ANALYTICAL
97, 438-449 (1979)
BIOCHEMISTRY
Analysis
STEPHEN *Department
of Glycopeptides as Borate Complexes Polyacrylamide Gel Electrophoresisl
WEITZMAN,*
VICTOR SCOTT,*
of Microbiology, State University TDepartment of Botany, University
by
AND KENNETH KEEGsTmt
of New York at Stony of Wisconsin-Madison,
Brook, Stony Brook, Madison, Wisconsin
New York 53706
11794;
Received March 8, 1979 A new method using polyacrylamide Tel electrophoresis in a Tris-borate buffer to analyze Pronase-derived glycopeptides is described. Examination of immunoglobulin, Sindbis virus, and ovalbumin-radiolabeled glycopeptides by this system demonstrates a pattern similar to that seen after Bio-Gel P-6 chromatography and, in addition, exposes a heterogeneity in the immunoglobulin and Sindbis virus glycopeptides not apparent after gel filtration. The resolution of glycopeptides by gel electrophoresis depends on the inclusion of borate ions in the sample, the gel, and the electrophoresis buffer. The borate ions react with neutral sugars, converting them to charged complexes which migrate during electrophoresis. The number of borate ions bound to a glycopeptide is a function of the composition, sequence, and linkages of the carbohydrates. Gel electrophoresis of glycopeptides in a borate buffer has several advantages: (1) The method requires no new equipment or special skills beyond those necessary for conventional polyacrylamide gel electrophoresis; (2) when performed on a slab gel, up to 24 samples can be analyzed simultaneously; and (3) since detection is by radioautography, small amounts of radiolabeled glycopeptides can be visualized by prolonging the exposure time. These characteristics are advantageous for studies of glycopeptides based on digestion products resulting from incubations with specific exo- and endo-glycosidases. Untreated glycopeptides have been compared on the same gel with glycopeptides sequentially treated with different glycosidases to gain structural information.
Polyacrylamide gel electrophoresis has been a valuable analytical and preparative tool for resolving individual components from complex mixtures of biological macromolecules. This technique has been used for years to study proteins from complex mixtures and, more recently, to do peptide mapping of purified proteins using specific proteases (1). Polyacrylamide gels have also been combined with specific chemical and enzymatic reactions to sequence DNA (2). Both peptide mapping and DNA sequencing take advantage of a similar principle-the availability of specific degradative enzymes and the ease with which multiple samples can be compared on a single slab gel. r This work was supported by funds from a National Science Foundation grant award, PCM 760 1975. 0003-2697/79/120438-12$02.00/O Copyright D 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Such techniques have played an important role in the recent advances in molecular biology since they offer an inexpensive, rapid method for protein and nucleic acid analysis which can be undertaken by most laboratories without large investments in specialized equipment or prolonged training. In the field of glycoprotein biochemistry, however, analyses of the oligosaccharide portions of these molecules remain cumbersome. Most of the structural characterizations of oligosaccharides attached to proteins have depended upon the isolation of decigram or gram quantities of purified glycoproteins and have, therefore, tended toward studying a limited number of serum glycoproteins, myeloma immunoglobulins, and animal viruses (3,4). Even techniques which can be used for analysis of radio438
GEL ELECTROPHORESIS
