3Institute of Biotechnology, Laboratory of Glycobiology, FIN-00014 University of Helsinki, Finland; 4Protein Glycosylation, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany; 5Rational Drug Design Program, Biomedicum Helsinki, PB 63, FIN-00014 University of Helsinki, Finland; 6Department of Bacteriology and Immunology, Haartman Institute, FIN-00014 University of Helsinki, Finland; and 7Department of Biological Sciences, Division of General Microbiology, FIN-00014 University of Helsinki, Finland
Received on November 25, 2001; revised on January 7, 2002; accepted on January 13, 2002.
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Abstract |
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Key words: polylactosamine/glycosyltransferase/synthetic/saccharide/glycoconjugate
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Introduction |
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Polylactosamine-type elongation reactions of the LdN determinant, generating GlcNAcß1-3GalNAcß1-4GlcNAc-OR, have not been reported, and the presence of truly internal LdN determinants in natural polylactosamine chains has not been established either. However, the closely related internal GalNAcß1-4Glc determinant with distal polylactosamine extensions has been described in Schistosoma mansoni glycolipids (Wuhrer et al., 2000). The apparent scarcity of information concerning internal LdN is slightly surprising because the synthesis of the distal LdN determinant is not uncommon in the animal kingdom, as discussed, and the generation of the GlcNAcß1-3GalNAc linkage is also well established, though only in the context of O-glycan core 3 biosynthesis (Brockhausen et al., 1985
).
Here, we describe enzyme-assisted conversion of unconjugated GalNAcß1-4GlcNAcß1-OR oligosaccharides and methyl glycosides into products of GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR type. Pure products of definite structures were obtained from these reactions and were fully identified by nuclear magnetic resonance (NMR). The novel trisaccharide determinant GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR proved to be a versatile acceptor for a number of glycosyltransferase reactions, which generated several types of polylactosamine analogs with internal LdN units. The distal GlcNAcß1-3'LdN determinant was cleaved quite slowly by jackbean ß-N-acetylhexosaminidase. This observation will be helpful in searching for putative internal LdN determinants among natural polylactosamines.
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Results |
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The NMR signals of the structural reporter groups of Glycan 10 are recorded in Table IV. A series of 2D NMR experiments analogous to those of Glycan 2 were performed with Glycan 10, and the 1H and the 13C resonances were assigned (Table III). The data show a great similarity between the analogous resonances of the distal e-GlcNAc of Glycans 2 and 10. For example, the e-GlcNAc H1 resonance of Glycan 10 showed a J1,2 of 8.4 Hz, establishing the presence of a ß-linkage also in Glycan 10. The analogous resonances of the d-GalNAc units in Glycans 2 and 10 were similar, too. A concluding HMBC experiment with Glycan 10 revealed a long range correlation between the distal e-GlcNAc H1 and the d-GalNAc C3 (data not shown), identifying the novel linkage of Glycan 10 also as GlcNAcß1-3GalNAc.
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Reactions of GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR glycans with glycosyltransferases
An overview in Figure 4 shows enzymatic reactions that converted the novel GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR glycans into several types of polylactosamine analogs carrying internal LdN determinants.
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Enzyme-assisted ß1,4-galactosylation of Glycan 10 gave the tetrasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-OMe (Glycan 11). In the MALDI-TOF mass spectrum, the major molecular ion signal at m/z 826.2 was assigned to [M+Na]+ of Hex1HexNAc3OMe (calculated m/z 826.3). The 1H-NMR spectrum of Glycan 11 (Table IV) revealed the H1 and H4 doublets of the acceptor and an additional doublet at 4.475 ppm (J1,2 = 7.9 Hz) that was assigned to the H1 of the ß4-linked f-Gal unit. Even here, ß1,4-galactosylation caused a downfield shift of the e-GlcNAc H1 resonance from 4.573 ppm in Glycan 10 to 4.595 ppm in Glycan 11.
