2 Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603, Japan; and 3 INSERM Unité 255, Centre de Recherches Biomedicales des Cordeliers, 15 rue de IEcole de Medecine, 75270 Paris, France
Received on March 6, 2002; revised on April 22, 2002; accepted on April 22, 2002
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Abstract |
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Key words: BHK cell/N-glycan/soluble Fc receptor/3D HPLC mapping
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Introduction |
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In mice and humans, three classes of FcRsFc
RI, Fc
RII, and Fc
RIIIhave been described (Fridman et al., 1992
; Hulett and Hogarth, 1994
). Fc
RI has high affinity for monomeric IgG, whereas Fc
RII and Fc
RIII exhibit low affinity for monomeric IgG but bind IgG-containing immune complexes with high avidity. Fc
RII and Fc
RIII possess similar extracellular regions composed of two Ig-like domains. There exist a variety of isoforms of the individual classes of Fc
Rs (Fridman et al., 1993
). For example, isoforms for human Fc
RIII are transmembrane Fc
RIIIa and glycosylphosphatidylinositol (GPI)-linked Fc
RIIIb, coded by two separate genes. Some of those isoforms are expressed as soluble form (sFc
R) that consist of their extracellular domains produced by proteolytic cleavage (Huizinga et al., 1990
; Sautès et al., 1991
).
Human FcRIIIb and its soluble counterpart interact not only with IgG but also with cell surface receptors, such as complement receptor type 3 (CR3; CD11b/CD18) (Petty and Todd, 1993
; Galon et al., 1996
). It has been reported that the glycosylation of (s)Fc
RIII is involved in its interactions with classical (IgG) and nonclassical (CR3) ligands. Glycosylation of sFc
RIII plays an inhibitory role in the interaction with human IgG3 but not IgG1 (Galon et al., 1997
; van den Nieuwenhof et al., 2000
). On the other hand, the interaction with CR3 is mediated by carbohydrate moieties expressed on (s)Fc
RIII molecules (Sehgal et al., 1993
; Galon et al., 1996
). Although structural basis of the interactions between IgG and Fc
Rs has been provided by nuclear magnetic resonance spectroscopy (Kato et al., 2000
) and X-ray crystallography (Sondermann et al., 2000
; Radaev et al., 2001
) by use of recombinant sFc
Rs, the role of Fc
RIII glycosylation in its binding of ligands has not been fully elucidated from structural aspects.
Therefore, to gain deeper insights into this question, human sFcRIII expressed by an appropriate mammalian expression system are required. Expression systems to produce high amounts of recombinant sFc
Rs of eukariotic cell lines have been developed (Teillaud et al., 1994
; Sautès et al., 1994
). We have shown that recombinant human sFc
RIII (rhsFc
RIII), which is produced in baby hamster kidney (BHK) cells, conserve the ability to interact with CR3, which is comparable to that of native sFc
RIII purified from human serum (Galon et al., 1996
). This strongly indicates that rhsFc
RIII and the native sFc
RIII glycoproteins carry common glycan structure(s) essential for interaction with CR3.
Here we report the N-glycosylation profile of rhsFcRIII produced in BHK cells. By use of a 3D high-performance liquid chromatography (HPLC) mapping technique (Takahashi et al., 1995a
, 1998; Tomiya et al., 1988
), structures of N-glycans expressed on rhsFc
RIII have been determined throughout, discriminating possible isomers resulting from partial galactosylation and sialylation. The N-glycosylation profile thus obtained has been compared with that of recombinant mouse sFc
RII (rmsFc
RII) produced in BHK cells. Similarity and difference of the N-glycosylation profiles between rhsFc
RIII and rmsFc
RII are discussed in light of their function.
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Results |
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Identification of N-glycan structures
Structural assignment of all neutral and sialyl N-glycans from sFcRIII was performed by 2D mapping technique, as described elsewhere (Takahashi et al., 1995a
). Except for six compounds (monosialyl MS1, MS4, and MS5 and disialyl DS2, DS3, and DS5), coordinates of all N-glycans from sFc
RIII coincided with those of known N-glycans on the map. Therefore, they were tentatively assigned to the known N-glycans as shown in Table TI. Then, cochromatography on ODS and amide-silica columns of each of the sample PA-glycans with the corresponding reference compound confirmed those assignments. The coordinate sets of six compounds (monosialyl MS1, MS4, and MS5; disialyl DS2, DS3, and DS5) do not match, by direct comparison, with any of the reference compounds available on the 2D map accumulated prior to this study.
