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Molecular & Cellular Proteomics 5:2146-2157, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Unusual N-Glycan Structures in -Mannosidase II/IIx Double Null Embryos Identified by a Systematic Glycomics Approach Based on Two-dimensional LC Mapping and Matrix-dependent Selective Fragmentation Method in MALDI-TOF/TOF Mass Spectrometry^*,S

Megumi Hato, Hiroaki Nakagawa, Masaki Kurogochi, Tomoya O. Akama, Jamey D. Marth^¶, Michiko N. Fukuda^,|| and Shin-Ichiro Nishimura^,**

From the Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan, The Burnham Institute, La Jolla, California 92037, and ^¶ Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Mannosidase IIx (MX) is an enzyme closely related to -mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of -mannosidase II and -mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983–8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097–6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.

The in vivo role of N-glycans has been evaluated using mouse mutants: the GnT-I null mutation leads to embryonic lethality (4), demonstrating that the high mannose-type N-glycans cannot support embryogenesis. GnT-II null embryos survive, but GnT-II nulls die within several weeks after birth, phenotypes attributable to congenital disorders of glycosylation type IIa (5). On the other hand, -mannosidase II (MII) null mice show mild phenotypes or dyserythropoiesis (6, 7). Structural analysis of N-glycans from MII null mouse tissues indicates the existence of an alternate pathway for detouring defective MII. -Mannosidase IIx (MX) is an enzyme closely related to MII as indicated in Fig. 1 (8, 9). Although MX null mice show no gross abnormalities, MX null males are infertile (10). MII/MX double knock-out mice show neonatal lethality. We have profiled major N-glycan structures from MII/MX double knock-out mice at embryonic day (E) 15.5 by means of a two-dimensional (2D) mapping based on a general approach using HPLC with pyridylamination (11, 12). The results of 2D LC mapping analysis and MALDI-TOF MS of the per-O-methylated N-glycan derivatives indicated that MII/MX double nulls do not synthesize the complex-type N-glycans (13).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Steps of N-glycan processing. MI, -mannosidase I; Glc I, II, glucosidase I, II.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—
Trypsin, sodium cyanoborohydride, and -lactoalbumin were purchased from Sigma. Chymotrypsin and Pronase were purchased from Calbiochem. -Mannosidase and ß-galactosidase from jack bean were purchased from Seikagaku Co. (Tokyo, Japan). 2,5-Dihydroxybenzoic acid, -cyano-4-hydroxycinnamic acid, human angiotensin II, bombesin, ACTH, and somatostatin 28 were purchased from Bruker Daltonics GmbSH (Bremen, Germany). Other materials were purchased from the sources indicated: Sephadex G-15, Amersham Biosciences; Bio-Gel P-4, Bio-Rad; peptide-N-glycosidase F, Roche Applied Science; ß1,4-galactosyltransferase, Toyobo Co. Ltd. (Osaka, Japan); TSKgel Amide-80 (4.6 x 250 mm), Tosoh (Tokyo, Japan); ShimPack HRC-ODS (6.0 x 150 mm), Shimadzu (Kyoto, Japan); and Develosil UG-C30 (4.0 x 10 mm), Nomura Chemical Co. Ltd. (Seto, Japan). 2-Aminopyridine and other chemical reagents were obtained from Wako Pure Chemical Co. Ltd. (Osaka, Japan). UDP-N-acetylgalactosamine was a gift from Yamasa Corp. (Choshi, Japan).

Generation of -Mannosidase II/IIx Double Knock-out Mice—
Mice lacking a functional MII allele and MX allele (10) were mated, and resulting heterozygotes were then crossed to obtain double-null animals as described previously (13). Genotypic proportions of pups born from MII^+/–MX^+/– parents were: MII^+/+MX^+/+:MII^+/+MX^+/–:MII^+/+MX^–/–:MII^+/–MX^+/+:MII^+/–MX^+/–:MII^+/–MX^–/–:MII^–/–MX^+/+:MII^–/–MX^+/–:MII^–/–MX^–/– = 20:47:20:30:85:33:15:33:1. Genotypic proportions of embryos (E15.5) produced from MII^–/–MX^+/– parents were: MII^–/–MX^+/+:MII^–/–MX^+/–:MII^–/–MX^–/– = 12:26:8, and those of E18.5 were 11:18:10, respectively.

