Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80075, 3508 TB Utrecht, The Netherlands, 2Institut für Physiologische Chemie, Universitätskrankenhaus Eppendorf, D-20246 Hamburg, Germany, and 3Gesellschaft für Biotechnologische Forschung mbH, Structure Research, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Received on February 7, 2000; accepted on April 3, 2000.
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Abstract |
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Key words: human epidermal growth factor receptor/human epidermoid carcinoma A431 cell line/glycoprotein/NMR spectroscopy/mass spectrometry
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Introduction |
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Earlier reports showed that the receptor domain is an 70.9 kDa single polypeptide having 10 to 11 potential N-glycosylation sites occupied with complex-type (predominantly tri- and/or tri'- and tetraantennary chains, with terminal Neu5Ac, Fuc, and -GalNAc) and oligomannose-type (mainly Man8GlcNAc2) oligosaccharides in a ratio of approximately 2:1. No evidence was found for O-linked oligosaccharides (Mayes and Waterfield, 1984
; Cummings et al., 1985
). The N-glycosylation and some glycan processing was suggested to be required for proper transport to the membrane and for establishing a functional conformation of the EGFR (Slieker and Lane, 1985
; Defize et al., 1988
).
The human epidermoid carcinoma A431 cell line, used as a model to study EGFR, has a high density of receptors (2 x 106 receptors/cell) and secretes a soluble 105 kDa glycoprotein, termed secreted EGF receptor (sEGFR), which is related to the cell surface domain of the EGFR (Mayes and Waterfield, 1984; Weber et al., 1984
). This protein represents the amino-terminal extracellular domain of the EGFR lacking its transmembrane and cytoplasmic parts, and has an additional potential N-glycosylation site (Ullrich et al., 1984
). sEGFR has an intact ligand binding site and dimerizes in the presence of EGF (Lax et al., 1991
). The soluble receptor protein can be isolated in relatively large quantities from an A431 variant cell culture supernatant by immunoaffinity chromatography under acidic conditions using monoclonal antibodies against the EGFR. Until now, analysis of the glycosylation of sEGFR has been limited to Con A Sepharose chromatographic data of radioactively labeled sEGFR glycopeptides (Mangelsdorf Soderquist et al., 1988
). Crystals of sEGFR could be obtained in the presence of the ligand (Günther et al., 1990
; Degenhardt et al., 1998
); the 3D structure, however, is still awaited. The protein accounted for less than one-third of the crystal volume indicating a high solvent content as might be expected for a highly glycosylated protein.
In this report, we present the fractionation and structure determination by high-resolution 1H-NMR spectroscopy and mass spectrometry of part of the enzymatically released oligosaccharides of sEGFR. For the first time, such a comprehensive glycan analysis has been performed on a human non-recombinant growth factor receptor. It shows one of the most heterogeneous and complex ensembles of N-glycans published so far.
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Results |
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The 1H-NMR data of the neutral diantennary fractions QdN.1, QdN.3, QdN.4 and QdN.5 (Figure 5) are presented in Table III, and the full structures are depicted in Table IV. The fraction QdN.2 is contaminated with Qd1 as is evident from the typical NMR signals of a Neu5Ac residue. The NMR data of fraction QdN.1 match those of a neutral complex-type (16)-fucosylated diantennary structure with terminal ß-Gal at both antennae (cf. compound CN4; Bergwerff et al., 1995
). Fraction QdN.3 consists of an equimolar mixture of two diantennary isomers, QdN.3A and QdN.3B, differing in the antennary location of the blood group A type 2 determinant, GalNAc(
13)[Fuc(
12)]Gal(ß14)GlcNAc. The occurrence of such an element is characterized by the structural-reporter-group signals of GalNAc (H-1,
5.180; H-2,
4.236; NAc,
2.039), Fuc (H-1,
5.350; H-5,
4.314; CH3,
1.254), and Gal (H-1,
4.598; H-4,
4.212) (Dua et al., 1986
; Kamerling and Vliegenthart, 1992
). The antennary position of the A type 2 determinant can be derived from the
values of Man-4' H-1 and H-2, and of terminal Gal-6/6' H-1. In QdN.3A, the determinant is linked to Man-4 (Man-4' H-1,
4.926 and H-2,
4.104; Gal-6' H-1,
4.474) and, consequently, in QdN.3B to Man-4' (Man-4' H-1,
4.909 and H-2,
4.091; Gal-6 H-1,
4.469). Fraction QdN.4 mainly consists of a diantennary oligosaccharide with the Lex determinant in each of the antennae (cf. compound GP22; Michalski et al., 1991
). The NMR spectrum of fraction QdN.5 shows a new combination of anomeric signals that fit the set of structural reporters of the ALey antigen, earlier established for this epitope in mucin-derived O-glycans (Strecker et al., 1992
): (
13)-linked GalNAc H-1 (
5.198), (
12)-linked Fuc H-1 (
5.295) and CH3 (
1.284), and (
13)-linked Fuc H-1 (
5.126) and CH3 (
1.274). When compared with the A determinant (see QdN.3), shifts for the GalNAc H-1 signal of +0.018 p.p.m. and for the (
12)-linked Fuc H-1 signal of 0.055 p.p.m. are due to the proximity of the (
13)-linked Fuc residue. The ALey determinant occurs exclusively in the lower branch as indicated by the shifts of Man-4 H-1 (
5.095) and H-2 (
4.157), when compared with the NMR data of QdN.1 (Table III).