ß1,4-Galactosylation of Glycan 15 gave the hexasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-4Glc (Glycan 17). The MALDI-TOF mass spectrum of the product revealed a major signal of m/z 1136.3 that was assigned to hexasaccharide Hex3HexNAc3 (calculated m/z 1136.4). The 1H NMR spectra of Glycans 15 and 17 (Table IV) confirmed successful transfer of ß4-bonded galactose to the distal GlcNAc of Glycan 15. The conversion of Glycan 15 to Glycan 17 was also accompanied by a downfield shift of the resonance of e-GlcNAc H1.
Enzymaticß 21,4-N-acetylgalactosaminylation of Glycan generated Glycan 7 with two successive LdN units.
Incubation of Glycan 2 with UDP-GalNAc and bovine milk ß1,4-galactosyltransferase gave a pentasaccharide (Glycan 7) in a yield that exceeded 95 mol%. (The reaction was performed by using a purified sample of ß1,4-galactosyltransferase from bovine milk [Palcic and Hindsgaul, 1991]. It appears that also ß1,4-N-acetylgalactosaminytransferase was present in the enzyme [van den Nieuwenhof et al., 1999
].) The MALDI-TOF mass spectrum revealed a major signal of m/z 1029.4 that was assigned as [M+Na]+ of the pentasaccharide Hex1HexNAc4OMe (calculated m/z 1029.4). The NMR spectrum (Figure 2F, Table II) confirmed that Glycan 7 represented the pentasaccharide GalNAcß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-OMe, a polylactosamine analog with two adjacent LdN units.
Enzymatic ß1,3-galactosylation of Glycan 2.
Glycan 2 was ß1,3-galactosylated by incubating it with UDP-Gal and a lysate of Colo 205 cells known to contain ß4GalT- and ß3GalT-activities, the latter representing ß3Gal T5 (Isshiki et al., 1999). The ß1,4-galactosylated product (Glycan 3) was removed from the desired ß1,3-galactosylated product, Glycan 8, by converting it back to Glycan 2 with the linkage-specific ß1,4-galactosidase from Diplococcus pneumoniae (Hughes and Jeanloz, 1964
; Renkonen et al., 1989
). Glycan 2, in turn, was successfully removed from Glycan 8 by hydrolysis catalyzed by a recombinant form of ß2,3,4,6-N-acetylglucosaminidase from Streptococcus pneumoniae, followed by chromatography (not shown). The MALDI-TOF mass spectrum of purified Glycan 8 revealed a major signal at m/z 988.3; it was assigned to [M+Na]+ of Galß1-3GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-OMe (calculated m/z 988.4). In high-pH anion exchange (HPAE) chromatography on a PA-1 column, known to separate the slowly eluting type 1 glycans from the type 2 isomers (Townsend et al., 1988
), Glycan 8 revealed a major component emerging in 40 mM NaOH at 4.77 min. In a similar run, purified Glycan 3 gave a major peak at 2.56 min. These experiments confirmed the identity of Glycan 8 as a pentasaccharide of type 1.
Enzymatic conversion of linear Glycan 3 into the doubly branched Glycan 6.
Mammalian blood serum contains ß1,6-GlcNAc transferase activity (cIGnT) that generates midchain branches to ordinary linear polylactosamines (Leppänen et al., 1991, 1997; Gu et al., 1992
). To see whether also internal LdN determinants participate in the branching reactions, we incubated the linear Glycan 3 with UDP-GlcNAc and rat serum and subjected the resulting saccharide mixture to gel filtration in a column of Bio-Gel P-4. The saccharide fraction emerging first from the column (Glycan 6) revealed in MALDI-TOF MS a major signal of m/z 1394.4 that was assigned to [M+Na]+ of Hex2HexNAc5OMe (calculated m/z 1394.5). Its 1H NMR spectrum (Figure 2E; Table II) revealed seven H1 resonances, two H4 doublets, and four singlet peaks arising from the five N-acetyl groups. The structural reporter group resonances of the branched trisaccharide determinant GlcNAcß1-3(GlcNAcß1-6)Galß1-OMe at the proximal end of Glycan 6 were identical with their counterparts in the synthetic trisaccharide GlcNAcß1-3(GlcNAcß1-6)Galß1-Ome (Maaheimo, 1998
). Importantly, the branch-bearing b-Gal of Glycan 6 revealed H1 and H4 resonances that were distinct from their analogs in the spectrum of linear Glycan 3. These resonances permitted a comparison of the kinetics of generation of the two branches of Glycan 6 as will be described.