Preparation and HPLC Analysis of PA-derivatized N-Glycans from Mouse Embryos—
PA-derivatized N-glycans from mouse embryos were prepared by the procedure reported previously (10). MII/MX double knock-out mice (n = 2), MII knock-out mice (n = 2), MX knock-out mice (n = 2), and wild-type (n = 1) mouse embryos (10 mg/sample) were homogenized in 0.1 M ammonium acetate and boiled for 15 min. After lyophilization, they were dissolved in 200 µl of 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM CaCl₂ and digested with 100 µg each of trypsin (Sigma) and chymotrypsin (Calbiochem) at 37 °C for 16 h. After heating at 90 °C for 10 min to inactivate the enzymes, the residues were treated with 10 units of N-glycosidase F (recombinant, Roche Applied Science) at 37 °C for 16 h to release N-glycans from glycopeptides/proteins. Reaction mixtures were then digested with 100 µg of Pronase (Calbiochem) for the purification by gel filtration chromatography (GFC). N-Glycans were fractionated by GFC using a Bio-Gel P-4 column (200–400 mesh, Bio-Rad) with H₂O as the eluant, and the carbohydrate-containing fractions were subjected to 2-aminopyridylation through the reductive amination with a large excess of 2-aminopyridine (Wako Pure Chemical Co. Ltd.) (pH 6.8) in the presence of sodium cyanoborohydride (Sigma) at 90 °C for 1 h according to the general protocol (17). The mixture of PA-derivatized N-glycans was fractionated by GFC on a Sephadex G-15 column (Amersham Biosciences) with 10 mM ammonium bicarbonate solution as eluant. The mixture was then subjected to acid hydrolysis with 0.01 M HCl (pH 2.0) to remove terminal sialic acid residues at 90 °C for 1 h. The mixture of PA-derivatized N-glycans was further purified by HPLC (D-7000 HPLC system equipped with L-7485 fluorescence detector, Hitachi High-Technologies Co., Tokyo, Japan) using a TSKgel Amide-80 column (4.6 x 250 mm, Tosoh) with stepwise elution in the following condition: at 40 °C at a flow rate of 1.0 ml/min; solvent A (mixture of 3% acetic acid-triethylamine buffer (pH 7.3) and acetonitrile at a ratio of 35:65 (v/v)); solvent B (mixture of 3% acetic acid-triethylamine buffer (pH 7.3) and acetonitrile at a ratio of 65:35 (v/v)). The column was equilibrated with solvent A. After injection of sample, the chromatography was performed stepwise using 100% solvent A for 0–7 min and 100% solvent B for 7–12 min. Then the elution of PA-derivatized N-glycans was monitored by using a fluorescence detector under an excitation at 320 nm and an emission at 400 nm, respectively, and each derivative was isolated.

Two-dimensional Mapping Analysis Using HPLC of the PA-derivatized N-Glycans Isolated from Mouse Embryos—
The two-dimensional mapping technique established by Tomiya et al. (11) was used to identify the structures of the PA-derivatized N-glycans isolated from mouse embryos. Major N-glycan structures were determined by plotting the elution positions under two independent chromatography modes of HPLC analysis on the basis of a database consisting of about 480 kinds of N-glycan standards as determined by Takahashi et al. (11). First the mixture of the neutral PA-derivatized N-glycans was subjected to and analyzed on a reverse-phase column (ShimPack HRC-ODS, 6 x 150 mm, Shimadzu). Elution was performed at a flow rate of 1.0 ml/min at 55 °C using a linear gradient elution system. Solvent C was 10 mM sodium phosphate buffer (pH 3.8), and solvent D was 10 mM sodium phosphate buffer (pH 3.8) containing 0.5% (v/v) 1-butanol. The column was initially equilibrated with a solvent (C:D = 80:20 (v/v)). After sample injection, elution was performed by using a linear gradient from the solvent C to a solvent (C:D = 50:50 (v/v)) in 60 min. Each PA-derivatized N-glycan fraction separated on the above mentioned ODS column was collected and subsequently applied to the second amide adsorption column (TSKgel Amide-80, 4.6 x 250 mm). The chromatography was performed at a flow rate of 1.0 ml/min at 40 °C using a gradient elution system of the solvents A and E. Here the solvent E was composed of 3% acetic acid-triethylamine buffer (pH 7.3) and acetonitrile (50:50 (v/v)). The column was first equilibrated with solvent A, and the elution was performed by using a linear gradient from solvent A to a solvent (A:E = 40:60 (v/v)) in 30 min. In both HPLC systems, the elution profile of the PA-derivatized N-glycans was monitored by a fluorescence detector using excitation at 320 nm and emission at 400 nm, respectively.