The 1H-NMR data of the neutral tri'antennary fraction QtN.2 (Figure 8) are presented in Table III, and the full structures are depicted in Table IV. Characteristic H-1 signals for Man-4 and Man-4' ( 5.127 and
4.870, respectively) combined with the Man-3, -4, and -4' H-2 signals show that fraction QtN.2 consists of structures having three antennae composed of N-acetyllactosamine (LacNAc) units [Gal(ß14)GlcNAc], including one antenna (ß16)-linked to Man-4' (cf. tri'antennary structure 11; Vliegenthart et al., 1983
). The major oligosaccharides in fraction QtN.2 have a tri'antennary structure with (QtN.2A; GlcNAc-2 H-1,
4.666 and NAc,
2.095) and without (QtN.2B; GlcNAc-2 H-1,
4.615 and NAc,
2.079) core fucosylation. Minor structures are present in fraction QtN.2 containing an (
12)-linked Fuc residue in a blood group H determinant (Michalski et al., 1991
; Kamerling and Vliegenthart, 1992
), which is reflected by the set of Fuc H-1 (
5.303), H-5 (
4.219), and CH3 (
1.235) structural-reporter-group signals (2D-TOCSY NMR analysis). Another set of Fuc H-1 (
5.351), H-5 (
4.312), and CH3 (
1.250) signals together with the GalNAc set (H-1,
5.182; H-2,
4.236; NAc,
2.039) indicates the presence of a blood group A determinant (see QdN.3). The attachment of both GalNAc and Fuc to terminal Gal results in a shift of the NAc signal of the adjacent GlcNAc residue to
2.061 for GlcNAc-5 (in QtN.2C) and to
2.056 for GlcNAc-5' (in QtN.2D) and/or GlcNAc-7' (in QtN.2E). The change in the chemical shift of Gal H-1 upon addition of an (
12)-linked Fuc residue (in an H epitope) is 0.063 p.p.m. downfield (Vliegenthart et al., 1983
) resulting in signals at
4.532 for both Gal-6 (in QtN.2F) and Gal-6' (in QtN.2G), and at
4.546 for Gal-8' (in QtN.2H). The presence of an (
12)-linked Fuc to a Gal residue, in both an A and H determinant, influences the H-1 and H-2 signals of the Man residue in the corresponding antenna, as is shown by a 2D-TOCSY experiment: Man-4' H-1 (
4.85) and H-2 (
4.07) for structures QtN.2D and QtN.2G, and Man-4 H-1 (
5.12) and H-2 (
4.18) for structure QtN.2C and QtN.2F. Based on these findings, the presence of structures QtN.2CQtN.2H is indicated (Table IV). For a discussion of the core-fucosylated analogues, that cannot be excluded as minor components, see the description of fraction QtN.3.
Fraction QtN.3 was analyzed by MALDI-TOF-MS showing overlap with fraction QtN.2. The mass spectrum (Figure 9) shows two major sodiated molecular ions at m/z 2175 [Hex6HexNAc5dHex + Na] and m/z 2321 [Hex6HexNAc5 dHex2 + Na] corresponding to glycan QtN.2A and glycans QtN.3AQtN.3C, respectively. Structures QtN.3AQtN.3C only differ in the position of the (12)-linked Fuc residue, but are all core-fucosylated. The peak at m/z 2175 can also originate from structures QtN.2F QtN.2H, although the NMR data showed that they cannot be major components. Minor sodiated molecular ions at m/z 2378 [Hex6HexNAc6dHex + Na] and m/z 2524 [Hex6HexNAc6dHex2 + Na] are in agreement with the structures QtN.2CQtN.2E and QtN.3DQtN.3F, respectively. Several other minor sodiated molecular ions (m/z 2159, 2201, 2305, 2363, and 2508) indicate the presence of glycans with antennae that lack one or two terminal Gal residues (structures QtN.3G through QtN.3K in Figure 9, proposed structures in Table V). 1H-NMR analysis (1D and 2D-TOCSY) revealed that fraction QtN.3 contains the same structural-reporter-group signals (Table III), including those of A and H determinants, as fraction QtN.2. However, the relatively low intensities of the GlcNAc-2 NAc (
2.080) and H-1 (
4.615) signals, specific for a fucose-free core, are indicative of a higher amount of core fucosylation in QtN.3, and the MALDI-TOF data have been interpreted accordingly. Glycans QtN.3AQtN.3C (Table IV) are the core-fucosylated forms of glycans QtN.2FQtN.2H, whereas oligosaccharides QtN.3DQtN.3F (Table IV) are the core-fucosylated forms of oligosaccharides QtN.2CQtN.2E. The positions of the H and A determinants are indicated by the H-1 and H-2 signals of the Man residue in the corresponding antenna, as is shown by a 2D-TOCSY experiment: Man-4' H-1 (
4.85) for structures QtN.3B and QtN.3E, and Man-4 H-1 (
5.116) and H-2 (
4.17) for structure QtN.3A and QtN.3D. It may be evident that on the basis of the present information structural variants of QtN.3AQtN.3F with an extra H determinant instead of core fucosylation cannot be excluded.
