4 Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands, and 5 Pharming Technologies BV, Archimedesweg 4, P.O. Box 451, NL-2300 AL Leiden, The Netherlands
Received on July 22, 2003; revised on September 10, 2003; accepted on September 11, 2003
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Abstract |
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Key words: C1 inhibitor / glycosylation / transgenic rabbit milk
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Introduction |
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Deficiency in the gene encoding hC1INH causes a condition called hereditary angioneurotic edema, which can be lethal when left untreated (Ebo and Stevens, 2000; Carugati et al., 2001
). Furthermore, the lack of hC1INH leads to excessive complement activation, and this in turn to severe symptoms characterized by sudden local swellings of soft connective tissue (Ebo and Stevens, 2000
). Current forms of treatment include hC1INH replacement therapy, which relies on hC1INH supplies prepared from donated human blood. However, there is a growing number of clinical conditions wherein hC1INH might be beneficial (Caliezi et al., 2000
), hence current supplies of hC1INH are not likely to meet clinical demands.
Recombinant glycoprotein production in mammary glands of transgenic animals with milk extraction is one of the most attractive approaches for the economic and large-scale production of therapeutic glycoproteins (Houdebine, 1995; Colman, 1996
). In addition to providing eukaryotic posttranslational modifications often required for biological activity, it is also the expression platform that provides the highest recombinant protein production capacity available to date, exceeding the capacity of the more established mammalian cell lines. Typical examples so far are human lactoferrin in mice (Nuijens et al., 1997
) and cows (Van Berkel et al., 2002
), human
-glucosidase in mice (Bijvoet et al., 1996
) and rabbits (Visser et al., 2000
), human granulocyte-macrophage colony stimulating factor in mice (Uusi-Oukari et al., 1997
), human erythropoietin in mice and rabbits (Korhonen et al., 1997
), human antithrombin in goats (Edmunds et al., 1998
), and human tissue-type plasminogen activator in goats (Denman et al., 1991
).
The posttranslational glycosylation of proteins is a species-, tissue-, cell-type-, and protein-specific phenomenon. Thus recombinant proteins can have different glycosylation patterns when compared with their native counterparts. Furthermore, in cell cultures, changes in growth media composition and in growth conditions may lead to changes in glycosylation patterns. These findings are of special interest when focusing on human therapeutic recombinant glycoproteins, because specific glycosylation patterns play important roles in the secretion, antigenicity, and clearance of glycoproteins (Jenkins and Curling, 1994; Jenkins et al., 1996
; Kamerling, 1996
; Varki et al., 1999
; Galet et al., 2001
).
Nowadays, much knowledge is available with respect to the glycosylation machineries of the two most popular mammalian cell lines for glycoprotein expression, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells (Kamerling, 1996). However, detailed glycan data and systematic studies on the glycosylation capabilities of the mammary glands of the different species, including the effects of stage of lactation, individual variation, and seasonal variation, are scarce. For human lactoferrin from cow milk (Van Berkel et al., 2002
) it was concluded that besides complex-type N-glycans, as in natural human lactoferrin, also oligomannose- and/or hybrid-type glycans do occur, whereas the presence of N,N'-diacetyllactosediamine units has been suggested. It has been reported that natural bovine lactoferrin contains complex- (both partially [
2-6]-sialylated N- acetyllactosamine and N,N'-diacetyllactosediamine units, and Gal[
1-3]Gal[ß1-4]GlcNAc units) and oligomannose-type N-glycans (Montreuil et al., 1997
). The occurrence of N,N'-diacetyllactosediamine units has also been suggested for human antithrombin produced in goat milk (Edmunds et al., 1998
). In the latter case, in contrast to plasma human antithrombin, the transgenic form also contained oligomannose-type structures, and in addition to N-acetylneuraminic acid, N-glycolylneuraminic acid was detected.
Because the glycosylation machinery of the rabbit mammary glands is rather unexplored, it is unpredictable what kind of glycans would predominate on recombinant glycoproteins from transgenic rabbit milk. In this study, a detailed analysis of the N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits (rhC1INH) is presented and discussed.