Another branched product generated from the linear Glycan 3 emerged from the gel filtration column soon after Glycan 6. It revealed in MALDI-TOF MS a major signal of m/z 1191.5 that was assigned to [M+Na]+ of Hex2HexNAc4OMe (calculated m/z 1191.5). The 1D 1H NMR spectrum (Figure 2D) suggested that this material represented largely a mixture of two hexasaccharide isomers (Glycans 4 and 5) with single GlcNAc branches at different sites of the linear acceptor. Particularly relevant resonances were those of H1 and H4 of the b-Gal. Based on the spectra of Glycans 3 and 6 we interpret the data as follows: in the spectrum of Glycan 4, where the b-Gal carries the c'-GlcNAc substituent at position 6, the b-Gal H4 resonates at 4.096 ppm, whereas in the spectrum of Glycan 5, where the b-Gal does not carry any GlcNAc branch, the b-Gal H4 resonance is observed at 4.124 ppm. Also in the H1-resonances of the branch-bearing and the branch-free b-Gal units, the differences in the chemical shifts were quite clear. Completely analogous distinction has been described in the H1 and H4 resonances of branch-bearing and branch-free galactose units of ordinary polylactosamines (Leppänen et al., 1997). By contrast, we could not establish in the present experiments any major differences between the H1 and the H4 signals of the branch-bearing GalNAc of Glycan 6 and the analogous signals of the linear Glycan 3.
The integrals of the H1 and H4 signals of the branch-bearing and the branch-free b-Gal units in the spectrum of the mixture of Glycans 4 and 5 suggest that about one third of the hexasaccharides of the mixture carry the c'-GlcNAc branch at the b-Gal (representing Glycan 4), whereas the majority of the hexasaccharides bear the e'-GlcNAc branch at the six d-GalNAc (representing Glycan 5). Hence, in the branching reaction of the linear Glycan 3, both the internal d-GalNAc and the proximal b-Gal served as acceptor sites, but the reaction proceeded preferentially at the internal d-GalNAc.
Purified recombinant human ß 1,6-GlcNAc transferase of cIGnT-type appeared to transfer to the internal GalNAc of Glycan 11.
The tetrasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-OMe (Glycan 11) was partially converted into a pentasaccharide product Hex1HexNAc4OMe (putative Glycan 13) on incubation with UDP-GlcNAc and a purified sample of a recombinant form of cIGnT from human embryonal carcinoma cells (Mattila et al., 1998). The MALDI-TOF mass spectrum of the desalted reaction mixture revealed that a product was formed that contained a new GlcNAc (Figure 5). The data suggest that in addition to rat serum cIGnT activity, the purified recombinant cIGnT also worked with the internal GalNAc of an LdN analog of polylactosamines.
