on molecular weight, susceptibility to exoglycosidase digestions, and linkage data. Putative O-glycans were released by reductive elimination, permethylated, purified on a C18 Sep-Pak cartridge, and analyzed by MALDI-MS.
Determination of the structure of N-glycans released from bovine LAMAN
Monosaccharide composition of PNGase F-released glycans The monosaccharide composition of the PNGase F-released N-glycans from bovine LAMAN was determined by GC-MS analysis of the heptafluorobutyrate derivatives of the methyl glycosides. The data revealed the presence of Fuc, Gal, Glc, Man, and GlcNAc residues at a molar ratio of approximately 2:1:0,3:6:1, respectively (data not shown). These results suggest that the major N-glycans of bovine LAMAN have compositions consistent with high-mannose-type structures. Moreover, the same experiment has been reproduced on the entire glycoprotein to determine the carbohydrate content which was of about 10% (w/w), and no GalNAc residues have been detected. This last observation suggests that no O-glycans are present within the polypeptide backbone of the bovine LAMAN.
MALDI-MS analysis of the PNGase F-released glycan The desalted PNGase F-released N-glycans were analyzed by MALDI-MS. The data from the MALDI-MS analysis are shown in Figure 1A and summarized in Table I. A very heterogeneous mixture of oligosaccharides was observed, affording about 20 pseudomolecular ions [M+Na]+. From their m/z ratio, monosaccharide compositions in terms of Hex, dHex, and HexNAc of each PNGase F-released oligosaccharide from bovine LAMAN have been determined and are summarized in Table I. Based on the MALDI-MS data and currently accepted models of eukaryotic N-glycan biosynthesis, these data indicate that bovine LAMAN contains glycans having a composition consistent with high-mannose structures (Fuc0–1Hex4–10HexNAc2; including the evolutionary conserved monoglucosylated oligomannosidic glycan Glc1Man9GlcNAc2), hybrid structures (Hex4–6HexNAc4), and complex structures (Fuc0–1Hex3–5HexNAc3–5). Notable features of these data are as follows: (1) oligomannosidic structures are more abundant than complex structures; and (2) most of the complex structures have compositions consistent with bi- and/or triantennary structures. It is important to note that compositions consistent with isobaric complex and hybrid structures were differentiated according to their endoH sensitivity (data not shown). Indeed, endoH releases only high-mannose and hybrid-type N-glycans from glycoproteins. The native N-glycans were also analyzed in the negative mode. No signals were detected, suggesting that no acidic structures (phosphorylated, sulfated, or sialylated) were present (data not shown).
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Sequential exoglycosidase digestion of the PNGase F-released glycan mixture of the bovine LAMAN To determine the carbohydrate sequence as well as the anomeric configurations, the PNGase F-released glycans were subjected to on-plate treatments with an array of exoglycosidases and then analyzed by MALDI-MS (Mechref and Novotny, 1998) (Figure 1B–D). After treatment with ß-galactosidase (Figure 1B), the pseudomolecular ions [M+Na]+ at m/z 1704 (Hex4HexNAc5), 1825 (Hex6HexNAc4), 1850 (dHex1Hex4HexNAc5), 1866 (Hex5HexNAc5), and 2012 (dHex1Hex5HexNAc5) were significantly reduced in intensity, with a significant concomitant increase in the abundance of the molecular ions at m/z 1542 (Hex3HexNAc5), 1663 (Hex5HexNAc4), and 1688 (dHex1Hex3HexNAc5), consistent with the removal of one or two terminal ß-Gal residues. The combined digestions with ß-galactosidase and ß-N-acetylhexosaminidase (Figure 1C) have greatly simplified the MALDI-MS spectrum, which is now characterized by several major pseudomolecular ions [M+Na]+ at m/z 933 (Hex3HexNAc2), 1079 (dHex1Hex3HexNAc2), 1257 (Hex5HexNAc2), 1419 (Hex6HexNAc2), 1581 (Hex7HexNAc2), 1743 (Hex8HexNAc2), and 1905 (Hex9HexNAc2). All these species were unaffected by the above exoglycosidase digestions, a result that is consistent with the assignment of high-mannose structures to these ions. Minor molecular ions were also observed at m/z 1095 (Hex4HexNAc2), 1403 (dHex1Hex5HexNAc2), and 2067 (Hex10HexNAc2). The molecular species at m/z 1339 (Hex3HexNAc4), 1501 (Hex4HexNAc4), 1542 (Hex3HexNAc5), 1663 (Hex5HexNAc4), and 1688 (dHex1Hex3HexNAc5) were efficiently digested by ß-N-acetylhexosaminidase to the products Fuc0–1Hex3HexNAc2 at m/z 1079 and 933, which are consistent with trimannosyl core with and without core fucosylation, respectively. It can then be concluded that the GlcNAc residues of the N-glycans antenna are ß-linked. The disappearance of pseudomolecular ions at m/z 1501 (Hex4HexNAc4) and 1663 (Hex5HexNAc4) is accompanied by a concomitant increase of the relative abundance of the pseudomolecular species at m/z 1095 (Hex4HexNAc2) and 1257 (Hex5HexNAc2), suggesting that these two initial species were consistent with the composition of hybrid structures. After -mannosidase treatment (Figure 1D), the molecular ions at m/z 1079 (dHex1Hex3HexNAc2), 1403 (dHex1Hex5HexNAc2), 1257 (Hex5HexNAc2), 1419 (Hex6HexNAc2), 1581 (Hex7HexNAc2), 1743 (Hex8HexNAc2), and 1905 (Hex9HexNAc2) were trimmed to Hex3HexNAc2 at m/z 933 and Fuc1Hex3HexNAc2 at m/z 1079. In addition, the molecular ion at m/z 2067 (Hex10HexNAc2) was abolished, concomitant with the appearance of a new signal at m/z 1743 (Hex8HexNAc2). This new molecular ion is consistent with the loss of two -Man residues from the original molecular ion at m/z 2067 (Hex10HexNAc2). After -glucosidase II digestion, the molecular ion at m/z 1743 (Hex8HexNAc2) disappeared (data not shown), and a new ion was observed at m/z 1581 (Hex7HexNAc2), confirming then the presence of the evolutionary conserved glucosylated oligomannose-type glycan (Glc1Man9GlcNAc2) on bovine LAMAN (Berg T, Hansen GM, et al., in preparation).