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Results |
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Release and fractionation of N-glycans of rhC1INH
The N-glycans of rhC1INH were released by peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F (PNGase F) digestion, separated as a pool from the remaining O-glycoprotein, and fractionated by anion-exchange chromatography on Resource Q. Three carbohydrate-containing fast protein liquid chromatography (FPLC) fractions, eluting at positions corresponding to neutral N0 (25%), monosialylated N1 (67%), and disialylated N2 (8%) structures were obtained (Figure 1). Neutral FPLC fraction N0 was further separated into six high-performance liquid chromatography (HPLC) fractions, denoted N0.1N0.6, by normal phase chromatography on LiChrospher-NH2 (Figure 2). The HPLC fractions N0.3 and N0.4 were subfractionated by high pH anion-exhange chromatography (HPAEC) on CarboPac PA-1 (Figure 3) after 2-aminobenzamide (2AB) labeling of the components present (to minimize high pHinduced epimerization of the reducing GlcNAc residues during HPAEC; Stroop et al., 2000), yielding four and five subfractions, respectively, denoted N0.3.12ABN0.3.42AB and N0.4.12ABN0.4.52AB. The mono-(N1) and dicharged (N2) FPLC fractions were subfractionated on CarboPac PA-1 (Figures 4 and 5), yielding fractions N1.1N1.8 and N2.1N2.3, respectively.
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Structural analysis of neutral N-glycans
The 1H NMR data of the identified neutral oligomannose-, hybrid-, and complex-type N-glycans are presented in Table I. The table includes also the coding system for the different monosaccharide constitutents. In the HPAEC subfraction N0.3.12AB no carbohydrate material could be detected, whereas HPAEC subfractions N0.4.12AB and N0.4.32AB did not contain sufficient material for NMR assignments. The same held for HPLC fraction N0.6.
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MALDI-TOF-MS analysis (positive-ion mode) of HPLC fraction N0.3 showed a major sodiated molecular ion at m/z 1419 (Hex6HexNAc2 + Na), and minor ones at m/z 1257 (Hex5HexNAc2 + Na) and 1460 (Hex5HexNAc3 + Na).
The 1H NMR spectrum of HPAEC subfraction N0.3.22AB contained the typical signals for a Gal(ß1-4)GlcNAc(ß1-2)Man(1-3) sequence attached to Man-3 (Man-4, H-1/H-2,
5.111/4.189; GlcNAc-5, H-1,
4.574; Gal-6, H-1,
4.466). These structural-reporter-group signals and those of terminal Man-A (H-1/H-2,
5.103/4.061) and Man-4' (H-1/H-2,
4.892/4.133) occur in equimolar proportions. In the N-acetyl methyl region only three signals were present, assigned to GlcNAc-5 (NAc,
2.045), GlcNAc-2 (NAc,
2.056), and GlcNAc-1 (NAc,
1.988). Such a combination of structural-reporter groups indicates the presence of a hybrid-type glycan, with Man-A extending Man-4' on the
1-6 branch, whereas Man-4 on the
1-3 branch carries an N-acetyllactosamine unit. 1H NMR data of a similar structure, but
(2-3)-sialylated and with GlcNAc-2 as reducing unit have been reported (compare with compound EH-1 in Spellman et al. [1991]
).
1H NMR analysis of the major component of HPLC fraction N0.3, HPAEC subfraction N0.3.32AB, revealed the presence of only two N-acetyl signals corresponding to GlcNAc-1 ( 1.987) and GlcNAc-2 (
2.060). Taking into account the MALDI-TOF MS data of fraction N0.3, an oligomannose-type N-glycan is indicated. The anomeric region of the 1H NMR spectrum showed a H-1 signal at
5.404, in accordance with the presence of a substituted Man-A residue (Vliegenthart et al., 1983
). The reporter signals at
5.058/4.074 for Man-D2 H-1/H-2 confirm this substitution. Furthermore, the occurrence of a terminal Man-B residue and an unsubstituted Man-4 residue are reflected by the presence of the H-1/H-2 signals at
4.909/3.978 and
5.088/4.088, respectively. An oligomannose-type glycan with the same arrangement of Man residues has been described earlier by Michalski et al. (1990)
, and its Man H-1/H-2
values are in good agreement with those of N0.3.32AB (compare with compound Man6GlcNAc2 II in Michalski et al., 1990
). The difference between the Man-4 H-1 values (
2AB 5.088 versus
5.094) is due to the 2AB labeling (compare N0.2 and N0.22AB, Table I). The assignments of the various Man H-1 and H-2 signals were verified by 2D total correlation spectroscopy (TOCSY) experiments. The structural assignment was further supported by 2D rotating-frame nuclear Overhauser enhancement spectroscopy (ROESY), showing cross-peaks between Man-D2 H-1 and Man-A H-2 and between Man-4 H-1 and Man-3 H-3.