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In another, shorter reaction, Glycan 11 gave 7% of the putative Glycan 13, and a parallel incubation under identical conditions converted 97% of the polylactosamine Galß1-4GlcNAcß1-3Galß1-4GlcNAc into the branched pentasaccharide Galß1-4GlcNAcß1-3(GlcNAcß1-6)Galß1-4GlcNAc as established by the MALDI-TOF mass spectrum of the reaction mixture (data not shown). These data imply that the recombinant cIGnT transferred the branch-forming GlcNAc about 30 times faster to an internal Gal of the ordinary i-type polylactosamine than to the internal GalNAc of the LdN-containing polylactosamine analog. The ß1,6-GlcNAc transferase activity of dIGnT type, present in hog gastric mucosal microsomes, converted the linear Glycan 10 into the branched Glycan 12. Incubation of the trisaccharide GlcNAcß1-3GalNAcß1-4GlcNAcß1-OMe (Glycan 10) with UDP-GlcNAc and hog gastric mucosal microsomes, which contain dIGnT activity (Piller et al., 1984; Seppo et al., 1990
; Helin et al., 1997
), gave a branched tetrasaccharide GlcNAcß1-3(GlcNAcß1-6)GalNAcß1-4GlcNAcß1-OMe (Glycan 12) that was isolated. MALDI-TOF mass spectrum of the purified product revealed a major signal of m/z 867.3 that was assigned to [M+Na]+ of HexNAc4OMe (calculated m/z 867.4). The NMR signals of the structural reporter groups (Table IV) confirmed the identity of the product as a tetrasaccharide with four HexNAc residues. In the acceptor, Glycan 10, both GlcNAc residues revealed NAc-proton singlets at 2.023 ppm. But in the product, Glycan 12, the new ß6-linked GlcNAc, revealed a "low field" NAc proton resonance at 2.061 ppm (Table IV). This signal was almost identical with the NAc proton resonance at 2.064 ppm of the ß6-linked GlcNAc in the closely related tetrasaccharide GlcNAcß1-3(GlcNAcß1-6)Galß1-4GlcNAcß1-OMe, synthesized from the trisaccharide GlcNAcß1-3Galß1-4GlcNAcß1-OMe in precisely the same manner than Glycan 12 was generated from Glycan 10 (Maaheimo, 1998
). Hence, we conclude that in Glycan 12, the novel e'-GlcNAc was probably ß6-linked to the internal GalNAc residue.
1,3-Fucosylation of internal LdN units.
Treatment of Glycan 15 (GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-4Glc) with GDP-fucose and a partially purified recombinant form of human Fuc TIV resulted in site-specific 1,3-fucosylation at the internal LdN unit, generating Glycan 16. The MALDI-TOF mass spectrum of the reaction mixture revealed that monofucosylated products were formed with an apparent yield of 88%; difucosylated products were not evident (data not shown). Chromatography in a column of immobilized wheat germ agglutinin (Niemelä et al., 1998
), gave pure Glycan 16. Its MALDI-TOF mass spectrum revealed a major signal of m/z 1120.4 that was assigned to [M+Na]+ of the hexasaccharide Hex2HexNAc3Fuc1 (calculated m/z 1120.4).
Pure Glycan 16 was ß4-galactosylated as described for the Glycans 2, 10, and 15, and the purified product, Glycan 18, was characterized. Its MALDI-TOF mass spectrum revealed a major signal of m/z 1282.5 that was assigned to [M+Na]+ of the heptasaccharide Hex3HexNAc3Fuc1 (calculated m/z 1282.5). The 1D 1H NMR spectrum of Glycan 18 (Table IV) suggested that the sample obtained was a reasonably pure specimen of the heptasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc. Comparison of the spectra of Glycans 17 and 18 in Table IV reveals that Glycan 18 contains the backbone of Glycan 17 and bears the LdN determinant in the
3-fucosylated form (Bergwerff et al., 1993
). The isomer, GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-4(Fuc
1-3)Glc, was absent or present only in very small amounts, as indicated, for example, by the absence of significant reporter group resonances of Glc-bonded fucose [de Vries, 1995 577] in the 1D NMR spectrum of Glycan 18.
Enzymatic cleavage of LdN analogs of polylactosamines
We studied two enzymatic degradation reactions, hoping they would prove promising for detection and isolation of putative natural LdN analogs among ordinary polylactosamines.
Endo-ß
-galactosidase cleaved the internal GalNAc
ß
1-4GlcNAc bond of Glycan 11.