Linkage analysis of the PNGase F-released glycans from the bovine LAMAN To identify the position of glycosidic bonds, PNGase F-released N-glycans from bovine LAMAN were permethylated (Ciucanu and Kerek, 1984), analyzed by MALDI-MS, and subjected to GC-MS analysis, as partially methylated alditol acetate derivatives, before and after sequential exoglycosidase digestions. According to the data summarized in Table II, some conclusions could be drawn which are as follows: (1) the abundant 2-linked Man indicates that most of the complex-type N-glycans are biantennary, but minor 2,4-branched Man and 2,6-branched Man suggest that minor tri- and/or tetraantennary structures are present; (2) Fuc, Gal, Man, and GlcNAc are the major nonreducing sugars, but minor terminal Glc was also detected; (3) the abundant terminal mannose is in accordance with high-mannose structures being the major components of the N-glycan population; (4) terminal Fuc and the 4,6-linked GlcNAc residues are consistent with the presence of core-fucosylated glycans; and (5) the high level of 3,4,6-linked Man indicates that most of the hybrid-type and/or the complex-type glycans are core bisected. After ß-galactosidase treatment, terminal Gal disappeared, while a decrease of 4-linked GlcNAc was observed, indicating that nonreducing Gal residues were attached to the 4-position of the GlcNAc residues, prior to ß-galactosidase digestion. Comparison of linkage data before and after ß-N-acetylhexosaminidase treatment indicated that loss of terminal ß-GlcNAc residues is accompanied by a decrease in 2-linked Man, 2,4-linked Man, 2,6-linked Man, and a concomitant increase in terminal Man. Besides, the 3,4,6-linked Man disappeared, and a concomitant increase of 3,6-linked Man was observed. These data indicate that GlcNAc residues were attached to the 4-position of the 3,4,6-linked Man and confirm the presence of core-bisected complex-type glycans and/or hybrid-type glycans. Comparison of linkage data before and after -fucosidase treatment indicated that loss of terminal Fuc is accompanied by a loss of the 4,6-linked GlcNAc and a concomitant increase of 4-linked GlcNAc, indicating that Fuc residues were attached to the 6-position of the GlcNAc residues. These data support the presence of (1,6)-core fucosylation.
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Assignment of the PNGase F-released glycans mixture of the bovine LAMAN Taking into consideration MALDI-MS, linkage, and sequential exoglycosidase data, we conclude that the major glycans released from bovine LAMAN fall into three classes, namely oligomannose-type (Fuc0–1Glc0–1Man4–9GlcNAc2), hybrid-type (Gal0–1Man4–5GlcNAc4), and complex-type (Fuc0–1Gal0–2Man3GlcNAc3–5) N-glycans (Figure 2). A minor monoglucosylated oligomannosidic N-glycan (Glc1Man9GlcNAc2) is also present and is consistent with the evolutionary conserved structure among LAMANs (Berg T, Hansen GM, et al., in preparation). High-mannose structures are more abundant than hybrid and complex structures. The major complex-type oligosaccharides have compositions consistent with biantennary structures with a bisecting GlcNAc residue and/or triantennary structures without (1,6)-core fucosylation. The major nonreducing epitopes in the complex-type glycans are GlcNAc and Gal(ß1–4)GlcNAc. The very low abundance of the minor compounds has to date precluded precise structural analysis.
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O-glycosylation analysis |
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Reductive elimination on de-N-glycosylated peptides did not give any monosaccharides when analyzed by GC-MS analysis of the heptafluorobutyrate derivatives of the methyl glycosides (data not shown). Putative O-glycans were also permethylated, purified on a C18 Sep-Pak cartridge, and analyzed by MALDI-MS. No signals corresponding to O-glycans were observed (data not shown). Thus, we could find no evidence that O-linked glycans were an important component of the bovine LAMAN.
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Strategy for investigating site-specific glycosylation of the bovine LAMAN |
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After elucidating the structures of the major PNGase F-released N-glycans from the bovine LAMAN, the structure of the N-glycans at each glycosylation site has been determined. Bovine LAMAN consists of five peptide chains, namely a- to e-peptides, each containing one N-glycosylation site, except for the d-peptide that contains four N-glycosylation sites. To obtain information about the glycosylation within each peptide, peptide chains of the bovine LAMAN were electrophoretically separated according to their molecular weight, using SDS–PAGE. Bands of interest were excised, reduced, carboxamidomethylated, and in-gel deglycosylated by treatment with PNGase F, according to the procedure developed by Küster et al. (1997)
. After desalting on nonporous graphitized carbon solid-phase extraction cartridge, the extracted glycans were finally analyzed by MALDI-MS, before and after on-plate sequential exoglycosidase digestions. After in-gel deglycosylation, identification of each peptide was confirmed by the use of in-gel tryptic digestion followed by MALDI-MS and matching of the mass fingerprint of the peptides obtained to a sequence database (ExPASy). |
Glycosylation of the peptide chains of the bovine LAMAN using SDS–PAGE |
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The peptide chains of the reduced and carboxymethylated bovine LAMAN were first electrophoretically separated, using SDS–PAGE, followed by in-gel deglycosylation using PNGase F and MALDI-MS analysis of the extracted products, as described previously (Küster et al., 1997
). The results of the SDS–PAGE obtained were in accordance with those obtained by Tollersrud and co-workers (1997). Figure 3A shows the different peptides constituting the bovine LAMAN. Because of their similar molecular mass, the a- and d-peptides were observed as a mixture. The abc-, c-, b-, and e-peptide chains were mostly well separated and showed a great heterogeneity due probably to the heterogeneity of their carbohydrate moieties. The abc-peptide originates from an incomplete proteolytic cleavage of the mature 110 kDa precursor of the bovine LAMAN (Nilssen et al., 1997; Tollersrud et al., 1997).
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Figure 3B depicts the MALDI-MS spectrum of the in-gel-released glycans from the unprocessed abc-peptide of the bovine LAMAN, which contains the N133, N367, and N497 glycosylation sites. This spectrum is dominated by a strong signal at m/z 1257, corresponding to the Hex5HexNAc2 high-mannose-type glycan, accompanied by lower signals at m/z 1403 (dHex1Hex5HexNAc2), 1419 (Hex6HexNAc2), 1542 (Hex3HexNAc5), 1581 (Hex7HexNAc2), 1663 (Hex5HexNAc4), 1688 (dHex1Hex3HexNAc5), 1704 (Hex4HexNAc5), 1743 (Hex8HexNAc2), 1809 (dHex1Hex5HexNAc4), 1825 (Hex6HexNAc4), and 1850 (dHex1Hex4HexNAc5). All these species were in agreement with those expected and summarized in Figure 2. Owing to its low abundance, the core-fucosylated hybrid structure (dHex1Hex5HexNAc4) was not detected in the total pool of PNGase F-released N-glycans and was only observed when the individual peptide chains were studied. The expected evolutionary conserved Glc1Man9GlcNAc2 among LAMANs, which is located to the c-peptide, has not been detected because of its low abundance within the total pool of the abc-peptide-carried glycans. The N-glycans belong to the following three classes: high-mannose structures, hybrid structures, and complex-type structures. High-mannose structures are more abundant than hybrid-type structures and complex-type structures. The structures of the N-glycans were elucidated using on-target sequential exoglycosidase digestions (data not shown). The site-specific distribution of these structures cannot be elucidated since their respective peptide chains were unprocessed and then electrophoretically unseparated.
As depicted in Figure 3C, the MALDI-MS spectrum of the PNGase F-released oligosaccharides from the bands containing the mixed a- and d-peptides shows the presence of a great diversity of pseudomolecular ions [M+Na]+, corresponding to a monosaccharide composition consistent with oligomannosidic (Hex5–9HexNAc2), hybrid (Hex4–6HexNAc4), and complex (Fuc0–1Hex3–5HexNAc3–5) structures. These structures are in keeping with those observed from the total pool of the N-glycans. These data indicate that most of the N-glycans of the bovine LAMAN are on the a- (N133) and d- (N645, N651, N692, and N766) peptides.