HPAEC subfraction N0.3.42AB turned out to contain a mixture of a hybrid- and an oligomannose-type N-glycan. Its 1H NMR spectrum showed H-1 signals for terminal GlcNAc-5 ( 4.551), terminal Man-A (
5.090), terminal Man-B (
4.907), and terminal Man-C (
5.051) with dominating signal intensities for H-1 of Man-B and Man-A. The peak intensity ratio of GlcNAc-5 H-1:Man-C H-1 is similar to that of Man-4 H-1 (
5.106):Man-4 H-1 (
5.341), whereby the first
value is indicative for Man-4 substituted with GlcNAc-5 (N0.3.4A2AB) and the second one for Man-4 substituted with Man-C (N0.3.4B2AB) (Vliegenthart et al., 1983
). Therefore the structural reporters clearly indicate two structures with the same upper arm and variations in the substitution of Man-4 in the lower arm (molar ratio A:B, 5:8). The downfield shifts of Man-4 H-1/H-2, caused by the attachment of GlcNAc-5, going from N0.22AB to N0.3.4A2AB, is comparable with such shifts reported in the literature (Mulder et al., 1995
). Compound N0.3.4B2AB is the conventional Man6GlcNAc2 structure, and taking into account the influence of the 2AB labeling, the structural reporters are in agreement with those of the nonlabeled compound (Tseneklidou-Stoeter et al., 1995
).
MALDI-TOF-MS analysis (positive-ion mode) of HLPC fraction N0.4 revealed m/z values of 1580, 1619, 1660 (major), and 1742, in accordance within 3 mass units with the sodiated adducts of Hex7HexNAc2, Hex6HexNAc3, Hex5HexNAc4, and Hex8HexNAc2.
HPAEC subfraction N0.4.22AB contains a digalactosylated diantennary complex-type N-glycan. The 1H NMR structural-reporter groups are in accordance with earlier published data for such a glycan (compare with compound 11 in Bendiak et al. [1988]), except for the values affected by the 2AB labeling. Note the difference in
value between GlcNAc-2 NAc for complex-type (
2.072) and hybrid-/oligomannose-type (
2.0552.062) 2-AB-labeled N-glycans (Table I).
The 1H NMR spectrum of HPAEC subfraction N0.4.42AB showed the occurrence of one major compound in a mixture of hybrid and/or oligomannose-type N- glycans, a hybrid-type N-glycan (>85% as judged from the ratio of the intensities of the N-acetyl methyl signals of GlcNAc-5 and GlcNAc-1). The compound is an extension of structure N0.3.4A2AB with a Gal-6 residue. The structural-reporter-group signals of the lower arm match those of the lower arm of N0.4.22AB, the ones of the upper arm those of the upper arm of N0.3.4A2AB.
The 1H NMR spectrum of HPAEC subfraction N0.4.52AB indicated the presence of one major compound in a mixture of oligomannose-type N-glycans, a Man8GlcNAc2 structure. For this structure holds that Man-4 is extended with a terminal Man-C residue (H-1/H-2, 5.054/4.07), and Man-4' with
(1-2)-substituted Man-B (H-1,
5.141) and Man-A (H-1,
5.402) residues, as evidenced by the presence of Man-D3 and Man-D2 H-1 signals at
5.040 and 5.054, respectively (compare with compound 71 in Vliegenthart et al. [1983]
; see also Van Halbeek et al. [1981]
).