Digestion of PA-derivatized N-Glycans Using -Mannosidase and ß-Galactosidase—
PA-derivatized N-glycans isolated by two successive HPLC modes were subjected to digestion with 50 milliunits of -mannosidase (jack bean) (Seikagaku Kogyo Co., Tokyo, Japan) in 0.1 M sodium acetate (pH 5.0), 40 mM ZnCl₂ (final volume, 20 µl) at 37 °C for 15 h. In some cases, PA-derivatized N-glycans were digested with 5 milliunits of ß-galactosidase from jack bean (Seikagaku Kogyo Co.) in 0.1 M citrate-phosphate buffer (pH 4.1) (final volume, 15 µl) at 37 °C for 15 h.

MALDI-TOF/TOF MS of PA-derivatized N-Glycans Using the MDSF Method—
Isolated PA-derivatized N-glycans were analyzed by MALDI-LIFT-TOF/TOF MS using MDSF, a method reported by Kurogochi and Nishimura (14). Matrix solutions were prepared as follows: DHB (Bruker Daltonics) was dissolved in water at 10 mg/ml, and CHCA (Bruker Daltonics) was prepared as a saturated solution in 3:1 (v/v) of acetonitrile:water. Samples were dissolved in water, applied on the target spot of an Anchorchip^TM plate (Bruker Daltonics), mixed with 1 µl of matrix solution, and dried at room temperature. Approximately a 1–2 pmol scale of the sample was used for the preparation of the test solution. PA-derivatized N-glycans were desalted by HPLC by using a Develosil UG-C30 column (4.0 x 10 mm, Nomura Chemical Co. Ltd.) at a flow rate of 0.2 ml/min at 25 °C. The elution was performed stepwise with water for 0–5 min and 20% methanol:water (v/v) for 5–20 min.

All measurements were performed using an Ultraflex TOF/TOF mass spectrometer equipped with a reflector and controlled by the FlexControl 2.2 software package (Bruker Daltonics). In MALDI-TOF MS reflector mode, ions generated by a pulsed UV laser beam (nitrogen laser, = 337 nm, 5 Hz) were accelerated to a kinetic energy of 23.5 kV. Metastable ions generated by laser-induced decomposition of the selected precursor ions were analyzed without any additional collision gas. In MALDI-LIFT-TOF/TOF mode, precursor ions were accelerated to 8 kV and selected in a timed ion gate. The fragments were further accelerated by19 kV in the LIFT cell (LIFT means "lifting" the potential energy for the second acceleration of ion source), and their masses were analyzed after the ion reflector passage. Masses were automatically annotated by using the FlexAnalysis 2.2 software package. External calibration of MALDI mass spectra was carried out using singly charged monoisotopic peaks of a peptide mixture of human angiotensin II (m/z 1046.542), bombesin (m/z 1619.823), ACTH-(18–39) (m/z 2465.199), and somatostatin 28 (m/z 3147.472).

Enzymatic Introduction of N-Acetyl-D-galactosamine into PA-derivatized N-Glycan Using ß1,4-Galactosyltransferase in the Presence of -Lactoalbumin (-LA)—
To prepare a model oligosaccharide structure as a matching standard compound, a N-acetyl-D-galactosamine (GalNAc)-modified PA-derivatized N-glycan, termed as HF5.12 (this structure is shown in Fig. 6), was obtained from MII knock-out mouse serum according to the method described above. PA-derivatized N-glycan (HF5.12) was then digested with 5 milliunits of ß-galactosidase from jack bean (Seikagaku Kogyo Co.) to afford HF5.11 (this structure is represented as J-2:HF5.11 in Fig. 3). Removal of the terminal D-galactose residue by ß-galactosidase was carried out in 0.1 M citrate-phosphate buffer (pH 4.0) at 37 °C for 15 h in a total volume of 10 µl. Finally GalNAc residue was transferred in a regio- and stereoselective manner to the intermediate HF5.11 by treating with ß1,4-galactosyltransferase (human, recombinant, Toyobo Co. Ltd.) and UDP-GalNAc (Yamasa Corp.) in the presence of bovine -LA (Sigma) (18, 19). Approximately 70 pmol of HF5.11 was incubated with 8 milliunits of ß1,4-galactosyltransferase, 500 pmol of UDP-GalNAc (a gift from the Yamasa Corp.), and 4 µg of -LA in 10 µl of 25 mM Tris-HCl buffer (pH 7.4) containing10 mM MnCl₂ at 37 °C for 20 h. To thoroughly transfer GalNAc residue into HF5.11, an additional 500 pmol of UDP-GalNAc was added to the reaction mixture and incubated for 48 h. Modified PA-derivatized N-glycan was isolated from the reaction mixture by HPLC using a Develosil UG-C30 column (4.0 x 10 mm, Nomura Chemical Co. Ltd.) at a flow rate of 0.2 ml/min at 25 °C. The column was first equilibrated with water, and the chromatography was performed stepwise using 100% water for 0–5 min and 20% methanol for 5–20 min. The target compound, a PA-derivatized N-glycan bearing a terminal GalNAc residue (termed L-2; represented in Fig. 6), was finally isolated by using HPLC under the condition described above. The structure of this standard material (L-2) was elucidated by MALDI-TOF/TOF MS analysis, and the detailed result is summarized in the supplemental information.