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Similarly, the resultant compounds from the ß-galactosidase and ß-N-acetylhexosaminidase digestion of MS4 and MS5 were expected to be one of the following four monosialyl monoantennary compounds: 1A2-110.8, 1A2-110.4, 1A2-110.3, and 1A2-110.9 found in Figure 2B.
On the enzymatic treatments, the coordinates of MS4 and MS5 changed from (15.2, 9.7) and (15.6, 9.7) to (11.8, 5.6) and (14.3, 5.5), respectively (Figure 2). The strongest candidates among the four possible aforementioned structures were 1A2-110.4 (11.8, 5.6) for the digested MS4, and 1A2-110.3 (14.2, 5.5) for the digested MS5. By cochromatography, we confirmed that the digestion products of MS4 and MS5 are identical with 1A2-110.4 and 1A2-110.3, respectively, among the four possible candidates. On the basis of these data we conclude that the position of sialylation in these compounds, specifically, Galß4GlcNAcß2Man3 branch in MS4 and Galß4GlcNAcß2Man
6 branch in MS5. The structures of MS1, MS4, MS5 thus identified are included in Table TI.
DS5 was converted into the known tetraantennary reference 410.16 on releasing sialic acids. ß-Galactosidase and ß-N-acetylhexosaminidase digestion of DS5 gave rise to two fractions, DSx (14.5, 6.6) and DSy (16.1, 6.6) at a molar ratio of 1:1 on the 2D map. Because any possible disialyl biantennary compounds as references were not available, the structures of DSx and Dsy could be only estimated to be that shown in Scheme S1 on the basis of GU differences computed from the contributions of each component monosaccharide unit in a specific position expressed in GU (Tomiya and Takahashi, 1998). These results indicated that DS5 was a 1:1 mixture of two disialyl tetraantennary compounds; see Scheme S2.
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Discussion |
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In a previous publication, we determined 18 major N-glycan structures of rmsFcRII (Takahashi et al., 1998
). Figure 3 compares the N-glycosylation profile of rhsFc
RIII determined in the present study with that of rmsFc
RII by plotting all sample coordinates on the 2D map. Much more divergent N-glycan structures are exhibited by rhsFc
RIII than by rmsFc
RII. In particular, high-mannose type N-glycans (from N1 to N5 in Table TI, and Figure 3A) are expressed only by rhsFc
RIII. Furthermore, larger multiantennary complex type oligosaccharides are predominant in the rhsFc
RIII. Indeed, for rhsFc
RIII, the most abundant glycan is a typical tetraantennary N12 (18%) and the total content of tetraantennary group (including N12) is 43%. On the contrary, most of the oligosaccharides of rmsFc
RII are biantennary N-glycans and prevalence of tetraantennary glycan is only 0.9%.
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The lectin-like site of CR3 has been shown to be responsible for binding to GPI-anchored FcRIII as well as to rhsFc
RIII (Stöckl et al., 1995
; Galon et al., 1996
). The lectin-like site of CR3 binds mannose-rich compounds such as zymosan (Ross et al., 1985
), and the Fc
RIII-CR3 cocapping is inhibited by N-acetyl-D-glucosamine,
-methyl-D-mannoside, and D-mannose, but not by glucose, galactose, N-acetyl-neuraminic acid, fucose, sorbitol, fructose, nor sucrose (Yamamoto et al., 1989
). Inhibitory effects by
- or ß-methyl-D-mannoside have been observed for the rhsFc
RIII-CR3 interaction (Galon et al., 1996
). One intriguing possibility is that the interaction of CR3 with Fc
RIIIb involves the lectin-like region of CD11b and the high-mannose type oligosaccharide(s) characteristically exhibited by Fc
RIII.
The human sFcRIII used in the present study possesses six possible N-glycosylation sites (positions 37, 44, 63, 73, 162, and 169), whereas mouse sFc
RII has four sites (positions 36, 63, 138, and 145). Thus most of the glycosylation sites are not conserved, although these two glycoproteins share 50% amino acid identity with each other. We suggest that the local environment (e.g., local conformational factors) surrounding the individual glycosylation sites affect the processing of the expressed glycans, resulting in differential overall glycosylation profiles between the two glycoproteins with close structural similarlity of polypeptide chains. If this is true, it is quite likely that different glycosylation sites in human Fc
RIII molecules exhibit different N-glycosylation profiles. Analyses of site-specific glycosylations of the recombinant human Fc
RIII is under way in our laboratory.
