NewN-Glycans in Horseradish Peroxidase

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

255, 183–187 (1998)

AB972463

New N-Glycans in Horseradish Peroxidase Noriko Takahashi,*,1 Kyung Bok Lee,†,2 Hiroaki Nakagawa,* Yoshinori Tsukamoto,* Katsuyoshi Masuda,‡ and Yuan Chuan Lee† *GlycoLab, Nakano Vinegar Co., Ltd., 2-6 Nakamura-cho, Handa City Aichi 475, Japan; ‡Meijo University, Tempaku-ku, Nagoya 468, Japan; and †Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218

Received August 14, 1997

We describe here the structures of several new Nglycan oligosaccharides from horseradish peroxidase (HRP). Glycopeptides from HRP were digested with glycoamidase A (from sweet almond) to release the N-glycans. The oligosaccharides were reductively aminated with 2-aminopyridine, and separated by highperformance liquid chromatography on octadecylsilyl– and amide–silica columns for two-dimensional mapping (Tomiya et al., Anal. Biochem. 171, 73–90, 1988). The N-glycans were also analyzed by electrospray ionization mass spectrometry. Two different lots of HRP showed a considerable difference in their Nglycan composition. © 1998 Academic Press Key Words: horseradish peroxidase; N-glycans; Nlinked oligosaccharides; glycoamidases A.

Horseradish peroxidase (HRP)3 and its conjugates are widely used in many areas of biomedical research, and its N-glycan is often simply regarded as solely composed of Xyl1Man3Fuc1GlcNAc2 (000.1FX) (1). In many usages of HRP, the N-glycan part may play a significant role such as in endocytosis and antigenic reactions. We found that HRP N-glycans contain many other structures than Xyl1Man3 Fuc1GlcNAc2. Moreover, two different batches of HRP from the same supplier showed a considerable difference in their compositions of N-glycan structures. Yang et al. also found the heterogeneity of N-glycans in HRP, but in a different way from our results (2). Our finding of the presence of different 1 To whom correspondence should be addressed. Fax: 181-569-245028. 2 Present address: Department of Chemistry, Konyang University, Nonsan Korea. 3 Abbreviations used: DP, polymerization degree; Fuc, L-fucose; HRP, horseradish peroxidase; PA, 2-pyridylaminated; ESI, electrospray ionization; MS, mass spectrometry, ODS, octadecylsilica.

0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

N-glycans in HRP should serve as a warning that the care must be used in interpreting the results obtained with HRP or its conjugates. Glycoamidase A from almond (3, 4) and glycoamidase F from Flavobacterium meningosepticum (5) are most frequently used to release N-linked oligosaccharides from glycopeptides or glycoproteins (4, 5). During our studies on the HRP N-glycans, we compared the action of five different commercial glycoamidase F preparations by analysis of the oligosaccharides liberated. MATERIALS AND METHODS

Materials. Glycoamidase A (glycopeptidase A) from sweet almond (3, 4) was from Seikagaku Kogyo (Tokyo, Japan). Glycoamidase F (from F. meningosepticum) (5) was purchased under different trade names: as N-glycanase and N-glycanase recombinant from Genzyme (Cambridge, MA); as N-glycosidase F and N-glycosidase F recombinant from Boehringer-Mannheim (Indianapolis, IN); and as PNGase F recombinant from New England BioLabs (Beverly, MA). Trypsin, chymotrypsin, and HRP (two different lots) were from Sigma Chemical Co. (St. Louis, MO). a-Mannosidase from jack bean and the pyridylamino (PA) derivatives of isomalto-oligosaccharides (4 –20) glucose residues) and of reference oligosaccharides (Code Nos. M3.3, M1.1FX, M2.1FX, 000.1, 000.1FX) were from Seikagaku Kogyo. The following oligosaccharides were prepared by the known methods: 000.1F and 000.1X from BG60 of Bermuda grass pollen (6), and 100.2FX from laccase of sycamore cells (7). M1.1 and M2.1 were obtained by a-mannosidase digestion of 000.1, and similarly M1.1F and M2.1F, by the digestion of 000.1F. Removal of the fucose a-(1,3)-linked to the PA-modified GlcNAc was carried out with neat trifluoroacetic acid at 20°C for 20 h (8) to yield M1.1X from M1.1FX and M2.1X from M2.1FX (see the structures in Fig. 2). 183