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1H-NMR spectroscopy, including 2D-TOCSY, indicates that fraction Qd1.6 consists of the (16)-fucosylated analogues of the glycans in Qd1.5 (Table VI). Each of the two fractions includes four structures, in which a Neu5Ac residue is (
23)-linked to one of the antennae. The other antenna carries the blood group A (Fuc H-1,
5.343, H-5,
4.309, and CH3,
1.249; GalNAc H-1,
5.179, H-2,
4.236, and NAc,
2.040; Gal H-1,
4.600 and H-4,
4.222) or Lex (Fuc H-1,
5.125, H-5,
4.827, and CH3,
1.176) determinant.
In oligosaccharide Qd1.6A, the presence of the Lex determinant in the (16) branch is indicated by the structural-reporter-group signals of Gal-6' (H-1,
4.450) and Man-4' (H-1,
4.909) (compare with QdN.4 and Qd1.4). Not only the presence of a Fuc residue on GlcNAc-5' influences the chemical shift of Man-4' H-1, but also Fuc (
12)-linked (in an A epitope) to Gal-6' has such an effect (see QdN.3B), resulting in
4.909 for Man-4' H-1 in structure Qd1.6B. Neu5Ac in structures Qd1.6C and Qd1.6D is (
23)-linked to Gal-6', as is shown by the chemical shift of Gal-6' H-1 at
4.549 (cf. glycan N2.5; Hård et al., 1992
). The Gal-6 H-1 chemical shifts,
4.445 for Qd1.6C and
4.600 for Qd1.6D, indicate the Lex and the blood group A epitope, respectively.
The reduced and permethylated fraction Qd1.7 shows a major sodiated molecular ion in MALDI-TOF-MS at m/z 2970 [Neu5AcHex5HexNAc3dHex3HexNAc-ol + Na] and a minor sodiated molecular ion at m/z 2796 [Neu5AcHex5 HexNAc3dHex2HexNAc-ol + Na] differing only by the mass of a dHex residue (Fuc, see monosaccharide analysis). The composition of the antennae in reduced and permethylated fraction Qd1.7 was determined in more detail by methylation analysis and ESI-MS-MS. Methylation analysis revealed the presence of terminal Fuc, 3,4-disubstituted GlcNAc, terminal Gal, and 3-substituted Gal residues, supporting structure Qd1.7A (with the sialyl-Lex and the Lex determinant). In ESI-MS, the major disodiated molecular ion at m/z 1496 [Neu5AcHex5HexNAc3dHex3HexNAc-ol + 2 Na] was selected for further fragmentation. The presence of a fragment at m/z 490 [dHexHexNAc-ol + Na] shows that the major molecular-ion peak corresponds to core-fucosylated glycans. Fragments at m/z 1021 [Neu5AcHexHexNAcdHex + Na] (and m/z 646: loss of Neu5Ac) and m/z 660 [HexHexNAcdHex + Na] indicate the presence of the sialyl-Lex determinant and a fucosylated Gal-GlcNAc unit in different antennae. A vertical bar is used to indicate that the position at the antennae is not specified.
Other fragments in the ESI-MS-MS spectrum of m/z 1496 of fraction Qd1.7 at m/z 847 [Neu5AcHexHexNAc + Na] and m/z 472 (loss of Neu5Ac from m/z 847) originate from a sialylated, non-fucosylated antenna, whereas the peaks at m/z 834 [HexHexNAcdHex2 + Na] and m/z 433 [HexdHex + Na] represent a Gal-GlcNAc unit bearing two Fuc residues. In the methylation analysis, apart from the before-mentioned residues, also a 2-substituted Gal residue is present. This structural information gives rise to structure Qd1.7B, containing the Ley antigen.
Methylation analysis shows that 3-substituted Gal (the attachment site for Neu5Ac) is present in a ratio of 2:1 to terminal Gal, implying that structure Qd1.7A, with a terminal Gal, accounts for 50% of the structures in fraction Qd1.7. The fragment ions at m/z 834 (typical for Qd1.7B) and m/z 660 (typical for Qd1.7A) are equally intense, therefore, a structural isomer of glycan Qd1.7A with the blood group H determinant, instead of the Lex epitope, is excluded as a major component of fraction Qd1.7.