labeled glycopeptides such as gel filtration, ion exchange, and paper chromatography provide limited information and are restricted in the number of samples which can be compared simultaneously. A polyacrylamide gel technique which resolves glycopeptides generated by proteases could provide an important analytical tool for studying multiple samples of these molecules. In addition, partial sequencing of the oligossaccharide portions could be carried out relatively easily in such a system using highly specific endo- and exoglycosidases. Since borate esterification of carbohydrates has proved valuable in separating monosaccharides by paper electrophoresis (5), an attempt was made to exploit this same chemical reaction for a&amide gel analysis. The Pronase-derived glycopeptides from ovalbumin, Sindbis virus, and an IgGzb mouse myeloma immunoglobulin were used as “markers” to determine conditions for optimal resolution of glycopeptides in acrylamide gels. These glycopeptides have been, and are continuing to be, characterized by us and others with regard to heterogeneity and carbohydrate composition, sequence, and linkages (6-9). They offer a test of a system’s ability to resolve and analyze a complex mixture of glycopeptides that are representative of serum and membrane glycopeptides in the molecular weight range of 1800 to 3500. EXPERIMENTAL
PROCEDURES
Acrylamide gel electrophoresis. A stock solution of 1 M Tris was titrated to different pH levels with either H,BO, or HCl. Dilutions were made from these stocks for sample buffer, reservoir buffer, and gel buffer. Three loo-ml stock solutions each with 30 g of acrylamide (Bio-Rad) and either 1.5,0.75, or 0.375 g ofbis(N, N’-methylenebis-acrylamide) (Bio-Rad) were used for gels with 5- 10, 15, or 20% acrylamide (w/v), respectively. Gels were polymerized by the
OF GLYCOPEPTIDES
439
addition of 0.001% N, N, N’, N’-tetramethylethylenediamine (w/v) and 0.1% ammonium persulfate (w/v). For radioautography, acrylamide gels were cast in a standard slab gel apparatus. This resulted in a gel 13 cm wide x 13 cm long x 1 mm thick. After the addition of lo-20 ~1 of a solution of 0.1% bromophenol blue in 60% sucrose to radiolabeled glycopeptide samples, 25-50 ~1 of the sample was applied to a well and subjected to electrophoresis toward the anode at 100 V (approximately 15 mA) until the dye front was 1 cm from the bottom of the gel (approximately 3 h). After electrophoresis, the slab gels were immediately dried and radioautographed. The radiolabel must be one, such as carbon-14, which can be detected by autoradiography. Attempts to detect tritiumlabeled glycopeptides by fluorography were not successful, due to the loss of the glycopeptides by diffusion during treatment of the gel to introduce the fluorescent compounds. The glycopeptides could not be precipitated in the gel, and any attempt to stain or fix them led to their diffusion out of the gel. (Although not used in these experiments, LKB has recently made commercially available sensitive radioautography paper that will develop 3H-labeled isotopes without any pretreatment of the gel.) For experiments using 3H-labeled glycopeptides in this report gels were cast in Pyrex tubing (0.5 mm in diameter x 12 cm long). After electrophoresis, carried out as previously described at 100 V, the gels were removed from the tubes and sliced at l-mm intervals with a Mickle gel slicer. Each slice was shaken for 16-24 h in 0.5 ml of H,O at 37°C and then mixed with 3-5 ml of tolueneTriton X-100 scintillation fluid for determination of radioactivity in a Searle scintillation counter. Radiolabeled glycopeptide preparation.
Radiolabeled immunoglobulin was isolated as previously described (4). An IgGPbsecreting mouse myeloma cell line derived
440
WEITZMAN,
SCOTT,
