Analysis of Oligosaccharide Structures of Glycoproteins in Polyacrylamide Gel

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Acknowledgments. The authors thank Dr. Andrew C. Y. Shum and Dr. Y. H. Wei for their invaluable assistance in preparation of the manuscript. This study was supported by Grants NSC88-2314B-010-118 from the National Science Council and NRICM-88204 from the National Research Institute of Chinese Medicine of R.O.C.

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Analysis of Oligosaccharide Structures of Glycoproteins in Polyacrylamide Gel Shin-ichi Nakakita, Daisuke Ama, Shunji Natsuka, 1 and Sumihiro Hase 2 Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received October 10, 2001; published online February 13, 2002

Glycoproteins used to analyze sugar chains are purified from various tissues before analysis of their chemical structures. However, glycoproteins possessing biological functions are usually obtained in very small amounts and they are sometimes difficult to be purified. Since proteins are often detected by SDS– PAGE as the final step of the purification procedures, a simple yet sensitive method for analyzing sugar structures from glycoproteins in the gel is desired. Analytical methods using enzyme digestion and fluorescence labeling for sugar structures of glycoproteins in polyacrylamide gel have been reported (1–3). However, enzymes have substrate specificities, and in some cases sugar chains are not always released from nonsoluble samples like cell membranes, and no enzyme that can release most O-linked sugar chains is reported. To overcome these problems, we have developed a sensitive method of analyzing N- and O-linked sugar chains of glycoproteins purified by SDS–PAGE combined with fluorescence labeling of sugar chains with 2-aminopyridine. 1

Present address: Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan. 2 To whom correspondence and reprint requests should be addressed. Analytical Biochemistry 303, 206 –209 (2002) doi:10.1006/abio.2002.5583 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

NOTES & TIPS

207

FIG. 1. An elution profile by reversed-phase HPLC of a PA-sugar chain fraction from fetuin. The PA-sugar chain fraction was prepared from the PVDF membrane extract with hydrazine. One-third of the reaction mixture was injected. The arrowhead A indicates the elution position of Core1-PA; B, Core2-PA; C, Bi-PA; D, 224Tri-PA. Fractions 1– 4 were collected as indicated by bars.

Materials and Methods Materials. Mouse cerebrums were excised from 20 eight-week-old ICR mice. Fetuin was purchased from Sigma (St. Louis, MO). Shodex Asahipak NH2P-50 was obtained from Showa Denko (Tokyo); Cosmosil 5C18-P, from Nacalai Tesque (Kyoto); anhydrous hydrazine, from Tokyo Chemical Industry (Tokyo); PVDF 3 membrane, from Bio-Rad (Hercules, CA); and 2-aminopyridine, from Wako Pure Chemicals (Osaka). Established procedure for preparation of PA-sugar chains from glycoproteins in polyacrylamide gel. Proteins were separated by SDS–PAGE using the method of Laemmli (4) with 7.5% gel (10 ⫻ 10 cm) and transferred to a PVDF membrane by electroblotting at 1 mA/cm 2 membrane area for 90 min by the method of Matsudaira (5). The protein-loaded membrane was stained with Coomassie brilliant blue R-250. Stained bands were cut out, freeze-dried, and added to 0.2 ml anhydrous hydrazine. The solution was slowly mixed at 25°C for 1 min, after which the membrane was removed. The solution was then heated at 60°C for 50 h (6), and sugar chains liberated were N-acetylated twice with 200 ␮l saturated sodium bicarbonate solution and 8 ␮l acetic anhydride at room temperature for a total reaction time of 30 min. The mixture was poured onto a Dowex 50W ⫻ 8 column (H ⫹ form, 0.5 ⫻ 3 cm), which was washed with 5 ml water. The washing and passthrough fractions were combined and lyophilized. The residue was heated with 20 ␮l of a coupling reagent (prepared by mixing 552 mg 2-aminopyrydine and 200 3 Abbreviations used: PVDF, polyvinylidene difluoride; PA, pyridylamino; Glc, D-glucose; Tris, tris(hydroxymethyl)aminomethane; Fuc, L-fucose; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetylD-glucosamine; Man, D-mannose; Xyl, D-xylose. The oligosaccharides used in this study and their abbreviations are listed in Table 1.

FIG. 2. SDS–PAGE of a “brain-type” sugar chain enriched fraction. A fraction enriched in brain-type sugar chains (40 ␮g protein) was separated by SDS–PAGE under the nonreducing condition. Proteins were stained with Coomassie brilliant blue R-250. Arrowheads indicate the molecular mass in kDa, and the arrowhead A, the 80-kDa protein.