The minor disodiated molecular ion in the ESI-MS spectrum at m/z 1409 [Neu5AcHex5HexNAc3dHex2HexNAc-ol + 2 Na] in Qd1.7 was also further fragmented. The presence of 4-substituted GlcNAc-ol in the methylation analysis indicates the absence of core fucosylation. ESI-MS-MS peaks at m/z 1021, 646, 660, and 316 [HexNAc-ol + Na] confirm the presence of structure Qd1.7C as an analogue of Qd1.7A without core fucosylation.
The MALDI-TOF mass spectrum of fraction Qd1.8 (after reduction and permethylation) displays a sodiated molecular ion at m/z 3216 [Neu5AcHex5HexNAc4dHex3HexNAc-ol + Na]. Methylation analysis yields 2,3- and 3-substituted Gal, terminal Fuc, 3,4-di- and 4-substituted GlcNAc, and terminal GalNAc residues, confirming structure Qd1.8. Fragmentation of the disodiated ESI-MS molecular ion at m/z 1620 [Neu5AcHex5HexNAc4dHex3HexNAc-ol + 2 Na] results in fragments at m/z 847 and 472 representing an antenna composed of Neu5AcHexHexNAc. Fragments at m/z 1079 [HexHexNAc2dHex2 + Na] and 678 [HexHexNAcdHex + Na] correspond to an antenna ending in the ALey determinant. The presence of a fragment at m/z 490 and the absence of a fragment at m/z 316 show that glycan Qd1.8 is core-fucosylated.
It should be noted that for fractions Qd1.7 and Qd1.8 the MS data indicating a fucosylated Gal-GlcNAc sequence have been interpreted as Gal(ß14)[Fuc(13)]GlcNAc and not as Gal(ß13)[Fuc(
14)]GlcNAc. This choice is based on the fact that all structures analyzed by NMR only contain Gal(ß14)GlcNAc sequences.
Disialylated oligosaccharides
The 1H-NMR data of fraction Qd2 (Figure 4) are presented in Table VI, and the numbering of the residues is depicted in structure Qd1.5D above. The structural-reporter-group signals of the major component of fraction Qd2, glycan Qd2A (60%), indicate a core-fucosylated (23)-disialylated diantennary structure (cf. glycan N2.4; Hokke et al., 1995
), and about 25% of fraction Qd2 consists of Qd2B, the non-fucosylated analogue of glycan Qd2A (cf. glycan N2B; Damm et al., 1987
). The remaining 15% of the structures have the sialyl-Lex determinant (Fuc H-1,
5.108, H-5,
4.816, and CH3,
1.166) linked to one of the antennae. The Gal-6 H-1 and Gal-6' H-1 signals at 4.510 and 4.519 p.p.m., respectively, show that the sialyl-Lex epitope is linked to the (
13) branch in glycan Qd2C and to the (
16) branch in glycan Qd2D. Because the intensity of the Gal-6 H-1 signal is higher than that of Gal-6' H-1, a structure with sialyl-Lex epitopes linked to both branches is excluded as a prominent minor fraction. In view of the fact that mixtures were analyzed, the occurrence of relatively small amounts of non-core-fucosylated analogues of Qd2C and Qd2D cannot be excluded.
Tetrasialylated oligosaccharides
1H-NMR data of the HPAEC fractions Q4.4, Q4.5, Q4.7, and Q4.8 (Figure 2) are presented in Table VII, and a full structure and its residue numbering is depicted in structure Q4.4A below.
NMR analysis shows that all fractions contain tetraantennary oligosaccharides, being evident from the typical set of values of the Man-4 and Man-4' H-1 signals and the Man-3, Man-4, and Man-4' H-2 signals (Vliegenthart et al., 1983; Hokke et al., 1995
). All glycans in the Q4 fractions have an (
16)-linked Fuc residue at GlcNAc-1, as follows from the typical set of Fuc H-1, H-5, and CH3
values, and the GlcNAc-2 NAc signal (Hokke et al., 1995
). Neu5Ac residues are (
23)-linked to Gal residues at all nonreducing termini, as indicated by the combined set of
values of the Neu5Ac H-3a/3e, Neu5Ac NAc, and Gal H-3 signals (Vliegenthart et al., 1983
).
Oligosaccharide Q4.8, the major constituent in Q4, is a tetrasialylated tetraantennary glycan with all four antennae composed of Neu5Ac(23)Gal(ß14)GlcNAc(ß1- (cf. glycan N4.4; Hokke et al., 1995
). The NMR spectra of fractions Q4.8 and Q4.9 are alike indicating that the two fractions are derived from the same glycan. Considering the alkaline conditions (pH 13) during the HPAEC separation, epimerization at C-2 of the reducing GlcNAc into ManNAc is obvious (Toida et al., 1996
). This structural change is confirmed by monosaccharide analysis, and by incubation of comparable compounds in 0.1 M NaOH followed by reinjection on an HPAEC column (data not shown).