Incubation of the tetrasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-OMe (Glycan 11) with endo-ß-galactosidase from B. fragilis (Boehringer, Germany) resulted in significant cleavage. The MALDI-TOF mass spectrum of the reaction mixture revealed a distinct signal that was assigned to [M+Na]+ of a hydrolysis product, the trisaccharide Hex1HexNAc2, that is, Galß1-4GlcNAcß1-3GalNAc (Figure 6A). Also the presence of another cleavage product was obvious; the signal at m/z 683.3 was assigned to [M+Na]+ of Galß1-4GlcNAcß1-3GalNAcß1-O-glycerol (calculated m/z 683.2). This compound probably represents a product from a transglycosylation reaction with free glycerol that was carried to the reaction milieu with the enzyme. The signal at m/z 826.2 represents the substrate (calculated m/z of [M+Na]+, 826.3); two-thirds of Glycan 11 apparently had survived the enzyme treatment in intact form. In a parallel control reaction, the pentasaccharide methyl glycoside Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-OMe of the ordinary polylactosamine type was cleaved completely. The MALDI-TOF mass spectrum of this digest (Figure 6B). revealed no signal of the intact substrate (calculated m/z of [M+Na]+, 947.3) but revealed signals of the hydrolysis products GlcNAcß1-3Galß1-OMe (observed m/z 419.9) and Galß1-4GlcNAcß1-3Gal (observed m/z 568.1), as well as the signal of a transglycosylation product Galß1-4GlcNAcß1-3Galß1-O-Glycerol (observed m/z 642.1). The proximal Galß1-OMe linkage of the substrate and of the cleavage product GlcNAcß1-3Galß1-OMe apparently resisted the enzymes action, implying an important role for the adjacent, proximal GlcNAc in the normal cleavage of the internal galactosidic linkages of i-type polylactosamines.
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The GlcNAcß1-3GalNAc linkage of LdN-saccharides was unusually resistant toward jackbean ß-N-acetylhexoxaminidase.
The LdN-hexasaccharide Galß1-4GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-4Glc (Glycan 17) was treated with a mixture of ß-galactosidase and ß-N-acetylhexosaminidase of jackbean. MALDI-TOF mass spectrum of the resulting digest revealed large signals that were assigned to the molecular ions [M+Na]+ and [M+K]+ of the pentasaccharide Hex2HexNAc3 (Figure 7A), representing probably GlcNAcß1-3GalNAcß1-4GlcNAcß1-3Galß1-4Glc (Glycan 15). Small signals (6%), assigned to the tetrasaccharide Hex2HexNAc2, representing probably GalNAcß1-4GlcNAcß1-3Galß1-4Glc (Glycan 14), were also present, but smaller cleavage products of the original Glycan 17 could not be detected. In a similar experiment, Glycan 18, Galß1-4GlcNAcß1-3GalNAcß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc, was also completely degalactosylated, but the resulting product (Glycan 16) was de-N-acetylglucosaminylated only to a very limited degree (data not shown).
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In a parallel control experiment, the heptasasaccharide Galß1-4GlcNAcß1-3Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4GlcNAc of the ordinary polylactosamine type was treated in the same way as Glycans 17 and 18. The resulting digest showed in the MALDI-TOF mass spectrum (Figure 7B) a complete loss of the distal galactose and also an almost complete loss of the peridistal GlcNAc of the heptasaccharide substrate. Considered together, the cleavage data obtained with the jackbean enzymes imply that the distal GlcNAcß1-3GalNAc bond of the LdN-containing Glycans 15 and 16 are much more resistant toward the ß-N-acetylhexosaminidase than the distal GlcNAcß13Gal linkage of the ordinary polylactosamine GlcNAcß1-3Galß1-4(Fuc
1-3)GlcNAcß1-3Galß1-4GlcNAc. The difference in the reaction rates is so large that desialylated and defucosylated polylactosamine backbones of the ordinary type can probably be eliminated by the combined action of ß-N-acetylhexosaminidase and ß-galactosidase of jackbean, leaving putative LdN analogs largely intact at the GlcNAcß1-3'LdNß1-OR stage of the "erosive" reaction.