from the MPC-11 tumor and previously adapted to tissue culture (10) was grown in 5% CO, suspended in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co.) supplemented with 20% horse serum, nonessential amino acids, glutamine, penicillin, and streptomycin. Cells, 5ml at 0.8-1.0 x lo6 cells/ml, were incubated in their usual growth media with 10 &i of [l-14C]glucosamine per milliliter (New England Nuclear Corp., 56.5 mCi/mmol) for 24-48 h. The cells were pelleted at 400g for 10 min, and the supernatant (secretions) was removed. The secreted, radiolabeled immunoglobulin was immunoprecipitated with rabbit antisera to MPC- 11 immunoglobulin. The immunoprecipitate was washed twice in l-2 ml of 0.1 M NH,HCO,. Radiolabeled Sindbis virus was grown in either BHK-21/13 cells or chick embryo cells, as previously described (7). Cells were infected with Sindbis virus at a multiplicity of lo-50 plaque-forming units/cell. After 1 h, they were incubated in medium containing either 2.5 PCi of [ lJ4C]glucosamine per milliliter (New England Nuclear Corp., 56.5 mCi/mmol) or 10 &i of [6-3H]fucose per milliliter (New England Nuclear Corp., 5-15 Wmmol). At 12 h after infection, the labeled virus was harvested and purified by sucrose gradient centrifugation, as previously described (10). The immunoprecipitated immunoglobulin or pelleted Sindbis virus was suspended in 0.5 ml of 0.1 M NH4NC03. Glycopeptides were generated by Pronase digestion for 30 h at 60°C. Pronase (Calbiochem, A grade) was dissolved in Hz0 at a concentration of lOmg/mlandO.l-0.2ml(l-2mg)wasadded at 0, 8, and 20 h. The samples were then placed in a boiling water bath for 5 min to inactivate the Pronase and lyophilized. Ovalbumin (Sigma, grade V) was digested exhaustively with Pronase. The glycopeptides were purified by gel filtration and further fractionated on Dowex 50, as described by Huang et al. (8). The identity of each glycopeptide was confirmed by deter-
AND
KEEGSTRA
mining the sugar composition of each of the pooled peaks. Neutral and amino sugar analyses were performed by gas chromatography using the alditol acetate derivatives, as previously described (3). Pools C, D, and E, using the nomenclature of Huang et al. (8), were acetylated using [14C]acetic anhydride, as described by Tai et al. (9). Gel jiltration chromatography. Pronasederived glycopeptides were suspended in 0.2 ml of 0.1 M NH,HCO, and chromatographed on Bio-Gel P-6 (200-400 mesh, Bio-Rad). The column (0.9 x 125 cm) was equilibrated and eluted with 0.1 M NH4HC03. Fractions (0.5 ml) were collected and SO-p1 aliquots were removed for determination of radioactivity. Glycosidase digestion. Neuraminidase (Vibrio cholera) was purchased from General Biochemicals. p-Galactosidase, pN-acetylglucosaminidase, and endo-p-Nacetylglucosaminidase D were prepared from S. pneumoniae as described by Koide and Muramatsu (11). Endo+-ZV-acetylglucosaminidase H was a generous gift of Dr. Phillip Robbins (Massachusetts Institute of Technology). Glycopeptide samples were dissolved in 0.20.5 ml of 0.1 M NH,HCO, and titrated to pH 5.5-6.0 with acetic acid. For neuraminidase digestion, 0.02 units were added for 3 h at 37°C. For /3-galactosidase and P-Nacetylglucosaminidase digestion 220 and 37.5 units, respectively, were added for 16 h at 37°C. Endo-p-N-acetylglucosaminidase H and D digestions were carried out as described previously (6,12). RESULTS Conditions for Gel Electrophoresis of Glycopeptides
A 1 M Tris solution was titrated to pH 8.3 with boric acid. Radiolabeled glycopeptide samples were dissolved in 100-200 ~1 of 0.01 M Tris-borate buffer, pH 8.3. Bromophenol blue in sucrose was added and the sample subjected to electrophoresis
GEL ELECTROPHORESIS
on a 10% acrylamide gel. The gel and the reservoir buffer both contained 0.1 M Trisborate buffer, pH 8.3. The results of electrophoresis and radioautography on this gel are presented in Fig. 1. The immunoglobulin and Sindbis virus glycopeptides have been resolved into a number of discrete bands. In a parallel experiment where Tris-hydrochloride was substituted for Tris-borate buffer, the glycopeptides were not resolved, giving a single broad band (data not shown). This establishes the importance of the borate ion in obtaining the pattern shown in Fig. 1. Other electrophoretic conditions were examined to determine their effect on the resolution of the glycopeptides. Gels with 5, 10, 15, and 20% acrylamide were comTRIS - BORATE
SBV
Ig
TOP 0 +
OF GLYCOPEPT’IDES
441