Fraction Q4.7 contains a mixture of isomeric structures with an extra LacNAc unit in one of the antennae of structure Q4.8. First of all, this is shown by the reporter signals of the Gal residue extended with the LacNAc unit: Gal-8' H-1 ( 4.468) and H-4 (
4.161) in Q4.7A. The Gal-8' H-1 signal in case of the absence of an extra LacNAc unit (
4.557) differs from those of Gal-6, -6', and 8, and can therefore be used for the assignment of the location of the extra LacNAc element in structure Q4.7A (Hokke et al., 1991
, 1995). The other isomers, with a LacNAc unit linked to Gal-6, 6', or 8, are summarized in structure Q4.7B (Table VII). The 1H-NMR data fit those of structure C in Hokke et al. (1991)
, in which the extra LacNAc element is linked to Gal-6' (H-1,
4.542), but since no data are available on the other two isomers, their absence or presence cannot be confirmed. As has been reported before (Hermentin et al., 1992
), the extra LacNAc unit in a sialylated oligosaccharide leads to a decrease of its retention time on HPAEC, as is the case for fraction Q4.7 that elutes 1 min earlier than Q4.8 (Figure 2).
The second major fraction, Q4.5, differs from Q4.8 only by the presence of an extra (13)-linked Fuc residue in one of the antennae (H-1,
5.103; CH3,
1.169) constituting the sialyl-Lex determinant. Based on a comparison of the
values of the NAc signals of antennary (
13)-fucosylated GlcNAc and nonfucosylated GlcNAc residues (e.g. QdN.4 versus QdN.1 and Qd1.5A versus Qd1.4), a shift of about 0.008 p.p.m. is expected. Comparing the NAc regions of Q4.5 and Q4.8 shows the presence of an additional signal for Q4.5 at
2.065, indicating an (
13)-fucosylated GlcNAc-7 (
0.009). The intensities of the GlcNAc-7 NAc signals at
2.074 and
2.065 show that fraction Q4.5 consists of an isomeric mixture of about 65% Q4.5A (extra Fuc residue linked to GlcNAc-7) and 35% Q4.5B, having the sialyl-Lex determinant at one of the other antennae. Shifts in the GlcNAc-5, -5', and -7' NAc signals due to an extra Fuc residue cannot be distinguished because of overlap with other NAc signals. Further evidence for glycan Q4.5A is shown by the Gal-8 H-1 signal at
4.517 (Maaheimo et al., 1995
). Because of the signal of Gal-8' H-1 at
4.530, Q4.5B contains at least the isomer with the Fuc (
13)-linked to GlcNAc-7'. The addition of a Fuc residue to an oligosaccharide also results in the decrease of HPAEC retention time, as is seen for Q4.5, having an elution time of 3.8 min lower than Q4.8, in Figure 2 (Hardy and Townsend, 1988
; Basa and Spellman, 1990
).
After permethylation, fractions Q4.2 and Q4.4 have been analyzed by FAB-MS. The spectra do not contain molecular ions, but typical fragments of the antennae can be identified. In the spectrum of fraction Q4.4 peaks are present at m/z 825 [Neu5AcHexHexNAc + H] and m/z 1448 [Neu5AcHex2 HexNAc2dHex + H] showing that one of the antennae consists of a sialylated dimeric LacNAc element with one Fuc residue attached. The absence of a fragment at m/z 999 [Neu5AcHexHexNAcdHex + H] indicates that the Fuc residue is linked to the inner of the two LacNAc units. 1H-NMR analysis of fraction Q4.4 shows the presence of an (13)-linked Fuc residue and an extra LacNAc unit (Table VII). FAB-MS shows that the two elements are located on one antenna, and NMR data provide the positions of the Fuc residue and the LacNAc unit. In glycan Q4.4A, the position of Fuc at GlcNAc-7 is determined by the characteristic shift of the GlcNAc-7 NAc signal (
2.066, see Q4.5A), and by the Gal-8 H-1 signal due to extension with a LacNAc element (
4.438). On the other hand, in glycan Q4.4B the position of the extra LacNAc unit at Gal-8' is characterized by the Gal-8' H-1 signal (
4.450), which differs from the value for Gal-8' in Q4.7A (
4.468) because of the Fuc residue linked to GlcNAc-7'.
The FAB-MS spectrum of fraction Q4.2 contains fragment ions at m/z 825, 999, and 1448, which demonstrates that one antenna contains a dimeric LacNAc element and a Fuc residue linked at one of the two LacNAc units. The elution time on HPAEC (Figure 2), 6.9 min lower than that of fraction Q4.8, implies that fraction Q4.2 includes a glycan with two (13)-linked Fuc residues and one extra LacNAc unit (Basa and Spellman, 1990
; Hermentin et al., 1992
). The 1H-NMR data show that the ratio between the CH3 signals of the (
16)-linked Fuc at GlcNAc-1 and the (
13)-linked Fuc is 1.0:1.5, indicating that, next to components with one (
13)-linked Fuc, structures with two (
13)-Fuc residues are present. Furthermore, 80% of the GlcNAc-7 NAc signal in fraction Q4.2 resonates at
2.064, which indicates that in 80% of the glycans a Fuc residue is (
13)-linked to GlcNAc-7. Combining these data with those from FAB-MS and the HPAEC profile leads to a major component (4050% of fraction Q4.2) with an (
13)-fucosylated GlcNAc-7 and an (
13)-fucosylated dimeric LacNAc element (Q4.2A). Fucosylation at the inner of the two LacNAc units cannot be excluded. The position of the extended antenna is not specified, as is indicated by the vertical bar.