The MALDI-MS spectrum, depicted in Figure 3D, of the PNGase F-released glycans from the c-peptide, which only contains the N497 site, was dominated by a strong signal at m/z 1905, which is consistent with the monosaccharide composition of Hex9HexNAc2, accompanied by lower signals at m/z 1257 (Hex5HexNAc2), 1419 (Hex6HexNAc2), 1581 (Hex7HexNAc2), 1743 (Hex8HexNAc2), and 2067 (Hex10HexNAc2). These oligomannosidic structures were confirmed by MALDI-MS analysis after on-plate -mannosidase and -glucosidase II treatments (data not shown). These data indicate that the c-peptide of the bovine LAMAN contains glycans having compositions consistent with high-mannose structures (Hex5–10HexNAc2), including the evolutionary conserved Glc1Man9GlcNAc2 glycan.
The MALDI-MS spectrum (Figure 3E) of the oligosaccharides released by PNGase F from the e-peptide, containing the N930 site, yielded two major pseudomolecular ions [M+Na]+ at m/z 1079 and 1257, compatible with a core-fucosylated trimannosyl structure dHex1Hex3HexNAc2 and an oligomannosidic structure Hex5HexNAc2, respectively. Three other pseudomolecular ions [M+Na]+ were observed at m/z 1095 (Hex4HexNAc2), 1419 (Hex6HexNAc2), and 1403 (dHex1Hex4,6HexNAc2). The in-gel-released oligosaccharides from the e-peptide were further submitted to on-plate sequential -fucosidase digestion followed by MALDI-MS analysis (Figure 3F). After this treatment, both components dHex1Hex3HexNAc2 (m/z 1079) and dHex1Hex5HexNAc2 (m/z 1403) were efficiently digested to Hex3HexNAc2 (m/z 933) and Hex5HexNAc2 (m/z 1257), respectively. Taking into consideration the linkage analysis data, these results suggest that dHex1Hex3HexNAc2 and dHex1Hex5HexNAc2 are (1,6)-core fucosylated.
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Site-specific glycosylation of the bovine LAMAN using concanavalin A immobilized affinity chromatography |
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To identify the site-specific glycosylation at the remaining N133, N367, N645, N651, N692, and N766 glycosylation sites, and to confirm the results obtained for the other N-glycosylation sites, glycopeptides obtained by tryptic digestion of the reduced and carboxamidomethylated bovine LAMAN were enriched by Con A immobilized affinity chromatography. Con A agglutinin is a plant lectin which binds to high-mannose-type and hybrid-type with high affinity. Complex-type N-glycans are also weakly bound to this lectin. Two Con A-Sepharose-bound fractions were obtained. Low-affinity fraction (F1), eluted with 10 mM
-methylglucopyranoside, containing glycopeptides with complex-type N-glycans, and high-affinity fraction (F2), eluted with 500 mM -methylglucopyranoside, containing glycopeptides with oligomannosidic and hybrid-type N-glycans, were desalted on a C18 Sep-Pak cartridge. The glycopeptides from these fractions were finally separated by reverse-phase high-performance liquid chromatography (HPLC) (Figure 4). The fractions were collected, and each purified fraction was analyzed by MALDI–MS, in conjunction with on-target exoglycosidase digestions. Tryptic glycopeptides were then identified, based on theoretical masses of tryptic peptides from the reduced and carboxamidomethylated bovine LAMAN with various additional N-glycans. Based on MALDI-MS profiles, fractions F1-I and F1-II from the low-affinity (F1) and F2-I, F2-II, and F2-III from the high-affinity (F2) fractions bound by Con A-Sepharose correspond to glycopeptide fractions. Figures 5 and 6 show the MALDI mass spectra of fractions F1-I, F1-II, F2-I, F2-II, and F2-III. Subtraction of the calculated average mass of the predicted tryptic peptide from the average molecular mass of the glycopeptides observed provided the oligosaccharide residue compositions, summarized in Table III. As expected for most of them, the monosaccharide compositions of glycopeptides were consistent with those identified by the MALDI-MS analysis of the PNGase F-released glycans from the bovine LAMAN (Table I). However, the site-specific glycosylation, within the N766 and N692 glycosylation sites, for example, indicated the presence of some minor additional structures like dHex1Hex4HexNAc2 (m/z 3125) and dHex1Hex6HexNAc4 (m/z 4277) not listed in Table I.
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To confirm the structure of their carbohydrate moiety, each glycopeptide was subjected to on-plate sequential exoglycosidase digestions, followed by MALDI-MS analysis, as illustrated in the case of glycopeptide from fraction F2-II in Figure 5. Finally, amino acid sequence of each glycopeptide was then obtained, after their deglycosylation using PNGase F, by nanoESI-MS/MS analysis (Figure 7).
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For example, the MALDI-MS spectrum of the native glycopeptide from the high-affinity fraction F2-II, bound to Con A-Sepharose, depicted in the Figure 5A, shows molecular ions [M+H]+ at m/z 1797, 1943, 1959, 2121, 2267, and 2283, spaced by dHex units (146 Da) and by Hex units (162 Da). These signals were assignable to the predicted tryptic peptide 366ANLSWSVK373 with additional high-mannose structures, having compositions Hex3HexNAc2 (m/z 1797), dHex1Hex3HexNAc2 (m/z 1943), Hex4HexNAc2 (m/z 1959), Hex5HexNAc2 (m/z 2121), dHex1Hex5HexNAc2 (m/z 2267), and Hex6HexNAc2 (m/z 2283). To confirm the structure of the carbohydrate moiety, the glycopeptide was submitted to on-plate sequential exoglycosidase digestions, using -mannosidase and/or -fucosidase, followed by MALDI-MS (Figure 5B and C). After, -mannosidase treatment, molecular ions [M+H]+ at m/z 1943, 1959, 2121, 2267, and 2283 disappeared, with a concomitant increase of protonated species [M+H]+ at m/z 1797 (dHex1Hex2HexNAc2) and a concomitant appearance of protonated species [M+H]+ at m/z 1634 (Hex2HexNAc2), 1618 (dHex1Hex1HexNAc2), and 1472 (Hex1HexNAc2). This experiment confirmed that all glycoforms present on the N367 glycosylation site corresponded to oligomannose-type N-glycans. These results are in good agreement with the results obtained by Tollersrud et al. (1997). Moreover, the protonated species [M+H]+ at m/z 1618 (dHex1Hex1HexNAc2) and 1802 (dHex1Hex2HexNAc2) corresponded to products from the digestion of fucosylated high-mannose structures. To confirm the presence of a fucose residue, glycoforms of the tryptic peptide 366ANLSWSVK373 were then digested using -mannosidase and -fucosidase. As depicted in the MALDI-MS spectrum of the Figure 5C, molecular ions [M+H]+ at m/z 1618 and [M+Na]+ at m/z 1802 disappeared. Taking into consideration the linkage analysis data, these results indicate that both molecular species at m/z 1943 (dHex1Hex3HexNAc2) and 2267 (dHex1Hex5HexNAc2) were core fucosylated. Then, to identify the glycosylation site, the glycopeptide from the high-affinity fraction F2-II was deglycosylated using PNGase F, and the resulting aglycone was analyzed by nanoESI-MS/MS. The product ion spectrum CID-MS/MS (Figure 7) obtained from the nanoESI-MS spectrum was dominated by an intense signal at m/z 905 and is consistent with the theoretical mass of the predicted tryptic peptide 366ADLSWSVK373, containing the N367 glycosylation site. The parent ion was selected by the quadrupole and was subsequently fragmented in the collision cell, under 40 eV as collision energy. The CID-MS/MS spectrum in Figure 7 shows two series of singly charged B- and Y-type fragment ions, which fit to the theoretical masses of the product ions calculated from the predicted sequence 366ADLSWSVK373 containing the N367 glycosylation site. The asparagine residue was converted to an aspartic acid residue after PNGase F treatment.