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tation, 320 nm; emission, 400 nm), and the elution positions of the PA-oligosaccharides were expressed in the glucose unit, in reference to the PA-derivatized isomalto-oligosaccharides of DP 4 –12 (11, 12), and analyzed by the two-dimensional mapping technique. Digestion with a-mannosidase. Each oligosaccharide isolated on the ODS and amide columns was digested with the a-mannosidase under the conditions previously described (13). The elution coordinates of the a-mannosidase-trimmed oligosaccharides were examined on the two-dimensional map to verify the structural identity. Electrospray ionization (ESI)–mass spectrometry. The ESI mass spectra were measured with a doublefocusing mass spectrometer JMS HX-110 (JEOL, Tokyo, Japan) equipped with an ESI ion source (Analytica, Branford, MA), as described previously (14). RESULTS AND DISCUSSION

FIG. 1. Comparison of PA-oligosaccharide profiles on the amide– silica column, by digestion of glycopeptides from HRP with glycoamidase A (A) and glycoamidase F (F). The left and right panels show the profiles obtained from two different lots of HRP.

Preparation, derivatization, and characterization of the oligosaccharides (9). Briefly, 1 mg of HRP was digested with a mixture of trypsin and chymotrypsin. The peptide mixture from 200 nmol of HRP glycopeptides was then digested with 0.2 mU of glycoamidase A in 50 mM citrate–phosphate buffer (50 ml, pH 5.0) at 37°C overnight. The mixture was finally digested with pronase to facilitate separation of oligosaccharides from peptidic materials. The oligosaccharide mixture was passed successively through a column of Dowex-50WX8 (H1, 1 ml) and a column of Dowex-1 (CO22 3 , 1 ml), and throughly dried. After reductive amination with 2-aminopyridine using sodium cyanoborohydride (10), the resultant pyridylamino (PA)-oligosaccharides were purified by gel filtration on a Sephadex G-15 column (1.0 3 40 cm). About a 1/200 portion from each of the PA-oligosaccharide mixture was separated and characterized by HPLC using the two-dimensional sugar mapping technique (9, 11, 12), except the amide column was used first, followed by the ODS column. In both cases, PAoligosaccharides were monitored by fluorescence (exci-

Oligosaccharide profiles of HRP glycopeptides. These are shown in Fig. 1. The left and right panels of Fig. 1 show the difference in the amide–silica column profiles of PA-oligosaccharide from two different lots of HRP. Structural characterization of the oligosaccharides from HRP. Oligosaccharides a– g were further chromatographed on the ODS column. Each peak was found to be homogeneous (data not shown). The twodimensional mapping analysis showed that all oligosaccharides except for e coincided (65%) with one of the known oligosaccharides. Thus oligosaccharides a, b, c, d, f, and g were assigned as M2.1, M2.1X, 000.1, 000.1X, 000.1FX, and 100.2FX, respectively

FIG. 2. Sequential digestion of PA-oligosaccharides from HRP with a-mannosidase. Each arrow indicates the changes after each trimming by a-mannosidase. The oligosaccharide designation of a, b, c, d, e, and f correspond to those in Fig. 1.

185

STRUCTURE ANALYSIS OF N-GLYCANS TABLE 1

Chromatographic Properties and the Proposed Structures of PA-Oligosaccharides Obtained from HRP Peak name, Code No.

Structure of PA-oligosaccharides Mana6 {

a M2.1

Mana6 {

b M2.1X

Mana6 {

c 000.1

Manb4GlcNAcb4GlcNAc

Manb4GlcNAcb4GlcNAc P Xylb2

Manb4GlcNAcb4GlcNAc

Glc units ODS amide

Relative quantity (mol%)