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Discussion |
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In this study, oligomannose-type structures (5) account for about 17% of the glycans and range from Man5GlcNAc2 to Man8GlcNAc2, but being mainly Man6GlcNAc2 and Man7GlcNAc2. This is different from the 33% oligomannose-type glycans on membrane-bound EGFR (Cummings et al., 1985) and the 31% on sEGFR (Mangelsdorf Soderquist et al., 1988
). The complex-type glycans on membrane-bound EGFR were described by Cummings et al. (1985)
as predominantly asialo tri- and/or tri'- and tetraantennary. If sialic acid was present, its occurrence was limited to one or two residues per glycan chain. The predominance of tri- and/or tri'- and tetraantennary glycans is confirmed for sEGFR, but 59% of the total glycan pool (Q1Q4 in Table I) is sialylated up to four sialic acids per oligosaccharide chain. Man-6-phosphate (Todderud and Carpenter, 1988
) and LacNAc repeats (Cummings et al., 1985
) are other structural elements of the membrane-bound form. Only the LacNAc repeats are found in sEGFR and account for about a third of the Q4 fraction representing 2% of the total carbohydrate moiety. The absence of Man-6-phosphate in sEGFR shows that phosphorylation of oligomannose-type glycans is specific for membrane-bound EGFR (Todderud and Carpenter, 1988
).
The sialyl-Lex epitope is present in all sialylated glycan pools of sEGFR (Qd1, Qd2 and Q4), but has not been reported before. On the other hand, Lea and Leb determinants reported by Childs et al. (1984), for the membrane-bound form, have not been found. The diversity in expressed carbohydrate antigens finds its origin in the A431 cells. sEGFR expressed in insect cell line Sf9 does not show this variety in epitopes and only shows two bands on isoelectric focusing gel electrophoresis (Hurwitz et al., 1991
). The high structural diversity of the N-glycans linked to sEGFR reflects the high number of expressed and active glycosyltransferases in A431 cells. Since this cell line is derived from carcinoma cells, it is interesting to compare the glycosylation of sEGFR (and EGFR) with that of EGFR from normal cells. Especially, since EGFR lacking the blood group A antigen has a different receptor functioning, including increase in receptor turnover and the number of high-affinity receptors (Defize et al., 1988
; Engelmann and Schumacher, 1993
). However, detailed information on the glycosylation of EGFR in normal cells is not yet available.
It was concluded earlier that the glycosylations of EGFR and sEGFR are very similar (Mangelsdorf Soderquist et al., 1988). Although the glycan structures clearly show many common features, there are some marked differences. The transport routes from the ER to the cell exterior and the kinetics of the transport are similar for both forms (Mangelsdorf Soderquist et al., 1988
). Therefore, EGFR and sEGFR are exposed to the same glycosylation machinery. Hence, differences in glycosylation can be traced back to the differences between secreted and membrane-bound forms.
Since more than 15 years, attempts are being made to elucidate the 3D structure of the extracellular domain of the receptor and its complex with EGF. Electron microscopic images (Lax et al., 1991) and small angle X-ray scattering (Lemmon et al., 1997
) support a 3D model of sEGFR as a four-lobed, doughnut-like overall shape. However, X-ray crystallography is a necessity to determine in detail the 3D structure, with and without the ligand. Progress has been made on the crystallization (Günther et al., 1990
; Degenhardt et al., 1998
), but the structure remains to be resolved. The immense heterogeneity of the glycosylation of sEGFR, as presented here, clearly points towards the application of glycan processing inhibitors for obtaining less heterogeneous sEGFR molecules.
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Materials and methods |
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In order to remove buffer salts, glycoprotein samples were passed over a Bio-Gel P-2 column (1.5 x 45 cm, Bio-Rad) eluted with 5 mM NH4HCO3. The void volume was collected and lyophilized.
Monosaccharide analysis
Desalted sEGFR samples (400 µg) were subjected to methanolysis (1.0 M methanolic HCl, 24 h, 85°C) followed by re-N-acetylation and trimethylsilylation. The trimethylsilylated (methyl ester) methyl glycosides were analyzed by GLC on a capillary DB-1 fused silica column (30 m x 0.32 mm, J & W Scientific). The value of GlcNAc has been corrected for the relatively stable GlcNAc-Asn linkage (Kamerling and Vliegenthart, 1989).