Using the same experiments, site-specific glycosylation within the N766 and N692 glycosylation sites from the d-peptide of the bovine LAMAN was also determined, and both sites were occupied by broad glycan heterogeneity, since the corresponding glycopeptides were found in both the low- and high-affinity fractions. Figure 6A and C depicts MALDI-MS spectra of the native glycopeptides, containing the N766 site, isolated from the F1-I and F2-I affinity fractions, respectively. MALDI-MS spectrum of the native glycopeptides from the F1-I fraction (Figure 6A) is dominated by a strong signal at m/z 3545, corresponding to the predicted tryptic sequence 765LNQTEPVAGNYYPVNSR781 with additional Hex5HexNAc4 structure. About 10 molecular ions were also observed and correspond to the same predicted tryptic peptide with additional structures, ranging from agalactosylated biantennary to core-fucosylated and bisected biantennary structures, and/or triantennary structures. This result is consistent with the low affinity of the Con A lectin for the complex-type N-glycans. The MALDI-MS spectrum of the native glycopeptides from the F2-I fraction (Figure 6C) shows three strong signals at m/z 3141, 3303, and 3545. These molecular ions correspond to the predicted tryptic peptide 765LNQTEPVAGNYYPVNSR781 with additional Hex5HexNAc2, Hex6HexNAc2, and Hex5HexNAc4, respectively. About eight minor molecular species were also observed and correspond to the same peptide with additional high-mannose-type (dHex0–1Hex3–7HexNAc2) and hybrid-type (Hex4–6HexNAc4) N-glycans. The molecular species at m/z 3545 consists in the major component within the N766 site, since it is found with high abundance in both the high- and low-affinity fractions. All these structures were confirmed by on-target exoglycosidase digestions (data not shown).
The site-specific glycosylation within the N692 site has also been determined after MALDI-MS analysis of both the F1-II (Figure 6B) and F2-III (Figure 6D) affinity fractions, containing glycopeptides bearing the predicted amino acid sequence 683ASLVQEVHQNFSAWCSQVVR702. The MALDI-MS spectrum, depicted in Figure 6B, has two main species at m/z 3848 and 4010, corresponding to the predicted peptide attached respectively to agalactosylated and galactosylated bisected biantennary glycan. It is important to note that these molecular ions can also correspond to the predicted peptide attached to minor triantennary structures. Minor molecular ions were also observed and were consistent with biantennary glycoforms, ranging from agalactosylated to core-fucosylated and bisected structures, and/or triantennary structures, with two terminating galactose. The MALDI-MS analysis of the glycoforms from the F2-III affinity fraction (Figure 6D) showed the presence of abundant and heterogenous high-mannose-type glycans (Hex4–8HexNAc2) and hybrid-type glycans (dHex0,1Hex4–6HexNAc4). The presence of the original structure dHex1Hex6HexNAc4 was not detected because of its low abundance within the total pool of PNGase F-released N-glycans. These structures were also confirmed by on-target exoglycosidase digestions (data not shown).
No tryptic glycopeptides, corresponding to the N645 and N651 glycosylation sites (639QAFYWYNASTGNNLSSQASGAYIFRPNQNKPLFVSHWAQTHLVK682), from the d-peptide of bovine LAMAN, were found by the analysis of both affinity fractions. Therefore, the Con A-unretained material was also fractionated using reverse-phase HPLC, and the fractions were analyzed by MALDI-MS. No glycopeptides corresponding to these sites were found, suggesting that these sites may be unglycosylated or sparsely glycosylated.
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Distribution of the major PNGase F-released N-glycans at each N-glycosylation site |
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Taking into consideration mass spectrometric data obtained from the analysis of each peptide chain of the bovine LAMAN and tryptic Con A-Sepharose-enriched glycopeptides, we conclude that (1) most glycosylation sites are occupied by a broad diversity of carbohydrates; (2) the N645 and N651, from the d-peptide, appear to be sparsely glycosylated or unglycosylated; (3) The N367 site, from the b-peptide chain, is only glycosylated with oligomannosidic structures (Fuc0–1Man3–5GlcNAc2); (4) the N497site, from the c-peptide, is only occupied by high-mannose structures (Glc0–1Man5–9GlcNAc2), including the evolutionary conserved Glc1Man9GlcNAc2 glycan; (5) N692 and N766 sites from the d-peptide chain both bear glycans consisting in oligomannosidic (Man3–7GlcNAc2), hybrid (Fuc0–1Gal0–1Man4–5GlcNAc4), and complex (Fuc0–1Gal0–2Man3GlcNAc4–5) structures; and (6) the N930, from the e-peptide, contains only oligomannosidic-type glycans (Fuc0–1Man3–5GlcNAc2).
With respect to the N133, from the a-peptide, site-specific glycosylation can be conducted, taking into consideration the data obtained from the analysis of the in-gel-released glycans from the abc- and c-peptides and the tryptic glycopeptide containing the N367 site, belonging to the b-peptide. Indeed, among the pool of in-gel-released glycans from the abc-peptide (Figure 3B), complex- (Fuc0–1 Gal0–1Man3GlcNAc5) and hybrid-type (Fuc0–1Gal0–1 Man5GlcNAc4) N-glycans were present, while they were not found on both the N497 and N367 sites (Figures 3D and 5A). These structures can be assigned to the N133 glycosylation site. Besides, Tollersrud et al. (1997) demonstrated that the N-glycans on the N133 glycosylation site can be released using the endoH enzyme. Thus, the N133 site (a-peptide) is glycosylated as represented in Scheme 2, which also illustrates the site-specific distribution of the major PNGase F-released glycans from the bovine LAMAN within each glycosylation site.
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Discussion |
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Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked oligosaccharide chains. Carbohydrate moieties can play important structural and functional roles in a glycoprotein such as immunogenicity, solubility, protein conformation, molecular recognition, intracellular trafficking, or protease resistance (Varki, 1993
; Helenius and Aebi, 2001, 2004; Trombetta, 2003). They are involved in the proper folding of the newly synthesized nascent proteins within the ER, by interacting with the components of the control quality machinery (for reviews see Spiro, 2002; Helenius and Aebi, 2004; Trombetta and Parodi, 2005). It has been reported that glycosylation could also be essential for lysosomal stability and activity in some lysosomal hydrolases by maintaining correct protein conformation (Ferlinz et al., 2001; Wujek et al., 2004; Berg T, Hansen GM, et al., in preparation).