MALDI-TOF-MS analysis (positive-ion mode) of HLPC fraction N0.5 showed a major m/z value of 1812, in accordance within 3 mass units with the sodiated adduct of Hex5HexNAc4dHex. 1H NMR analysis revealed as the major compound a fucosylated complex-type diantennary N-glycan, but the fucosylation does not occur at the Asn-bound GlcNAc-1 (GlcNAc-1 H-1, 5.191; GlcNAc-2 NAc,
2.083). The structural-reporter-group signals indicate that the Man-4' residue (H-1,
4.93) is extended with an N-acetyllactosamine unit, and the Man-4 residue (H-1,
5.109) with a Lewis x epitope (compare with Qd1.5A and Qd1.5C in Stroop et al., 2000
; see also asialo-afuco diantennary structure Q0-F in Spellman et al. [1991]
). The (
1-3)-fucosylated GlcNAc-5 is reflected by the typical Fuc signals at
5.126 (Fuc H-1) and 1.174 (Fuc CH3) (Kamerling and Vliegenthart, 1992
).
Structural analysis of monosialylated N-glycans
The 1H NMR data of the identified monosialylated hybrid- and complex-type N-glycans are presented in Table II. The table includes also the coding system for the different monosaccharide constituents. HPAEC fractions N1.7 and N1.8 did not contain sufficient material for NMR assignments. In all presented structures the lower, (1-3) arm is Neu5Ac(
2-6)Gal (ß1-4) GlcNAc(ß1-2)Man(
1-3): Neu5Ac H-3e,
2.6672.671; Neu5Ac H-3a,
1.7171.718; Gal-6 H-1,
4.4444.446; GlcNAc-5 H-1,
4.6034.609; Man-4 H-1,
5.1325.137; Man-4 H-2,
4.1944.207 (slightly influenced by the type of upper arm) (compare with compound HST in Damm et al. [1987]
).
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MALDI-TOF-MS analysis (negative-ion mode) of HPAEC fraction N1.5 revealed two pseudomolecular ions, one at m/z 1931 [Hex5HexNAc4Neu5Ac H], denoted N1.5A, and one at m/z 1727 [Hex5HexNAc3Neu5Ac H], denoted N1.5B. From the 1H NMR spectrum of N1.5 it could be deduced that the major (2-6)-sialylated nonfucosylated N-glycan N1.5A has an N-acetyllactosaminylated upper arm, being the nonfucosylated analogue of N1.3 (compare with compound N1.4 in Hård et al. [1992]
). Component N1.5B is an (
2-6)-sialylated analog of N0.3.22AB, a hybrid-type N-glycan with Man-4' extended with a Man-A residue. The molar ratio A:B amounts 3.6:1, as calculated from the two Man-4' H-1 signal intensities.
MALDI-TOF-MS analysis (negative-ion mode) of HPAEC fraction N1.6 showed a pseudomolecular ion at m/z 1890 [Hex6HexNAc3Neu5Ac H]. However, the 1H NMR spectrum gave evidence for the occurrence of two components, major N1.6A and minor N1.6B. The major component was indicated to be an (2-6)-sialylated hybrid-type N-glycan with terminal Man-A (H-1/H-2,
5.091/4.066) and terminal Man-B (H-1,
4.910) residues attached to Man-4' (H-1/H-2,
4.875/4.146) (compare with N0.4.42AB). A second set of H-1 signals of lower intensity observed at
4.898 (H-1 of monosubstituted Man-4'; compare with N1.5B), 5.410 (H-1 of substituted Man-A), and 5.056 (H-1 of terminal Man-D2) supports the presence of isoform N1.6B, with a different arrangement of Man-4'-linked Man residues. The ratio of terminal to substituted Man-A is approximately 8:3, reflecting the relative abundance of these structures.
Structural analysis of disialylated N-glycans
1H NMR analysis of HPAEC fraction N2.1 revealed the presence of an (2-6)-disialylated, (
1-6)-fucosylated diantennary glycan (compare with compound Q2.2 in Van Rooijen et al. [1998]
). Its nonfucosylated analog turned out to be present in HPAEC fraction N2.2 (compare with compound HST in Damm et al. [1987]
). The 1H NMR data of both compounds are included in Table II. HPAEC fraction N2.3 did not contain sufficient material for NMR assignments.