View larger version (17K): [in this window] [in a new window]   FIG. 6. A schematic structure of HF5.12 and peak L-2.

View larger version (39K): [in this window] [in a new window]   FIG. 3. N-Glycan structures determined by two-dimensional mapping technique. Peak numbers are indicated at left, while code numbers of N-glycans are at right. The code number of N-glycans follows the previous report (11).

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (23K): [in this window] [in a new window]   FIG. 2. Chromatograms of PA-derivatized N-glycans on ODS column. Analyses of PA-glycans released from mouse embryos were first performed on ODS columns. Each peak of glycans was fractionated and subsequently analyzed on an amide column. Peak labels correspond with Table I. Elution conditions were as follows: column, ShimPack HRC-ODS (6 x 150 mm); eluent, solvent C, 10 mM sodium phosphate buffer (pH 3.8); solvent D, 0.5% (v/v) 1-butanol in solvent C; gradient condition, a linear gradient from 20 to 50% solvent D in 60 min. KO, knock-out; DKO, double knock-out.

View larger version (32K): [in this window] [in a new window]   FIG. 4. LIFT-TOF/TOF spectra of PA-glycans containing bisecting GlcNAc in the presence of DHB. Fragment ion m/z 569 is typical for bisecting GlcNAc.

Precise Structural Characterization of Unknown Glycoforms—
These important and informative fragment ions generated by means of MDSF method allowed for further precise structural characterization of unknown oligosaccharide structures found in this study. For example, an unknown structure in peak O was identified by comparing TOF/TOF MS of this sample with that of standard materials, H5.1 and H5.3, containing a bisecting-type trisaccharide unit (data shown in Fig. 4 measured in the presence of DHB matrix) in addition to their TOF/TOF MS data measured in the presence of CHCA. In this case, the precise structure of peak O was successfully concluded by the existence of some key fragment ions detected at m/z 569 due to the bisecting trisaccharide component, GlcNAcß-4Manß-4GlcNAc (Fig. 4), and at m/z 833 and 1036 due to the pentamannooligosaccharide structures. Similarly it was suggested that MALDI-TOF MS of peak L-2 in the presence of two matrices can be used for the production of two distinct precursor ions at m/z 1866 as a protonated ion by DHB and m/z 1888 as a sodium adduct ion by CHCA corresponding to Hex₅HexNAc₄FucPA. It seemed that the LIFT-TOF/TOF fragmentation patterns derived both from [M + H]⁺ and [M + Na]⁺ are quite similar to those of the known HF5.12 (Fig. 5). In the LIFT-TOF/TOF spectra of m/z 1846 (HF5.12) and m/z 1888 (peak L-2) as [M + Na]⁺, fragment ions at m/z 525, 671, 833, 874, 1036, 1481, and 1684 were clearly observed in both spectra (Fig. 5A and Table II). Here it was demonstrated that ion peaks observed at m/z 833 and 1036 are characteristic fragment ions corresponding to the general hybrid-type N-glycans including five mannose residues. In addition, the LIFT-TOF/TOF spectra of HF5.12 and L-2 revealed that a terminal D-galactose residue found in HF5.12 must be N-acetyl-D-galactosamine residue as detected by the meaningful mass difference in the two materials. These significant ion pairs could be observed at m/z 388 and 429, 1198 and 1239, 1401 and 1442, and 1701 and 1742 with a respective +41 mass shift. In contrast, precursor ions at m/z 1824 (HF5.12) and m/z 1866 (peak L-2) detected as [M + H]⁺ in the presence of DHB produced fragment ions m/z 204, 300, 366, and 446 (Fig. 5B). Although the fragment ion at m/z 446 means that both structures have an L-fucose residue attached to the GlcNAc residue at the reducing end, a fragment ion at m/z 407 was observed only in the case of peak L-2 instead of a significant degree of decrease in the intensity of the ion peak at m/z 366 due to [HexNAc + Hex + H]⁺. This result also suggests that an oligosaccharide in peak L-2 includes a di-N-acetyllactosamine unit, GalNAcß-4GlcNAc, which has been reported in some glycoprotein hormones (20, 21).