We have used bovine LAMAN as an interesting model for understanding the human glycoprotein as well as the pathophysiology of its defect-related disease, namely -mannosidosis. This enzyme was shown to be glycosylated, and seven of the eight N-glycosylation sites were site occupied (Tollersrud et al., 1997; Heikinheimo et al., 2003). Apart from mediating lysosomal sorting and transport (Tollersrud et al., 1997; Heikinheimo et al., 2003) or maintaining lysosomal stability (Berg T, Hansen GM, et al., in preparation), the putative functions associated with its carbohydrates remained unknown. As a first step toward elucidation of N-glycan functions, we have initiated the complete structural elucidation of the major N-glycans as well as their site-specific distribution at each N-glycosylation site of bovine LAMAN. In this study, we first determined the structure of all N-linked glycans of the bovine LAMAN, summarized in Figure 2, as the major structures to be released by PNGase F. The major structures fall into three classes, namely high-mannose-type (Fuc0–1Glc0–1Man4–9GlcNAc2), hybrid-type (Gal0–1Man4–5GlcNAc4), and complex-type (Fuc0–1Gal0–2 Man3GlcNAc3–5) N-glycans, with or without core fucosylation and with or without bisecting GlcNAc (Figure 2). A minor monoglucosylated high-mannose N-glycan (Glc1Man9GlcNAc2) was also described and is consistent with the evolutionary conserved structure among LAMANs. High-mannose structures are more abundant than hybrid and complex structures. The major complex-type oligosaccharides have compositions consistent with biantennary structures with a bisecting GlcNAc residue or triantennary structures without (1,6)-core fucosylation. The major nonreducing epitopes in the complex-type glycans are GlcNAc and Gal(ß1–4)GlcNAc. Thus, in the lysosomal environment of the bovine LAMAN, the oligosaccharides may undergo degradation to shorter chains. Indeed, since the high-mannose glycans represent potential substrates for the enzyme itself, some of the truncated and high-mannose glycans (Fuc0–1Man3–5GlcNAc2) may not represent native structures and may arise from high-mannose structures with a greater number of mannose residues. For example, truncated high-mannose structure Man4GlcNAc2 may be essentially obtained from LAMAN-digested hybrid-type Man4–5GlcNAc4 structures, which further lose their GlcNAc-terminated residues. This hypothesis is confirmed by the presence of core fucosylation on both Man4GlcNAc2 and Man5GlcNAc2 structures, which requires an unsubstituted GlcNAc residue (ß1,4)-linked to the -Man residue of the (1,3)-antennae of the N-glycan for the transfer (Harpaz and Schachter, 1980; Kornfeld and Kornfeld, 1985). Although (1,6)-core fucosylation appears to be a terminal event occurring exclusively on complex or hybrid structures, there is growing evidence that core-fucosylated high-mannose structures can also occur in mammalian cells (Lin et al., 1994; Endo et al., 1996; Hoja-Lukowicz et al., 2000). In fact, some lysosomal glycoproteins, including porcine LAMAN, typically possessing a high content of high-mannose structures, were shown to carry core-fucosylated high-mannose-type N-glycans (Man5GlcNAc2 and smaller) (Howard et al., 1982; Takahashi et al., 1983, 1984; Taniguchi et al., 1985; Kozutsumi et al., 1986; Maley et al., 1989). Finally, the Fuc0,1Man3GlcNAc2 structures arise in the final catabolic products from the aspecific lysosomal exoglycosidase action on the native glycans. Also, no 6-phosphomannosyl high-mannose glycans, as the recognition signal for lysosomal proteins, were found among PNGase F-released N-glycans, probably due to their cleavage by lysosomal phosphatases.
The occurrence of high-mannose glycans, including the rare Glc1Man9GlcNAc2 structure, stably carried on a mannosidase, suggests that they may play some critical function for the proper protein folding as well as stability against proteolysis, as demonstrated on jack bean -mannosidase (Kimura et al., 1999). Indeed, Kimura and co-workers have shown that the high-mannose structures, including Glc1Man9GlcNAc2, could only be removed by endoH, under denaturing conditions; this last observation suggests that these glycans were buried within the folded polypeptide and, thus, protected from its hydrolytic activity. When the high-mannose-type glycans were removed by endoH prior to renaturation, recovery of -mannosidase activity failed, indicating that this glycan appears to be important for enzyme activity. Additionally, monoglucosylated N-glycan on the lepidopteran hemolymph arylphorin storage protein could not be released under native conditions by PNGase F (Kim et al., 2003). This last observation indicates that the structures were protein buried and thus protected against enzymatic activity. These observations confirm the role for this glycan as structural anchor for maintenance of the protein folding, promoting for example a high stability against proteolysis. The presence of monoglucosylated N-glycans in mature secreted glycoproteins suggests that these oligosaccharides are located in a protein region for which folding does not depend on calnexin/calreticulin lectin chaperones. This localization would prevent the removal of terminal glucose residue by ER--glucosidase II (for reviews see Spiro, 2002; Helenius and Aebi, 2004; Trombetta and Parodi, 2005).
Bovine LAMAN has previously been shown to contain eight potential N-glycosylation sites (N133, N367, N497, N645, N651, N692, N766, and N930), most of them being glycosylated (Tollersrud et al., 1997). In this study, we characterized the site-specific glycosylation of the bovine enzyme, analyzing each individual electrophoretically separated peptide chain and tryptic glycopeptides. The N133, N367, N497, and N930 glycosylation sites were not fully occupied, as judged by the results of SDS–PAGE (Figure 3A), which revealed the presence of both nonglycosylated and glycosylated forms of a-, b-, c-, and e-peptides. It is important to note that due to the low amount of protein sample available, and in view of the complexity of the structural studies of bovine LAMAN, no quantitative data with respect to the relative abundance of each glycan or the level of site occupancy of the six glycosylation sites have been generated. This is the first report on site-specific glycosylation analysis on a LAMAN. For all eight potential N-glycosylation sites, our results indicate that six are glycosylated, namely N133, N367, N497, N692, N766, and N930 (Scheme 2). However, we cannot exclude that the N645 and N651 of the peptide d are glycosylated. Structures deduced at these glycosylation sites were in agreement with those previously identified and summarized in Figure 2.
The N367 (b-peptide) and N930 (e-peptide) glycosylation sites are only glycosylated with truncated (Fuc0–1Man3–4 GlcNAc2) and high-mannose structures (Fuc0–1 Man5GlcNAc2). The occurrence of truncated and small high-mannose structures indicates that these glycans are located at the surface of the enzyme, as all putative -mannosyl residues from high-mannose structures were trimmed, as previously observed for some lysosomal glycoproteins (Howard et al., 1982; Takahashi et al., 1983, 1984; Taniguchi et al., 1985; Kozutsumi et al., 1986; Maley et al., 1989; Kimura et al., 1999). These structures, or those from which they were derived, were hypothesized to be potential sites for lysosomal sorting.
The N692 and N766 sites of the d-peptide show a broad microheterogeneity, resembling that of total PNGase F-released glycans from bovine LAMAN (Figure 1A, Scheme 2). These glycosylation sites bear glycans comprising in high-mannose (Fuc0–1Man3–7GlcNAc2), hybrid (Fuc0–1Gal0–1Man4–5GlcNAc4), and complex (Fuc0–1 Gal0–2Man3GlcNAc4–5) structures. Both these sites therefore represent the only sites bearing a majority of complex-type glycans, suggesting that these were surface exposed within the molecule, thus allowing accessibility of the oligosaccharides to various processing enzymes, including glycosidases as well as transferases, during the biosynthesis of the LAMAN. However, the N766 glycosylation site has been also hypothesized to be in the main site for lysosomal sorting (Heikinheimo et al., 2003).