Release, fractionation, and structural analysis of O-glycans of rhC1INH
Resource Q fractionation of the O-linked oligosaccharide-alditols, obtained after alkaline borohydride treatment of the N-deglycosylated rhC1INH, yielded three carbohydrate- containing fractions, denoted O.1 (45%), O.2 (33%), and O.3 (22%) (Figure 6). 1H NMR analysis of the neutral FPLC fraction O.1 showed the presence of the core 1 structure Gal(ß1-3)GalNAc-ol (compare with compound 2 in Kamerling and Vliegenthart [1992]) (Table III). The monocharged FPLC fraction O.2 contained a mixture of two monosialylated alditols, Neu5Ac(
2-3)Gal(ß1-3)GalNAc-ol (O.2A) (compare with compound 78 in Kamerling and Vliegenthart [1992]
) and Gal(ß1-3)[Neu5Ac(
2-6)]GalNAc-ol (O.2B) (compare with compound 9 in Kamerling and Vliegenthart [1992]
) in an approximate molar ratio of 1:2, as judged from the intensities of the H-1 signals of substituted (
4.546) and terminal (
4.474) Gal. The dicharged FPLC fraction O.3 contained Neu5Ac(
2-3)Gal(ß1-3)[Neu5Ac(
2-6)]GalNAc-ol (compare with compound 85 in Kamerling and Vliegenthart [1992]
).
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Discussion |
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Here, we focus on the N,O-glycosylation pattern of rhC1INH, the transgenic material expressed in the mammary gland of transgenic rabbits, excreted in the milk. A first inspection of the results learns that oligomannose-, hybrid-, and diantennary complex-type N-glycans do occur. When sialylated, only Neu5Ac is present, and only (2-6) linkages are found. Part of the complex-type N-glycans contains the Lewis x epitope. The small core 1-type O-glycans show the usual (
2-3)- and (
2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin. Neu5Gc, found in low amounts in CHO- and BHK-expressed recombinant glycoproteins (Hokke et al., 1990
; Nimtz et al., 1993
) and suggested to occur in transgenic human antithrombin produced in goat milk (Edmunds et al., 1998
), was not observed on the rhC1INH glycans. This sialic acid is not expressed in normal adult human cells; higher levels of Neu5Gc may lead to immune reactions in humans. In this context, it should be noted that N-glycans of CHO- and BHK-derived recombinant glycoproteins contain (
2-3)-linked sialic acid only. Another well-documented immunogenic carbohydrate antigen, Gal(
1-3) Gal(ß1-4)GlcNAc(ß1-(Hamadeh et al., 1992
), was also not detected in rhC1INH.
In Table IV a survey is presented of the amounts in molar percentages of the N- and O-glycans established. The neutral N-glycans in rhC1INH make up about 25% of the total N-glycan pool. The majority of these glycans were of the oligomannose- and hybrid-type, with Man5GlcNAc2 being the most abundant structure, accounting for about 10% of the total N-glycan pool. About 75% of the N-glycans were sialylated. The monocharged N-glycans (about 67% of the total N-glycan pool) were of the hybrid- and mono- or diantennary complex type. Here, the major structures were the monosialylated, partially (1-6)-fucosylated diantennary N-glycans (about 30% of the total N-glycan pool). The dicharged N-glycans (about 8% of the total N-glycan pool) comprised disialylated, partially (
1-6)-fucosylated diantennary N-glycans. In this context it is of interest to mention that with increasing expression levels of rhC1INH, the ratio of oligomannose-type:hybrid-type N-glycans increases and that a decrease in the extent of sialylation occurs with the progress of lactation (Koles et al., unpublished data).
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The finding of Man8GlcNAc2 isomer N0.4.5, containing Man-D2 and Man-D3 indicate that in the mammary gland of the rabbit the alternative endo-mannosidase pathway (More and Spiro, 1990) is competetively active. Here a Glc(
1-3)Man disaccharide is released by a Golgi-localized endo-
-mannosidase yielding Man8GlcNAc2 missing Man-D1, followed by a sequential cleavage of Man-D3 and Man-C by Golgi-localized
-mannosidases (Verbert, 1995
), yielding the Man6GlcNAc2 isomer N0.3.3, the second most abundant oligomannose-type structure in this study (a product that also could arise from the ER-
-mannosidase II processing). Interestingly, the endo-mannosidase pathway seems to be an active pathway in the mammary glands of cow, goat, sheep, and rhesus monkey, too, as lactoferrins from these animals contain, in addition to Man9GlcNAc2, the Man8GlcNAc2 structure N0.4.5 (Van Halbeek et al., 1981
; Coddeville et al., 1992
; Montreuil et al., 1997
). Usually the endo-mannosidase pathway, a processing with one exception so far only observed in vertebrates (Dairaku and Spiro, 1997
), is followed for circumventing
-glucosidase blockades (Moore and Spiro, 1990
; De Praeter et al., 2000
) but can also be induced by inhibiting ER/Golgi-localized
-mannosidases I, thereby blocking the formation of Man8GlcNAc2 missing Man-D2 (Weng and Spiro, 1996
). The reason why the mammary tissue is using partly the endo-mannosidase pathway remains to be clarified.