␮l acetic acid) at 90°C for 60 min. The Schiff base thus obtained was reduced by heating with 70 ␮l reducing reagent (freshly prepared by mixing 200 mg dimethylamine– borane complex, 50 ␮l water, and 80 ␮l acetic acid) at 80°C for 35 min as previously reported (7). The reaction mixture was added to 90 ␮l water, and the solution was extracted twice with 90 ␮l water-saturated phenol:chloroform (1:1, v/v) and once with 90 ␮l chloroform, as previously described (8). The aqueous phase was lyophilized, and the residue was dissolved in water. Most contaminating materials were removed by

FIG. 3. An elution profile by reversed-phase HPLC of PA-sugar chains prepared from the 80-kDa band. One-fifth of the reaction mixture was injected. Arrowheads A, B, and C indicate the elution positions of M5A-PA, BA-1-PA, and BA-2-PA, respectively.

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Structures and Abbreviations of PA-Sugar Chains Abbreviation

Structure

Core1

GalNAc Gal␤1}3

Core2

Gal␤1-4GlcNAc␤1{

6 GalNAc 3 Gal␤1}

224-Tri

Gal␤1-4GlcNAc␤1-2Man␣1{

6 Man␤1-4GlcNAc␤1-4GlcNAc 3 4 } Man␣1 }2

Gal␤1-4GlcNAc␤1

{

Gal␤1-4GlcNAc␤1

Bi

Gal␤1-4GlcNAc␤1-2Man␣1{

6 Man␤1-4GlcNAc␤1-4GlcNAc 3

Gal␤1-4GlcNAc␤1-2Man␣1} BA-1

GlcNAc␤1-2Man␣1{

Fuc␣1{ 6 6 GlcNAc␤1-4Man␤1-4GlcNAc␤1-4GlcNAc 3 Man␣1}

BA-2

GlcNAc␤1-2Man␣1{

M5A

Man␣1{

Fuc␣1{ 6 6 GlcNAc␤1-4Man␤1-4GlcNAc␤1-4GlcNAc 3 GlcNAc␤1-2Man␣1}

6 Man␣1{ 6 3 Man␣1} Man␤1-4GlcNAc␤1-4GlcNAc 3 Man␣1}

a newly introduced procedure using a Shodex Asahipak NH2P-50 column (0.46 ⫻ 5.0 cm) as follows. Two eluents, A and B, were used. Eluent A was acetonitrile: water:acetic acid (950:50:3, v/v/v) titrated to pH 7.0 with 7 M aqueous ammonia; Eluent B was acetonitrile: water:acetic acid (200:800:3, v/v/v) titrated to pH 7.0 with 7 M aqueous ammonia. The column was equilibrated with 3% Eluent B. After injecting a sample, Eluent B was increased linearly to 33% in 3 min and then to 100% in 5 min. After that, the column was washed with Eluent B in 5 min at a flow rate of 0.6 ml/min. The fraction between 3 and 13 min after the injection was collected and concentrated to dryness by a rotary evaporator followed by lyophilization. HPLC of PA-sugar chains. PA-sugar chains were detected by fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm). All the HPLC procedures were carried out at 25°C.

Reversed-phase HPLC using a Cosmosil 5C18-P column (0.15 ⫻ 25 cm) was performed with two eluents, C and D. Eluent C was 100 mM ammonium acetate buffer, pH 6.0, Eluent D was Eluent C supplemented with 1.0% 1-butanol. The column was equilibrated with 1% Eluent D. After injecting a sample, the proportion of Eluent D was linearly increased from 1 to 52% in 51 min and then to 100% in 12 min at a flow rate of 0.15 ml/min. Reversed-phase HPLC using a Cosmosil 5C18-P column (0.15 ⫻ 25 cm) was performed with two eluents, E and F. Eluent E was 20 mM ammonium acetate buffer, pH 4.0, Eluent F was Eluent E supplemented with 0.5% 1-butanol. The column was equilibrated with 15% Eluent F. After injecting a sample, the proportion of Eluent F was linearly increased from 15 to 85% in 90 min at a flow rate of 0.15 ml/min. Size-fractionation HPLC was performed on a Shodex Asahipak NH2P-50 column (0.46 ⫻ 5 cm) at a flow rate

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of 0.6 ml/min. Two eluents, A and B, were used. The column was equilibrated with 3% Eluent B. After injecting a sample, Eluent B was increased linearly to 33% in 3 min and then to 70% in 32 min.

2.