N-glycosylation site N497 (c-peptide) is conserved among all known LAMANs from mammalian to plants and was found to be fully glycosylated and occupied by Glc0,1Man9GlcNAc2 glycans. This site was reported to be mainly occupied by Man9GlcNAc2 and Glc1Man9GlcNAc2 structures (Berg T, Hansen GM, et al., in preparation). Although our results confirmed the presence of these structures, smaller high-mannose structures Man5–8GlcNAc2 were shown to be present. These N497-carried glycans were postulated to play a key role as a structural anchor for LAMANs. These high-mannose-type structures were shown to rest against the 3-helix bundle, joining b- and c-peptides (Heikinheimo et al., 2003). This configuration then reduces protein flexibility and allows a higher stability against proteolysis. Glycosylation site N497 and the two proximate cysteines (C493 and C501) form a ß-hairpin loop and were shown to be very important for the maintenance of lysosomal stability in LAMANs. These cysteine residues are essential for the formation of the Glc0,1Man9GlcNAc2 structure (Berg T, Hansen GM, et al., in preparation). Moreover, an identical structural organization has also been described on a cathepsin Z-related protein; it is composed of a high-mannose-type glycan associated with a surface-exposed peptide ß-hairpin loop (Appenzeller-Herzog et al., 2005). This pattern appears to be essential for the capture of secretory glycoproteins within the ER to target them at the ERGIC compartment through the COP II pathway.
The N133 glycosylation site was shown to be glycosylated with a majority of hybrid-type (Fuc0–1Gal0–1Man5 GlcNAc4) glycans with a low amount of truncated and high-mannose-type (Fuc0–1Man4–5GlcNAc2), complex-type (Fuc0–1Gal0–1Man3GlcNAc5) glycans. These structures can also be related to their topology within the polypeptide backbone. Indeed, this site should be surface exposed within bovine LAMAN to allow accessibility of the oligosaccharides to the ER- and Golgi-related processing enzymes, leading to the biosynthesis of core-fucosylated hybrid-type and complex-type glycans during LAMAN biosynthesis. This observation could also account for the low abundance of truncated and high-mannose structures within this site. Moreover, this surface-exposed location of N133 was previously shown through the three-dimensional structure of bovine LAMAN, solved by Heikinheimo and co-workers (2003). They have indeed shown that the N133 glycosylation site is located within a surface-exposed ß-turn between two helices and hypothesized a putative involvement of its carbohydrates in the proper folding of the enzyme.
N-glycans can be crucial for several biological processes and collectively ensure the proper operation of lysosomal hydrolases. The well-known implication is that of lysosomal targeting of acid hydrolases. The well-characterized sorting mechanism for lysosomal proteins is based on a selected phosphorylation of -mannosyl residue(s) of high-mannose structures by the UDP-N-acetylglucosamine: glycoprotein N-acetylglucosamine-1-phosphotransferase (phosphotransferase), followed by subsequent transport to lysosomes by Man-6-P receptors (MPRs) (for reviews see Kornfeld and Mellman, 1989; Kornfeld, 1990; Dahms and Hancock, 2002). Recognition of the site for lysosomal sorting by phosphotransferase was suggested to require distant lysine residues (Metcalf and Fusek, 1993), and the structural pattern in cathepsins suggests a separation of 34 Å between two lysine residues (Cuozzo et al., 1998). However, a small number of lysosomal enzymes, such as acid phosphatase (Waheed et al., 1988), cathepsin D, glucocerebrosidase, sphingolipid-activating protein (Rijnboutt et al., 1991), and -glucosidase (Wisselaar et al., 1993) have been shown to reach lysosomes by an MPR-independent pathway under normal conditions. It has recently been hypothesized that LAMAN may be sorted and carried to lysosomes via an MPR-independent pathway under normal conditions, as may be judged by its routing to lysosomes in NH4Cl-treated cells (data not published). Moreover, it has been shown that all N367, N766, and N930 glycosylation sites were located on the same side of the molecule and are about 40 Å apart within a positively charged region (Heikinheimo et al., 2003). This region consists of two groups of evolutionary conserved lysines among LAMANs and mainly surrounds the N766 glycosylation site. This observation confirms that these sites should be potential sites for phosphorylation and lysosomal sorting.
The detailed glycosylation pattern of bovine LAMAN, determined in this study, in combination with data from the elucidation of its three-dimensional structure, conducted by Heikinheimo et al. (2003), provides insights into the involvement of carbohydrate structures in the biological and biochemical properties of LAMANs.
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Materials and Methods |
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Materials
Bovine LAMAN was purified as described previously (Tollersrud et al., 1997
). Recombinant peptidyl-N-glycosidase F (PNGase F; EC 3.5.1.52
[EC]
) from Escherichia coli was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Sequencing-grade modified trypsin (EC 3.4.21.4
[EC]
) was purchased from Promega (Madison, WI). Jack bean -mannosidase (EC 3.2.1.24
[EC]
), bovine testis ß-galactosidase (EC 3.2.1.23
[EC]
), and bovine kidney -fucosidase (EC 3.2.1.51
[EC]
) were purchased from Sigma Chemicals (St. Louis, MO). Jack bean ß-N-acetylhexosaminidase (EC 3.2.1.30
[EC]
) was purchased for Calbiochem (La Jolla, CA). Con A-Sepharose 4B was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Sodium cyanoborohydride, dimethylsulfoxide, and iodomethane were from Fluka (Buchs, Switzerland). Dithiothreitol (DTT) and iodoacetamide (IAA) were from Bio-Rad (Hercules, CA). Sodium borodeuteride and 2,5-dihydroxybenzoïc acid were purchased from Sigma Chemicals. Methanol, acetonitrile, and trifluoroacetic acid (TFA) were HPLC reagent grade. All aqueous solutions were prepared using ultra-pure water.
Monosaccharide composition analysis
An aliquot of 10 µg of bovine LAMAN was methanolyzed with 0.5 mL methanolic HCl 0.5 M for 16 h at 80°C. After evaporation under a stream of nitrogen, the released methyl glycosides were dissolved in 200 µL anhydrous acetonitrile and peracylated by adding 25 µL heptafluorobutyric anhydride, the reaction being conducted at 150°C for 30 min. After evaporation under a stream of nitrogen, the perheptafluorobutyryl-1-O-methylglycosides were dissolved in anhydrous acetonitrile and then analyzed by gas chromatography (GC) on a Shimatzu instrument equipped with a 25 m x 0.32 mm CP-Sil5 CB Low bleed/MS capillary column and 0.25 µm film phase (Chrompack France, Les Illis, France). The samples were analyzed using a linear gradient of 1.2°C/min from 100 to 140°C (Zanetta et al., 1999).