Release of the carbohydrate chains
The N-linked carbohydrate chains were enzymatically released from the protein by recombinant peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F (PNGase F, EC 3.5.1.52; Boehringer Mannheim), according to a modified version of a previously described protocol (Damm et al., 1989b). sEGFR, in two batches of 30 mg, was dissolved in 10 ml 50 mM Tris, adjusted to pH 7.4 with HCl, containing 50 mM EDTA and 1% (v/v) ß-mercaptoethanol. After adding SDS (34 mg), the mixture was heated for 1 h at 40°C followed by cooling to room temperature. Then, the detergent decanoyl-N-methylglucamide (68 mg; MEGA-10, Boehringer Mannheim) was added and the solution was incubated with 6 U PNGase F for 24 h at room temperature in an end-over-end mixer. With a second portion of 6 U PNGase F the incubation was continued for another 24 h. The incubation was monitored with SDSPAGE, on a 10% slab gel, and Coomassie brilliant blue staining.
The incubation mixture was fractionated on a Superdex 75pg column (60 x 2.6 cm, Pharmacia) using 150 mM NH4HCO3 (pH 7 with HCl) at a flow rate of 4 ml/min (Pharmacia FPLC system). The elution was monitored by conductivity and UV detection (214 nm). Fractions were collected and aliquots stained for carbohydrate with orcinol/H2SO4. Carbohydrate-positive fractions were pooled and lyophilized twice, then desalted on three connected HiTrap Desalting columns (Pharmacia) at a flow rate of 3 ml/min, using 5 mM NH4HCO3 as eluent and subsequent lyophilization. Oligosaccharides were detected as a void-volume peak by monitoring the effluent at 206 nm.
FPLC fractionation
The Superdex carbohydrate-positive fraction derived from the first sEGFR batch was fractionated according to charge on a 1 ml Resource Q anion-exchange column (Pharmacia) at a flow rate of 4.0 ml/min (Pharmacia FPLC system). The column was first eluted with 2 ml H2O, followed by a linear concentration gradient of 050 mM NaCl in 8 ml H2O, a gradient of 50250 mM NaCl in 8 ml H2O, and, finally, by a steeper gradient of 250500 mM NaCl in 4 ml H2O. The eluent was monitored at 214 nm. After lyophilization, the five fractions obtained were desalted on a Bio-Gel P-2 column (45 x 1.6 cm), and lyophilized again. This gel permeation step was also used to separate the neutral fraction roughly into two fractions (complex- and oligomannose-type glycans).
Concanavalin A fractionation
The Superdex carbohydrate-positive fraction derived from the second sEGFR batch was subjected to Concanavalin A Sepharose (Pharmacia) lectin chromatography. For this purpose, a 3 ml column was used, equilibrated with TBS binding buffer (pH 8.0) containing 0.15 M NaCl, 10 mM Tris, 1 mM CaCl2, and 1 mM MgCl2. The column was first eluted with 20 ml binding buffer at a flow rate of 14 ml/h, yielding tri/tri'- and tetraantennary complex-type glycans. Elution with 20 ml binding buffer containing 10 mM methyl -D-glucopyranoside was used, at the same flow rate, to elute the diantennary complex-type glycans. Finally, 20 ml binding buffer containing 100 mM methyl
-D-mannopyranoside, at 60°C, eluted the oligomannose-type glycans. The elution was monitored at 206 nm. The three resulting fractions were separated from the salts and monosaccharides using four connected HiTrap Desalting columns (5 mM NH4HCO3, 3 ml/min) and subsequent lyophilization, followed by fractionation on Resource Q, as described above for the first sEGFR batch.
HPLC fractionation
The oligomannose-type glycan fraction was subfractionated using a Kratos SF 400 HPLC system (ABI Analytical, Kratos Division) equipped with a 100-µm LiChrospher-NH2 column (25 x 0.46 cm, Merck). The column was eluted with a linear concentration gradient of 30 to 38% (v/v) 10 mM phosphate buffer (K2HPO4/KH2PO4, pH 6.5) in acetonitrile for 40 min at a flow rate of 2 ml/min.
The complex-type glycan fractions (neutral and charged) were subfractionated on the same HPLC system equipped with a 10 µm LiChrosorb-NH2 column (25 x 0.46 cm, Merck). The column was eluted with an acetonitrile/phosphate buffer (10 mM potassium phosphate, pH 6.5) gradient program at a flow rate of 2 ml/min. For details concerning the solvent systems, see the legends to Figures 5, 6, and 8.
The eluents were monitored at 206 nm. The HPLC fractions were desalted on three connected HiTrap Desalting columns (flow rate 3 ml/min) using 5 mM NH4HCO3 as eluent and subsequent lyophilization.