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Materials and methods |
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The PA-derivatives of isomalto-oligosaccharides (420 glucose residues) and of reference N-glycans (code numbers, M5.1, M6.1, M8.2, 110.1, 110.3, 210.1, 210.2, 210.3, 210.4, 310.8, 410.9, 410.12, 410.13, and 410.16) were purchased from Seikagaku Kogyo. (The known N-glycan structures are coded in "The Elution Coordinate Database for 2-D/3-D Sugar Map" [http://www.gak.co.jp/FCCA], which contains information of more than 400 kinds of N-glycans.) The following PA-glycans were prepared by known methods: M6.10 and M7.7 were obtained from M9.1 by jack bean -mannosidase digestion (Tomiya et al., 1991); 410.42 was prepared from recombinant erythropoietin (Tomiya et al., 1993
); 410.15 was from 3A5-410.16 by ß-galactosidase then sialidase digestion; 210.4b, 1A2-210.3, 1A3-210.4, 1A4-210.4, 1A2-210.4a1, 2A4-210.4, 3A2-310.18, 3A3-410.16, 3A5-410.16, 3A6-410.16, and 4A2-410.16 were from mouse sFc
RII (Takahashi et al., 1998
); and 310.18, 2A3-310.18 and 3A4-410.16 were from human integrin (Nakagawa et al., 1996
).
Preparation of sFcRIII
The cDNA encoding the NA2 form of human FcRIII-B ectodomains (194-amino-acid-long including a 7-amino-acid C-terminal stop linker) was expressed in the BHK cell line. The rhsFc
RIII glycoprotein was purified by ion exchange and affinity chromatography as described (Sautès et al., 1994
). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis showed that the material was 99% pure and migrated with apparent molecular mass 47 to 56 kDa.
Preparation of PA N-glycans from sFcRIII and analysis by 3D HPLC mapping technique
One milligram of rhsFcRIII was used as starting material. Experimental procedures of liberation and PA-derivatization of N-glycans and chromatographic conditions have been described in detail elsewhere (Nakagawa et al., 1995
; Takahashi et al., 1995a
). Briefly, sFc
RIII was proteolyzed with trypsin and chymotrypsin mixture. The proteolysate was further digested with glycoamidase A to liberate N-glycans. After removing the peptidic materials, the reducing ends of N-glycans were reductively aminated with a fluorescent reagent, 2-aminopyridine using sodium cyanoborohydride (Yamamoto et al., 2001
).
The mixture of PA-oligosaccharides was separated by HPLC on a diethylaminoethyl (DEAE) column according to the sialic acid content. Each separated fraction was then applied to an ODS HPLC column. Then each of the fraction separated on the ODS column was applied to the amide-silica column for the separation on the basis of their sizes. The elution times of a given PA-oligosaccharides on ODS and amide-silica columns, both of which are calibrated in GUs, provide a unique set of coordinates on the 2D map. The coordinates of the PA-glycans originating from sFcRIII were compared with those of the authentic reference compounds.
The GUs of all unknown N-glycans were first compared with those of the reference N-glycans on the 2D map. We then select a few references from the data as candidates whose coordinates coincided with those of the sample within allowable error (± 5%). The sample PA-glycan and each of the selected references were coinjected onto ODS and amide-silica columns. Identification was made judging whether they are eluted simultaneously giving a single peak on both HPLC profiles.
For further confirmation, the sample PA-glycan was digested with one or more exoglycosidases. At each step of the trimming, the elution positions of the resultant PA-glycans were examined as to whether they could be identified with the expected reference compound. The trimming and comparison was continued until the product from the sample compound became coincidental with a reference PA-oligosaccharide on the map.
Each PA-glycan (approximately 50 pmol) isolated on the ODS and amide-silica columns was digested by exoglycosidases (-sialidase from A. ureafaciens, ß-galactosidase and ß-N-acetylhexosaminidase from jack bean under the conditions described elsewhere; Nakagawa et al., 1995
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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