Reduction and carboxymethylation
An aliquot of 2 mg of bovine LAMAN was dissolved in 1 mL of 0.6 M Tris/HCl pH 8.2 and denatured by 6 M guanidine hydrochloride. The sample was incubated at 50°C for 30 min. The sample was reduced using a fourfold molar excess of DTT over the number of disulfide bridges. The sample was flushed with argon and incubated at 50°C for 5 h. After addition of IAA (5 molar excess over the DTT), the sample was flushed with argon and incubated overnight in the dark at room temperature. The sample was then extensively dialyzed against 50 mM ammonium bicarbonate at 4°C and lyophilized.
Tryptic digestion
The tryptic digestion of DTT-reduced carboxymethylated LAMAN was carried out in 100 mM ammonium bicarbonate buffer (pH 8.4) containing trypsin with an enzyme-to-substrate ratio of 1:50 (w/w). The enzyme reaction was incubated for 24 h at 37°C. The reaction was terminated by boiling for 5 min before lyophilization.
Isolation of tryptic glycopeptides by Con A immobilized affinity chromatography
To isolate the N-glycopeptides, the tryptic digest from the reduced carboxamidomethylated bovine LAMAN was loaded onto Con A-Sepharose 4B column (2 mL) equilibrated in buffer A (5 mM sodium acetate pH 4.5, 100 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2). After washing with 20 mL of buffer A, the low-affinity N-glycopeptides were eluted with 20 mL of buffer B (Buffer A + 10 mM -methylglucopyranoside), and the high-affinity glycopeptides were eluted with 20 mL of buffer C (Buffer A + 0.5 M -methylglucopyranoside). Both fractions F1 and F2 eluted respectively with buffers B and C were lyophilized and desalted on a C18 Sep-Pak cartridge.
Reverse-phase HPLC separations of the tryptic glycopeptides
The Con A-purified tryptic glycopeptides were fractionated using reverse-phase HPLC on an XTerra MS C18 (2.1 x 150 mm; 3.5 µm) column. The column was equilibrated with 0.1% (v/v) TFA. After injection, isocratic conditions were applied for 10 min, with the initial solvent, followed by a linear gradient to 80% (v/v) acetonitrile in 0.1% (v/v) TFA for 180 min. The flow rate was 0.125 mL/min and at absorbance was measured at 214 nm. The collected fractions were lyophilized.
PNGase F-digestion of tryptic glycopeptides
PNGase F-digestion was performed in 50 mM ammonium bicarbonate buffer pH 8.4 at 37°C for 24 h. The reaction was terminated by lyophilization, and the products were purified on a C18 Sep-Pak (Waters, Milford, MA) to separate the N-glycans from the de-N-glycosylated peptides. After conditioning the C18 Sep-Pak by sequential washing with methanol (5 mL), water (5 mL), acetonitrile (5 mL), and 0.1% (v/v) TFA (2x 5 mL), the sample, dissolved in 0.1% (v/v) TFA, was loaded onto the C18 Sep-Pak, and the glycans were eluted with 3 mL of 0.1% (v/v) TFA. The released oligosaccharides were then lyophilized.
Reductive elimination
Putative O-glycopeptides remaining after PNGase F-digestion of the tryptic glycopeptides were subjected to reductive elimination according the procedure developed by Carlson (1968). After terminating the reaction with glacial acetic acid, the sample was purified on a column (7 x 0.5 cm) of Dowex 50 (8X; H+ form), and the unbound material was then lyophilized. Borate salts were removed by several evaporations with methanol containing 5% (v/v) acetic acid.
Clean-up procedure PNGase F-released N-glycans
The released N-glycans were desalted on columns of 150 mg of nonporous graphitized carbon (Alltech, Deerfield, IL) according to the procedure described previously (Packer et al., 1998). The columns were sequentially washed with 5 mL methanol and 2x 5 mL 0.1% (v/v) TFA. The glycans were dissolved in 300 µL of 0.1% (v/v) TFA, applied to the column, and washed with 2x 5 mL of 0.1% (v/v) TFA. The elution of glycans was conducted with the application of 5 mL of 60% (v/v) acetonitrile in water containing 0.1% (v/v) TFA. The fractions were freeze-dried.
Permethylation of PNGase F-released N-glycans
Permethylation using the sodium hydroxide procedure was performed according to Ciucanu and Kerek (1984). After derivatization, the reaction products were further purified on a C18-Sep-Pak. The C18 Sep-Pak was sequentially conditioned with methanol (5 mL), water (5 mL), acetonitrile (5 mL), and water (5 mL). The derivatized glycans dissolved in methanol were applied on the cartridge, washed with 3x 5 mL water, 2 mL of 10% (v/v) acetonitrile in water, and eluted with 3 mL of 80% (v/v) acetonitrile in water. Acetonitrile was evaporated under a stream of nitrogen, and the sample was freeze-dried.
Linkage analysis
The permethylated oligosaccharides were hydrolyzed in 500 µL of 4 M TFA at 100°C for 4 h. After removing the acid, the permethylated compounds were then reduced at room temperature overnight by adding 200 µL of 2 M ammonia solution containing sodium borodeuteride (5 mg/mL). The reduction was terminated by adding acetic acid, and borates were eliminated under a stream of nitrogen in the presence of methanol containing 5% (v/v) acetic acid. After adding 50 µL of pyridine and 200 µL of acetic anhydride, peracetylation of the samples was conducted at 100°C for 2 h. After evaporation under a stream of nitrogen, the partially methylated alditol acetates (PMAA) were dissolved in chloroform, and the chloroform phase was washed 10 times with water. This PMAA-containing phase was finally dried under a stream of nitrogen, and the PMAA were dissolved in methanol GC-MS analysis. GC separation of PMAA was performed using a Carbo Erba GC 8000 gas chromatograph fitted with a 25 m x 0.32 mm CP-Sil5 CB Low bleed capillary column, 0.25 µm film phase (Chrompack France, Les Upis Cedex, France). The temperature of the Ross injector was 260°C. Samples were analyzed using a temperature program starting by a gradient of 2°C/min from 130 to 180°C, after 2 min at 130°C, followed by a gradient of 4°C/min until 240°C. The column was coupled to a Finnigan Automass II mass spectrometer. Analyses were performed in the electron impact mode using an ionization energy of 70 eV. Quantification of the various PMAA derivatives was carried out using total ion current (TIC) of the MS detector in positive ion mode.
Sequential exoglycosidase digestions
These were carried out on PNGase F-released glycans onto the MALDI sample plate according to the procedure developed by Mechref and Novotny (1998) and in corollary in-solution using the following enzymes and conditions: ß-galactosidase, 10 mU in 50 mM ammonium formate pH 4.6; ß-N-acetylhexosaminidase, 0.2 U in 200 µL of 50 mM ammonium formate pH 4.6, and -mannosidase, 0.5 U in 200 µl of 50 mM ammonium acetate buffer, pH 4.5. All enzyme digestions were incubated at 37°C for 48 h with a fresh aliquot of enzyme being added after 24 h and terminated by boiling for 10 min before lyophilization. An appropriate aliquot was taken after each digestion and permethylated for MALDI-MS and GC-MS analysis.