View larger version (27K): [in this window] [in a new window]   FIG. 5. LIFT-TOF/TOF spectra of HF5.12 and peak L-2 using MDSF. Remarkable fragment ions are assigned in Table II. A, precursor ions are m/z 1846 (HF5.12) and m/z 1888 (peak L-2) as [M + Na]+. B, precursor ions are m/z 1824 (HF5.12) and m/z 1866 (peak L-2) as [M + H]+.

In a similar manner, other unknown N-glycans were also systematically characterized by means of the MDSF method. Including peaks O and L-2, seven of 10 unknown N-glycan structures indicated in Table I were fully characterized, and partial structures of the other three (peaks J-3, K-2, and Q-2) were estimated as shown in Fig. 7 (TOF/TOF MS data for all unknown glycoforms, see materials in supplemental information). Fig. 8 summarizes structures and relative molar ratios of N-glycans investigated by combined use of the two-dimensional mapping technique and the MDSF method of MALDI-LIFT-TOF/TOF mass spectrometry. The versatility of this novel analytical approach is evident because MALDI-LIFT-TOF/TOF analysis using MDSF method does not require any authentic samples and can be used for precise structural characterization of oligosaccharide materials obtained only in extremely small quantities.

View larger version (33K): [in this window] [in a new window]   FIG. 7. N-Glycan structures analyzed by MDSF of MALDI-LIFT-TOF/TOF spectrometry. Proposed N-glycan structures from mouse embryos are shown. Three novel glycoforms detected at peaks M-2, S, and W were identified by characteristic fragment ion peaks and enzymatic treatments as described in the supplemental information (Figs. S-1, S-5, and S-6).

View larger version (22K): [in this window] [in a new window]   FIG. 8. Relative ratios of each N-glycan from mouse embryos. Peak labels are the same as those used in Figs. 3 and 7. KO, knock-out; DKO, double knock-out.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As summarized in Figs. 8 and 9, the ratio of hybrid and complex types varies significantly depending on the genotypes of these mice (Fig. 9A), whereas the relative quantity of high mannose type is constant. This suggests that disruption of MII or MX does not affect the upstream products but does affect the downstream products. The ratio of complex-type oligosaccharides is 60% of total N-glycans both in wild type and MX nulls, and that of MII nulls is reduced to 20%. This observation suggests that at least 20% of complex-type N-glycans in MII nulls were produced by MX.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Relative ratios of specific N-glycan structures. A, relative ratios by type of N-glycan structures. B, relative ratio of N-glycans containing bisecting GlcNAc. C, relative ratio of -1,6 Fuc bound to N-acetylglucosamine at reducing end. KO, knock-out; DKO, double knock-out.

View larger version (33K): [in this window] [in a new window]   FIG. 10. N-Glycans identified by combining 2D LC mapping and MDSF-MALDI-TOF/TOF MS strategy.

	ACKNOWLEDGMENTS

 
We thank Drs. Toshitada Noguchi and Tomoki Hamamoto (Yamasa Corp.) for the kind gift of the UDP-GalNAc.

	FOOTNOTES

 
Received, June 6, 2006

Published, August 8, 2006

Published, MCP Papers in Press, August 9, 2006, DOI 10.1074/mcp.M600213-MCP200

¹ The abbreviations used are: ER, endoplasmic reticulum; MII, -mannosidase II; MX, -mannosidase IIx; GnT, N-acetylglucosaminyltransferase; LA, lactoalbumin; Man, mannose; Glc, glucose; Gal, galactose; Fuc, fucose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetyl-D-galactosamine; LacNAc, N-acetyllactosamine; Hex, hexose; HexNAc, N-acetylhexosamine; GFC, gel filtration chromatography; PA, pyridylamino; MDSF, matrix-dependent selective fragmentation; CHCA, -cyano-4-hydroxycinnamic acid; DHB, 2,5-dihydroxybenzoic acid; E, embryonic day; 2D, two-dimensional; ACTH, adrenocorticotropic hormone.

^* This work was supported in part by the National Project on Functional Glycoconjugate Research Aimed at Developing for New Industry from the Ministry of Education, Science, Sports and Culture of Japan.

^S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

^|| To whom correspondence may be addressed: The Burnham Inst., 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3143; E-mail: michiko{at}burnum.org

^** To whom correspondence may be addressed. Tel.: 81-11-706-9043; E-mail: shin{at}glyco.sci.hokudai.ac.jp

	REFERENCES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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