Comparing the N-glycosylation patterns of native serum hC1INH and rhC1INH (Table IV), it can be concluded that the total processing in native serum hC1INH (only N2.1/N2.2 and the [2-3]-sialylated isomers) is much more complete than in rhC1INH (N2.1/N2.2 make up about 10% of the total N-glycan pool). Comparing the O-glycosylation patterns of native serum hC1INH and rhC1INH (Table IV), it is remarkable that the only structure (O.2A) present in native material is a minor one (about 10% of the total O-glycan pool) in the recombinant material. In rhC1INH the (
2-6)-sialylated extensions of O.1 and O.2A are the major products (about 60% of the total O-glycan pool). Taking together, when compared with native serum hC1INH, in terms of sialylation the N-glycans of rhC1INH are (
2-6)-undersialylated [Gal(ß1-4)GlcNAc
-2,6-sialyltransferase/ST6Gal I], whereas the O-glycans are (
2-6)-oversialylated (GalNAc
-2,6-sialyltransferase/ST6GalNAc II]. Biological activity studies of rhC1INH are in progress and will be published elsewhere.
The detailed analysis of the N,O-glycosylation pattern of rhC1INH, revealing remarkable differences in glycosylation pattern between native serum hC1INH and rhC1INH, makes clear the importance of such studies for therapeutic glycoproteins when expressed in new biological systems. Earlier research has demonstrated the usefulness of studying the glycosylation machinery of CHO and BHK cells, and similar research has to be set up to explore the glycosylation machinery of the mammary gland of animals used for transgenic purposes.
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Materials and methods |
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Release and isolation of O-glycans
N-deglycosylated rhC1INH was subjected to alkaline borohydride treatment according to Piller and Piller (1993). Briefly, the concentrated N-deglycosylated glycoprotein solution (
0.4 ml) was mixed with 10 ml 0.1 M NaOH containing 1 M NaBH4, and kept for 16 h at 45°C, then cooled on ice and neutralized with 2 M aqueous HOAc. Boric acid was removed by repetitive coevaporation with MeOH, containing 1% (v/v) HOAc. Finally, the material was applied to a Dowex 50W-X8, H+ column (100200 mesh, Pharmacia), and O-glycans (oligosaccharide-alditols) were eluted with water and lyophilized. When required, samples were further purified on graphitized carbon columns (Packer et al., 1998
).
FPLC fractionation
The pools of N- and O-glycans were fractionated into neutral and charged species on a Resource Q column (6 ml, Pharmacia, Uppsala, Sweden) using a Pharmacia FPLC system. Elutions were performed with water followed by a linear concentration gradient of NaCl in water at a flow rate of 4 ml/min; for gradient details, see relevant figure captions. Fractionations were monitored by UV absorbance at 214 nm and conductivity measurements. Individual fractions were lyophilized, desalted on 5 connected HiTrap columns (5 ml, Amersham, Little Chalfont, U.K.) using 5 mM NH4HCO3 as eluent, and lyophilized again.
HPLC fractionation
The FPLC fraction of neutral N-glycans was further fractionated on a LiChrospher-NH2 5 µm (250 mm x 4.6 mm, Alltech) column equipped with a LiChrospher Amino 5 µm guard column (7.5 x 4.6 mm), using a Waters 600 HPLC system. Elutions were carried out with a linear gradient of water in acetonitrile at a flow rate of 1 ml/min; for gradient details, see relevant figure captions. The fractionation was monitored by UV absorbance at 206 nm. Individual fractions were lyophilized.