SDS–PAGE of bovine LAMAN
An aliquot of bovine LAMAN was denaturated in a Laemmli buffer at 100°C for 10 min and run on 12.5% gel (18 x 20 cm), as described previously (Laemmli, 1970). The electrophoresis was conducted in the running buffer (25 mM Tris /190 mM glycine /0.1% (w/v) SDS), using a PROTEAN II xi CELL (Bio-Rad) under constant current (20 mA for stacking step, 35 mA for running step). The gel was then fixed, stained with a Coomassie Brilliant Blue R-250 dye for 15 min, and partially destained with a methanol-acetic acid-water (25:7:68; v/v/v) overnight.
Processing of peptides LAMAN-corresponding bands excised from gels
The Coomassie-stained bands were excised from the gel using a sterile scalpel and transferred into 0.5 mL microcentrifuge tubes (Eppendorff). Each excised gel piece was further destained by washing several times with the two following solutions: 50 mM ammonium bicarbonate-acetonitrile (1:1, v/v) and acetonitrile for 20 min. The solution was then removed, and the gel pieces were dehydrated with acetonitrile for 20 min. After removing acetonitrile, the gel pieces were left to dry in a vacuum centrifuge for 30 min at room temperature. Each gel piece was further suspended in a 50 mM ammonium bicarbonate containing 20 mM DTT. Peptides-corresponding bands were then reduced at 56°C for 45 min. After having removed the DTT-containing supernatant, each gel piece was suspended with a 50 mM ammonium bicarbonate containing 110 mM of IAA and S-alkylation conducted overnight in the dark at room temperature. The alkylant reagent-containing solution was removed, and the gel pieces were washed several times in 100 mM ammonium bicarbonate for 20 min. The gel pieces were finally dehydrated with acetonitrile and dried in the vacuum centrifuge for 45 min.
In-gel PNGase F-digestion
The dried gel pieces were rehydrated in 20 mM ammonium bicarbonate buffer (pH 8.3) containing 1 U of PNGase F, and the enzymatic deglycosylation was performed at 37°C overnight. The PNGase F-released N-glycans were eluted from the gel pieces with water three times for 20 min. The pooled extracted glycans were dried in a vacuum centrifuge and then desalted on minicolumns of 10 mg of nonporous graphitized carbon. The columns were sequentially washed with 1 mL of methanol and 2x 1 mL of 0.1% (v/v) TFA. The glycans were dissolved in 300 µL of 0.1% (v/v) TFA, applied to the column, and washed with 2x 1 mL of 0.1% (v/v) TFA. The elution of glycans was conducted with the application of 1 mL of 60% (v/v) acetonitrile in water containing 0.1% (v/v) TFA. The fractions were dried in a vacuum centrifuge.
In-gel trypsin digestion and extraction of peptides
The dried gel pieces were suspended with a 50 mM ammonium bicarbonate containing 5 mM CaCl2 and trypsin with an enzyme-to-substrate ratio of 1:50 (w/w) and incubated at 37°C overnight. The enzymatic reaction was terminated in boiling water for 10 min, and the tryptic peptides were extracted from gels once with 25 mM ammonium bicarbonate, twice with 45% (v/v) acetonitrile in water containing 5% (v/v) formic acid, and once with 95% (v/v) acetonitrile in water containing 5% (v/v) formic acid for 20 min. The pooled extracted peptides were dried in a vacuum centrifuge. The isolated tryptic peptides were dissolved in 0.1% (v/v) TFA, and an aliquot was desalted using reverse-phase C18 Zip-Tip (Millipore). The C18 Zip-Tip was sequentially equilibrated with methanol, 0.1% (v/v) TFA, and the tryptic peptides were loaded onto. After washing with 0.1% (v/v) TFA, the elution was carried out in a 0.5 mL microcentrifuge tube (Eppendorff) containing 10 µL of 80% (v/v) acetonitrile in water containing 0.1% (v/v) TFA.
Matrix-assisted laser desorption/ionization mass spectrometry
MALDI-MS experiments were carried out on Voyager Elite DE-STR Pro instrument (PersSeptive Biosystem, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm) and a gridless delayed extraction ion source. The spectrometer was operated in positive reflectron mode by delayed extraction with an accelerating voltage of 20 kV and a pulse delay time of 200 nsec and a grid voltage of 66%. All spectra shown represent accumulated spectra obtained by 400–500 laser shots. Sample was prepared by mixing a 1 µL aliquot (5–10 pmoles) with 1 µL of matrix solution on the MALDI sample plate. The matrix solution was prepared by saturating methanol–water (1:1) with 2,5 dihydroxybenzoic acid (DHB) (10 mg/mL).
The on-target sequential exoglycosidase digestions were performed on desalted samples (glycopeptides and glycans), dissolved with ultra-pure water at 5–10 pmoles/µL. An aliquot of 1 µL of each sample was spotted on the MALDI sample plate and reconstituted in 1 µL of reaction buffer (10 mM sodium phosphate, pH 6.5). For enzymatic sequencing, several enzyme arrays, used in combination or not, were added on each spotted sample according to the following procedure: -glucosidase, 150 mU; -mannosidase, 23.75 mU; ß-galactosidase, 1.25 mU; ß-N-acetylhexosaminidase, 150 mU; -fucosidase, 7.7 mU. The MALDI plate was then placed in a crystallization beaker containing water at 37°C for 8 h. The enzymatic reactions were terminated by the addition of 1 µL of a matrix solution (DHB), and the samples were analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS).
Electrospray tandem mass spectrometry (nano ESI-MS/MS)
All analyses were performed using a Q-STAR Pulsar quadrupole time-of-flight (Q-TOF) mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Ontario, Canada) fitted with a nanoelectrospray ion source (Protana, Odense, Denmark). Derivatized glycans, dissolved in a solution of 80% (v/v) methanol and 1% (v/v) acetic acid and peptides, dissolved in an equal volume of methanol–water containing 0.1% (v/v) formic acid (5 fmoles/µL) were sprayed from a gold-coated "medium-length" borosilicate capillaries (Protana). A potential of 800 V was applied to the capillary tip, and the focusing potential was set at –100 V, the declustering potential varying between –60 and –110 V. For the recording of conventional mass spectra, time-of-flight data were acquired by accumulation of 50 MCA (multiple channel acquisition) scans. For generation of MS/MS data, the parent ion was selected by the quadrupole and was subsequently fragmented in the collision cell using nitrogen at a pressure of about 5.3 x 10–5 Torr and a appropriate collision energy. The CID spectra were recorded by the orthogonal TOF analyzer over a range of m/z 100–2000. For the recording of CID spectra, time-of-flight data were acquired by accumulation of 60 MCA scans.
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Conflict of interest statement |
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None declared.
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Acknowledgments |
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This research was supported by the Centre National de la Recherche Scientifique (Unité Mixte de Recherche CNRS/USTL 8576; Director: Dr Jean-Claude Michalski), the Ministère de la Recherche et de l’Enseignement Supérieur. The Mass Spectrometry facility used in this study was funded by the European Community (FEDER), the Région Nord-Pas de Calais (France), and the Université des Sciences et Technologies de Lille.
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Abbreviations |
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Con A, concanavalin A; DHB, 2,5 dihydroxybenzoic acid; ER, endoplasmic reticulum; HPLC, high-performance liquid chromatography; LAMAN, lysosomal
-mannosidase |
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