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Discussion |
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(1) GalNAcß1-4GlcNAcß1-OR + UDP-GlcNAc GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR + UDP
The purified oligosaccharide products were unambiguously identified by a series of 1D and 2D NMR experiments culminating in the measurement of some HMBC spectra, which revealed consistently the long-range heteronuclear correlation over the novel glycosidic bond, that is, between the distal GlcNAc-H1 and the peridistal GalNAc-C3 (cf. Figure 3). Previous work has established that human serum contains a ß1,3-GlcNAc transferase activity that catalyzes the reactions of Equation 2 (Piller and Cartron, 1983; Yates and Watkins, 1983
; Hosomi et al., 1984
; Seppo et al., 1990
).
(2) Galß1-4GlcNAcß1-OR + UDP-GlcNAc GlcNAcß1-3Galß1-4GlcNAcß1-OR + UDP
We suggest that the serum activity responsible for the reactions of Equation 2 may well be responsible also for the reactions of Equation 1. This notion is based on previous data showing that some purified glycosyltransferases are genuinely multifunctional, transferring efficiently to GalNAc as well as to Gal; pertinent examples include the sialyl 2,3-transferase known as ST3Gal II (Toivonen et al., 2001
) and the ß6-GlcNAc transferase generating O-glycan core 4 as well as branched polylactosamines of I-type (Ropp et al., 1991
; Yeh et al., 1999
). It will be interesting to see whether some of the purified recombinant ß1,3-GlcNAc transferases (GlcNAc to Gal) (Sasaki et al., 1997
; Zhou et al., 1999
; Shiraishi et al., 2001
) will transfer also to distal LdN units.
In the present experiments, human serum converted only 12.5% of the GalNAcß1-4GlcNAcß1-OR into GlcNAcß1-3GalNAcß1-4GlcNAcß1-OR. Under comparable conditions, serum catalyzes 4070% conversion of i-type polylactosamines Galß1-4GlcNAcß1-OR into GlcNAcß1-3Galß1-4GlcNAcß1-OR in our laboratory (Leppänen et al., 1997; Penttilä et al., unpublished data). These data suggest that internal LdN units may not be abundant in human polylactosamines. In vitro synthesis of the GlcNAcß1-3GalNAc linkage has been described in the context of O-glycan core 3 biosynthesis (Brockhausen et al., 1985
). We do not know whether the serum ß1,3-GlcNAc transferase activity catalyzing the reactions of Equation 1 can also generate core 3.
Whether internal LdN units are actually present among ordinary mammalian polylactosamines is not yet known, but some of the present observations may provide tools for enriching and recognizing putative glycoconjugates with internal LdN units. In particular, the erosive treatment of desialylated and defucosylated backbones of polylactosamines with ß-galactosidase and ß-N-acetylhexosaminidase of jackbean will destroy the ordinary polylactosamines (Niemelä et al., 1998) but will leave the GlcNAcß1-3'LdNß1-OR determinants virtually intact as shown by the present data. The "nude" polylactosamine backbones for this kind of erosive experiments can be obtained in many cases by enzymatic desialylation and mild acid defucosylation (Hounsell et al., 1985
). As shown in Figure 4, the synthetic GlcNAcß1-3'LdNß1-ORs proved to be functional acceptors for enzyme-assisted ß1,3-galactosyl-, ß1,4-galactosyl-, ß1,4-N-acetyl-galactosaminyl,
1,3-fucosyl- and various ß1,6-N-acetyl-glucosaminyl transfer reactions catalyzed by mammalian enzymes. Most of the products were purified and characterized adequately in the present experiments. Hence, the data imply that many mammalian enzymes working with backbones of ordinary i-type polylactosamines catalyze analogous reactions also with internal LdN units of polylactosamine analogs.
The apparent multifunctionality of polylactosamine-metabolizing enzymes may be partly responsible for the fact that internal LdN groups have not been observed in natural polylactosamines so far. Their (small) total amount may be distributed among too many distinct determinants. To conclude, the present experiments (1) represent the first successful synthesis of GlcNAcß1-3'LdN units, (2) show that all monomer units of this trisaccharide function as acceptor sites for various glycosyltransferases known to synthesize ordinary mammalian polylactosamines, and (3) provide methods for selective enzymatic removal of ordinary polylactosamines from putative mixtures containing polylactosamines and their analogs that carry internal LdN units.