Labeling of N-glycans with 2AB
Neutral N-glycans from the HPLC fractionation were treated with 0.35 M 2AB (Sigma)/1 M NaCNBH3 in Me2SOHOAc (7:3, v/v) for 2 h at 65°C (Bigge et al., 1995; Stroop et al., 2000
). The 2AB fluorescently labeled glycans were purified via paper chromatography on acid-pretreated QMA (Whatman) filter paper strips using acetonitrile (three times) as a mobile phase. Glycans (remaining at the base line) were eluted from the dried paper strips with water, concentrated, and subfractionated by HPAEC.
HPAEC fractionation
HPAEC was performed on a Dionex DX 500 workstation equipped with a pulsed amperometric detection (PAD) system. A series of N-glycan fractions were subfractionated on a CarboPac PA-1 (250 x 9 mm) column using linear gradients of 0.5 M NaOAc in 0.1 M NaOH at a flow rate of 4 ml/min; for gradient details, see relevant figure captions. The following pulse potentials and durations were used during the triple-pulse amperometric detection with a gold electrode at 300 mA: E1 0.05 V, 480 ms; E2 0.60 V, 120 ms; E3 -0.60 V, 60 ms. Fractions were immediately neutralized with 0.1 M HCl, then lyophilized, desalted on five connected HiTrap columns (5 ml, Amersham Pharmacia) using 5 mM NH4HCO3 as eluent, and lyophilized again.
Quantification of oligosaccharides
The molar ratio of the FPLC fractions was calculated from the FPLC peak areas on the basis of the weighted average number of C=O groups (responsible for UV absorption at 214 nm) being known after structural identification and determination of the relative amounts of each individual component. The molar ratio of the constituent oligosaccharides within each FPLC fraction was determined from the HPLC peak areas (corrected for the number of C=O groups) at 206 nm. When HPAEC/PAD was used for further fractionation of HPLC fractions, the PAD response was assumed to be equal for each oligosaccharide present within one HPLC fraction. For mixtures, molar ratios were determined on the basis of the 1H NMR spectra.
1H NMR spectroscopy
Prior to 1H NMR spectroscopy, samples were lyophilized twice from 99.9% 2H2O (Cambridge Isotope Laboratories, Andover, MA), then dissolved in 99.96% 2H2O (Cambridge Isotope Laboratories). 1H NMR spectra were recorded at 500 MHz on a Bruker AMX-500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University) at a probe temperature of 300K and p2H 7. 1D spectra of 5000 Hz spectral width were recorded in 16K complex data sets using a water eliminated Fourier transform pulse sequence as described by Hård et al. (1992). Chemical shifts are expressed in ppm relative to internal acetone (
2.225 in 2H2O) or acetate (
1.908 in 2H2O) (Vliegenthart et al., 1983
). 2D-TOCSY spectra at 500 MHz were recorded using Bruker software with MLEV-17 mixing sequence cycles between 20 and 100 ms. Data matrices of 512 x 2048 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. 2D-ROESY spectra were recorded with a mixing time of 300 ms. Phase-sensitive handling of data was performed by the time-proportional phase increment method implemented by the Bruker software. NMR data were processed using a locally developed software package (J. A. van Kuik, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University).
MS
For MALDI-TOF-MS in the positive-ion mode, samples (0.51 µl) were mixed in a 1:1 ratio with a mixture of 5 mg/ml 2,5-dihydroxybenzoic acid and 0.25 mg/ml 5- methoxysalicylic acid in 1 ml ethanol/10 mM NaCl (1:1, v/v) as a matrix. For the negative-ion mode, 2 mg/ml 2',4', 6'-trihydroxyacetophenone monohydrate in acetonitrile/13.3 mM ammonium citrate (1:3, v/v) was used as a matrix. In this case the sample matrix mixture was dried under reduced pressure (Papac et al., 1998). Measurements were performed on a PerSeptive Biosystems Voyager-DE MALDI-TOF mass spectrometer with implemented delayed extraction technique using an N2 laser (337 nm) with 3 ns pulse width. Spectra were recorded in a linear mode at an accelerating voltage of 24.5 kV using an extraction delay of 90 ns.
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Acknowledgements |
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Footnotes |
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2 Present address: GenMab BV, Jenalaan 18d, NL-3584 CK Utrecht, The Netherlands
3 To whom correspondence should be addressed; e-mail: j.p.kamerling{at}chem.uu.nl
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Abbreviations |
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References |
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