7.4 3.2

4.1

8.7 3.4

6.7

7.3 4.2

12.7

7.2 4.7

31.2

5.9 5.1

9.5

5.6 5.6

33.1

5.9 6.3

2.7

} Mana3 Mana6 {

d 000.1X

e M3.3F

f 000.1FX

g 100.2FX

Manb4GlcNAcb4GlcNAc } P Xylb2 Mana3

Mana3

Mana6 } {

Manb4GlcNAcb4GlcNAc P Fuca3

Mana6 {

Manb4GlcNAcb4GlcNAc } P P Xylb2 Fuca3 Mana3

Mana6 {

Manb4GlcNAcb4GlcNAc } P P Xylb2 Fuca3 GlcNAcb2Mana3

(Table 1). Cochromatography (on amide– and ODS– silica columns) of each of the sample PA-oligosaccharides a, b, c, d, f, and g with the corresponding reference PA-oligosaccharides confirmed the above assignment. Further confirmation of the structures by a-mannosidase digestion. After a-mannosidase digestion, each of c, d, e, and f led to one of the known compounds, M1.1, M1.1X, M1.1F, and M1.1FX (Fig. 2, right column). Moreover, small amounts of the intermediates, M2.1, M2.1X, and M2.1FX (the middle column of Fig. 2) were also observed in each case. The elution positions of the intermediates also helped the structural confirmation. ESI–MS of the oligosaccharides from HRP. The ESI– MS spectrum of oligosaccharides c, d, e, and f (Fig. 3)

showed that molecular mass of oligosaccharides c, d, and f agreed well with the calculated values based on the assigned structures. Structural determination of oligosaccharide e. ES– mass spectrometry gave the value of 1157.4 for oligosaccharide e, in agreement with the composition of Man3GlcNAc2Fuc1. One of the possible oligosaccharide structures for such a composition was 000.1F, obtained from BG60 (6). Mana6 {

000.1F:

Manb4GlcNAcb4GlcNAc } P Fuca3 Mana3

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FIG. 3.

Electrospray mass spectrum of PA-oligosaccharides c, d, e, and f.

Digestion of oligosaccharide e with a-mannosidase (Fig. 2) could not provide any useful information. However, when the oligosaccharide 000.1F was coinjected with the oligosaccharide e, the two peaks were clearly discernable (Fig. 4), and thus the peak e could not be 000.1F. The fucose residue of oligosaccharide e could not be released by bovine kidney a-fucosidase but was removed by hydrolysis with neat trifluoroacetic acid at 20°C for 20 h (8). Therefore, the fucose residue in oligosaccharide e could not

be a-(1,6)-linked but was a-(1,3)-linked to the PAmodified GlcNAc. This mode of fucose linkage is commonly found in N-glycans of plant glycoproteins (6, 7, 15). Having established the linkage mode of Fuc, the arrangement of Man residues must be elucidated. Based on the known N-glycan structures, there are four possible ways to arrange three mannosyl residues (Fig. 4). For 000.1 and M3.3, the elution data are already known, but for M3.1 and M3.2, only

STRUCTURE ANALYSIS OF N-GLYCANS

187

ACKNOWLEDGMENT The authors express their sincere appreciation to Mrs. K. Okumura for her excellent technical assistance.

REFERENCES

FIG. 4. Structural characterization of oligosaccharide e by the 2-D mapping. The coordinates of oligosaccharide e and its product by chemical defucosylation were compared with those of the reference compound (000.1F).

calculated (13) data are available. The elution positions of the defucosylated e coincided only with those of M3.3 by cochromatography on the two columns. Thus we can conclude that oligosaccharide e had a zigzag arrangement for the three Man residues as shown in Table 1. Different batches of HRP from the same commercial source showed considerable differences. It is not too surprising, therefore, that the HRP oligosaccharide profile reported by other workers (2) was somewhat different from our results. For example, we could not detect any oligosaccharides containing 4 –7 Man, while their presence was noted by Yang et al. (2). None of the commercial preparations of glycoamidase F (see Materials and Methods) could release N-linked oligosaccharides containing a-(1,3)-fucosylated at the Asn-linked GlcNAc. Thus, the inability for glycoamidase F to release peaks e, f, and g (lower panel of Fig. 1) is the innate property of this enzyme (16 –18). Since the Fuca(1,3)GlcNAcb-Asn is commonly found in glycoproteins from plant (6, 7, 15) and insects (14, 19), it is clearly advantageous to use glycoamidase A rather than glycoamidase F for structural studies or for total release of oligosaccharides from plant or insect glycoproteins.

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