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Materials and methods |
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ß1,3-N-Acetylglucosaminyltransferase reactions with distal LdN determinants
Typically, 120 nmol of the acceptor and 2400 nmol of UDP-GlcNAc (Sigma) were incubated with 500 µl of fresh human serum, which contains ß1,3-N-acetylglucosaminyltransferase activity (Piller and Cartron, 1983; Yates and Watkins, 1983
; Hosomi et al., 1984
; Seppo et al., 1990
). In some experiments, a concentrate of ß3-GlcNAc transferase activity from human serum was used; the concentrate was prepared by precipitation with ammonium sulfate as described elsewhere (Yates and Watkins, 1983
).
Glycosyltransferase reactions with GlcNAcß1-3'LdN acceptors
The ß1,6N-acetylglucosaminyltransferase reactions of dIGnT type, catalyzed by hog gastric mucosal microsomes, were performed essentially as in Seppo et al. (1990). The ß1,6N-acetylglucosaminyltransferase reactions of cIGnT type, catalyzed by the recombinant enzyme, were performed as described in Mattila et al. (1998)
; the reactions catalyzed by rat serum were performed as described in Leppänen et al. (1997)
. The ß1,4-GalT reactions were performed with purified ß1,4GalT from bovine milk (Sigma) essentially as described in Brew et al. (1968)
. This enzyme was also used as the catalyst for the ß1,4-N-acetylgalactosaminyltransfer reactions (Palcic and Hindsgaul, 1991
; van den Nieuwenhof et al., 1999
). The ß1,3-GalT reactions were performed essentially as described in Pykari et al. (2000)
. Colo 205 cell lysates were used as the enzyme, hence both ß1,3- and ß1,4-GalT reactions took place, requiring subsequent separation of the two major products. The
1,3-FucT reaction was performed by incubating the acceptor and GDP-fucose with a partially purified sample of recombinant human Fuc TIV from BHK-21 cells (Grabenhorst et al., 1998
).
Enzymatic degradation reactions
Incubations with the endo-ß-galactosidase (B. fragilis, Boehringer, Germany) were performed as in Leppänen et al. (1997). The exohydrolase reactions with mixed jackbean ß-galactosidase and jackbean ß-N-acetylhexosaminidase were performed essentially as described in Niemelä et al. (1998)
. Recombinant ß2,3,4,6-N-acetylglucosaminidase from Streptococcus pneumoniae (Calbiochem) was used according to the manufacturers recommendations.
Origin of reference oligosaccharides
LNß1-3'LNß1-3Gal-OMe was obtained as described in Niemelä et al., 1998), LNß1-3'(Fuc
1-3)LN ß13'LN was obtained as described (Niemelä et al., 1999
).
Chromatographic methods
Biogel P2 chromatography was performed essentially as described (Leppänen et al., 1997). Gel filtration HPLC on Superdex peptide HR 10/30 (Pharmacia) was performed as described in Maaheimo et al. (1995)
for the Superdex 75 HR column. The HexNAc content of oligosaccharide peaks was measured of UV absorption, standardized against external GlcNAc and GalNAc. HPAE chromatography was performed as described in Maaheimo et al. (1995)
, but 40 mM NaOH was used throughout as the eluant. Chromatography on immoblized wheat germ agglutinin was performed as described elsewhere (Renkonen et al., 1988
). Desalting of neutral oligosaccharides was carried out on mixed beds of AG-1 (AcO-) and AG-50W (H+) (Bio-Rad, CA) as described in Renkonen et al. (1988)
.
MS and NMR spectroscopy
Positive-ion MALDI-TOF MS was performed as in Leppänen et al. (1997). The m/z values are monoisotopic. The nano-NMR experiments were performed as described in Toivonen et al. (2001)
; the conventional NMR experiments were carried out as in Maaheimo et al. (1995)
.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed at Rational Drug Design Program, Biomedicum Helsinki
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References |
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