HPAEC fractionation
Fractionation of highly charged Resource Q fractions was carried out by high-pH anion-exchange chromatography with pulsed amperometric detection using a Dionex LC system consisting of a Dionex Bio LC quaternary gradient module, a PAD 2 detector, and a CarboPac PA-1 column (25 x 0.9 cm, Dionex) (Damm et al., 1989a). Elutions were carried out at a flow rate of 4 ml/min. Detection was performed with a gold electrode and triple-pulse amperometry, comprising the following pulse potentials and durations at 300 mA: E1 = 0.05 V, 480 ms; E2 = 0.60 V, 120 ms; E3 = 0.60 V, 60 ms. For further details concerning the gradients of the 0.1 M NaOH/0.5 M NaOAc solvent system, see caption to Figure 2. Fractions were immediately neutralized by addition of 1 M HCl and lyophilized. Desalting was carried out on three connected HiTrap Desalting columns (flow rate 3 ml/min) using 5 mM NH4HCO3 as eluent and subsequent lyophilization.
Permethylation and methylation analysis
Desalted and dried HPAEC samples were dissolved in 500 µl dry dimethyl sulfoxide, and freshly powdered NaOH (10 mg) was added. The solution was kept for 10 min at room temperature under an inert atmosphere, then 100 µl CH3I was added (Jay, 1996
). Extra portions of 100 µl CH3I were added after 10 and 20 min. Then, the reaction was quenched by the addition of 1 ml freshly prepared 4 mM sodium thiosulfate. The permethylated oligosaccharides or oligosaccharide-alditols were isolated by extraction with chloroform (3 x 1 ml). The organic phase was washed with water (3 x 0.5 ml) and concentrated.
For methylation analysis, oligosaccharides were permethylated, purified on Sepharose LH-20 (run with ethyl acetate), hydrolyzed, reduced, and peracetylated as described previously (Geyer et al., 1983). Separation and identification of partially methylated alditol acetates was performed on a Finnigan gas chromatograph (Finnigan MAT Corp.), equipped with a 30 m DB-5 capillary column, connected to a Finnigan GCQ ion-trap mass spectrometer (Structure Research, GBF Braunschweig).
1H-NMR spectroscopy
The oligosaccharide samples were exchanged twice in 2H2O (99.9 atom% 2H, Cambridge Isotopes Ltd.) with intermediate lyophilization, then dissolved in 2H2O (99.96 atom% 2H, Isotec Inc.). 1D 1H-NMR spectra were recorded at 500 MHz on a Bruker AMX500 instrument, and at 600 MHz on Bruker AMX600 (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University) or Bruker Avance600 (NSR Center, SON NMR facility, Nijmegen University, the Netherlands) spectrometers at probe temperatures of 300 K. Chemical shifts () are expressed in p.p.m. by reference to internal acetone (
2.225) in 2H2O (Vliegenthart et al., 1983
).
2D-TOCSY spectra at 500 or 600 MHz were recorded using Bruker software with MLEV-17 mixing sequence cycles of 100 ms. Data matrices of 512 x 2048 or 256 x 1024 points were collected representing a spectral width of 4800 Hz in each dimension. The 2HO1H signal was suppressed by presaturation for 1 s during the relaxation delay. Phase-sensitive handling of the data was performed by the time-proportional phase increment method implemented in the Bruker software. The time domain data were zero-filled to data matrices of 1024 x 2048 or 512 x 1024 points, respectively, prior to multiplication with a squared-bell function phase shifted by /3 (Hård et al., 1992
).
Mass spectrometry
For analysis by positive-ion MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry), 2,5-dihydroxybenzoic acid (10 mg/ml 10% v/v ethanol in water) was used as UV-absorbing matrix. One µl of a sample was spotted onto the target, mixed with the matrix solution in a 1:2 ratio and dried at room temperature. The concentrations of the analyte mixtures were approximately 150 pmol/ml. Measurements were performed on a PerSeptive Biosystems Voyager-DE MALDI-TOF mass spectrometer with implemented delayed extraction technique using a N2 laser (337 nm) with 3 ns pulse width. Spectra were recorded in the linear mode at an accelerating voltage of 24 kV using an extraction delay of 150 ns for enhanced resolution.
Positive-ion FAB (fast atom bombardment) mass spectra of permethylated oligosaccharides were obtained using MS1 of a JEOL JMS-SX/SX102A tandem mass spectrometer (Bijvoet Center, Department of Mass Spectrometry, Utrecht University) operated at an accelerating voltage set around 6 kV. The matrix used was thioglycerol and the bombarding gas was Xe.
For ESI-MS-MS (electrospray ionization tandem mass spectrometry), a Finnigan MAT TSQ 700 triple quadrupole mass spectrometer (Structure Research, GBF Braunschweig) equipped with a nanospray ion source (Protana) was used. The reduced and permethylated samples were dissolved in methanol saturated with NaCl (about 10 pmol/ml) and 3 µl of solution was filled into gold-coated nanospray glas capillaries (Protana). The tip of the capillary was placed directly in front of the entrance hole of the heated transfer line of the mass spectrometer and a voltage of 800 V applied leading to flow rates of
50 nl/min. For collision-induced dissociation experiments, parent ions were selectively transmitted by the first mass analyzer and directed into the collision cell (with argon as collision gas) with a kinetic energy set around 58 eV.
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Footnotes |
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References |
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