Results and Discussion Preparation of PA-sugar chain from glycoprotein in polyacrylamide gel. Fetuin (2 ␮g) was separated by SDS–PAGE, and the protein in the polyacrylamide gel was transferred to a PVDF membrane by electroblotting. PA-sugar chains were prepared from the stained band visualized with Coomassie brilliant blue R-250 as described under Materials and Methods. After digestion with arthrobacter sialidase (5 mU, pH 5.0, at 37°C for 16 h), a part of the fraction was analyzed by reversed-phase HPLC (Fig. 1). Elution positions of sugar chains were agreed well with those of standard Core1-PA, Core2-PA, Bi-PA, and 224Tri-PA. Fractions 1– 4 were collected and further analyzed by size-fractionation HPLC, and again their elution positions were the same as the standard Core1-PA, Core2-PA, Bi-PA, and 224Tri-PA, respectively (data not shown). These results are compatible with the reported results that Core1 and 224Tri are the major sugar chains, and Core2 and Bi, the minor sugar chains of fetuin (9, 10). The total yield of M5A-PA from Taka-amylase A obtained by the present method was 16% of that obtained without SDS–PAGE and electroblotting. Application of the established procedure to glycoproteins with “brain-type” sugar chains. The established method was applied to a 80-kDa glycoprotein which was previously reported to be enriched in “brain-type” sugar chains (11). A fraction (40 ␮g of proteins) containing the 80-kDa glycoproteins was separated by SDS–PAGE, and proteins in the polyacrylamide gel were transferred to a PVDF membrane by electroblotting (Fig. 2). The 80-kDa protein band was cut out and lyophilized. PA-sugar chains prepared from the band were analyzed by reversed-phase HPLC (Fig. 3). M5A-PA and the brain-type sugar chains, BA-1-PA and BA-2-PA, were detected. As the present method uses hydrazinolysis, sugar chains can be released from nonsoluble samples such as glycoproteins in cell membranes, and both N- and O-linked sugar chains, such as GalNAc-O-Ser/Thr-, Glc-O-Ser-, Fuc-O-Ser/Thr-, and Xyl-O-Ser-type sugar chains, can be analyzed. The structures of PA-sugar chains prepared by this method can be analyzed using a two-dimensional sugar mapping combined with exoglycosidase digestion as reported (8).

3.

4. 5.

6. 7.

8.

9.

10.

11.

matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. Anal. Biochem. 250, 82–101. Weitzhandler, M., Kadlecek, D., Avdalovic, N., Forte, J. G., Chow, D., and Townsend, R. R. (1993) Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. J. Biol. Chem. 268, 5121–5130. Anumula, K. R., and Du, P. (1999) Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins. Anal. Biochem. 275, 236–242. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10035–10038. Natsuka, S., and Hase, S. (1998) Analysis of N- and O-glycans by pyridylamination. Methods. Mol. Biol. 76, 101–13. Kuraya, N., and Hase, S. (1992) Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions. J. Biochem. 112, 122–126. Yanagida, K., Natsuka, S., and Hase, S. (1999) A pyridylamination method aimed at automatic oligosaccharide analysis of Nlinked sugar chains. Anal. Biochem. 274, 229 –234. Green, D. E., Adelt, G., and Baenziger, J. U. (1987) The asparagine-linked oligosaccharides on bovine fetuin. J. Biol. Chem. 263, 18235–18268. Edge, A. S., and Spiro, R. G. (1987) Presence of an O-glycosidically linked hexasaccharide in fetuin. J. Biol. Chem. 262, 16135–16141. Nakakita, S., Natsuka, S., Ikenaka, K., and Hase, S. (1998) Development-dependent expression of complex-type sugar chains specific to mouse brain. J. Biochem. 123, 1164 –1168.

Housekeeping Gene Variability in Normal and Carcinomatous Colorectal and Liver Tissues: Applications in Pharmacogenomic Gene Expression Studies 1 Carmelo Blanquicett,* Martin R. Johnson,* Marty Heslin,† and Robert B. Diasio* ,2 *Division of Clinical Pharmacology, Department of Pharmacology and Toxicology, Comprehensive Cancer Center, and †Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294 Received October 17, 2001; published online February 13, 2002

Early molecular studies examining cancer susceptibility and progression have now been expanded into 1

REFERENCES 1. Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: In-gel deglycosylation followed by

Supported by NIH Grant CA85381. To whom correspondence and reprint requests should be addressed at Department of Clinical Pharmacology, 1824 6th Avenue South, Wallace Tumor Institute, Room 558B, University of Alabama at Birmingham, Birmingham, AL 35294-3300. Fax: (205) 975-5650. E-mail: [email protected]. 2

Analytical Biochemistry 303, 209 –214 (2002) doi:10.1006/abio.2001.5570 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.