pared. The bands seen on a 5% gel were broader and more diffuse than those on the 10% gel seen in Fig. 1. The 15 and 20% gels, however, appeared indistinguishable from the 10% gel by visual inspection (data not shown). Since it was easier to handle and dry, the 10% gel was considered optimal and used in all future experiments. Since borate-ester formation is dependent on pH, 1 M Tris solutions were titrated to different pH values with H,BO,. Five different gels were examined with the Trisborate buffer adjusted to pH 5,6,7,8, and 9 (see Experimental Procedures). The acrylamide gel and the reservoir buffer again contained 0.1 M solution of the appropriate buffer. The effect of changing the concentration of Tris-borate in the sample buffer was also examined in these same experiments. Samples with undiluted Tris-borate buffer and serial dilutions down to 10e4 M were subjected to electrophoresis. The results of these studies (data not shown) indicated that when Tris-borate buffers at pH 5,6, or 7 were used the resolution was inferior to that at pH 8 and 9. In addition, sample buffer concentrations of Tris-borate higher than 0.01 M also decreased resolution. Concentrations lower than 0.01 M offered no improvement. The general pattern seen in Fig. 1 was similar despite variations in acrylamide concentration, pH, and sample buffer concentration. Optimal resolution, however, was obtained with a 10% acrylamidegelinO.1 ~Tris-borate,pH 8.3. Comparison of Borate Gel Electrophoresis and Gel Filtration Chromatography
BOTTOM 0 --j
FIG. 1. Polyacrylamide gel electrophoresis of Sindbis virus and immunoglobulin glycopeptides. Sindbis virus (SBV) and immunoglobulin (Ig) radiolabeled with [14C]glucosamine were purified, digested with Pronase, and subjected to electrophoresis on a Tris-borate gel as described under Experimental Procedures.
The basis for the multiple bands seen on Tris- borate acrylamide gel electrophoresis of Sindbis virus and immunoglobulin glycopeptides was investigated using glycopeptides purified by gel filtration chromatography. Immunoglobulin radiolabeled with [14C]glucosamine was digested with Pronase and applied to a Bio-Gel P-6 column (Fig. 2, top panel). Peaks were pooled, lyophilized,
442
WEITZMAN,
SCOTT, AND KEEGSTRA
1
40
f
1
I
I
52
I
I
64
I
76
,&a#!+
00
“0
FRACTION
Ig
A
B
NUMBER
C
FIG. 2. Comparison of Bio-Gel P-6 chromatography and Tris-borate gel electrophoresis using immunoglobulin glycopeptides. Top panel: Purified immunoglobulin radiolabeled with [i4C]glucosamine was digested with Pronase and applied to a Bio-Gel P-6 column (0.9 x 125 cm) equilibrated and eluted with 0.1 M NH,HC03. Fractions (0.5 ml) were collected and 50-4 aliquots were removed for determination of radioactivity. Peaks were pooled as in the figure and lyophilized to dryness. Bottom panel: T&-borate sample buffer was added to each pool, and each sample subjected to electrophoresis as described in the text. An unfractionated sample of immunoglobulin glycopeptides appears in the first lane (Ig).
dissolved in sample buffer, and subjected to electrophoresis on Tris-borate gels (Fig. 2, bottom panel). The major immunoglobulin glycopeptide peak seen after gel
filtration (pool C, top panel, Fig. 2) clearly results in a single band on Tris-borate gels. Likewise, pool B has a corresponding single band on the gel. Pool A, however, resolves
GEL ELECTROPHORESIS
OF GLYCOPEPTIDES
than peak C on the gel. Although one band is most likely the peak between fractions 75-80, not enough information is available on the immunoglobulin glycopeptides to resolve this problem. Since Sindbis virus glycopeptides have been characterized in much more detail (7,13,14) a series of experiments similar to those in Fig. 2 were
into three distinct bands. On the one hand, then, Tris-borate gel electrophoresis yields a glycopeptide pattern comparable to P-6 gel filtration. On the other hand, this gel system seems capable of exposing a heterogeneity in glycopeptides that is not revealed by gel filtration. A few bands can be seen migrating slower
L
1
?
I
I
I
,
64
52
I
76
88
“0
FRACTION
SBV TOP@
BOTTOM
443
NUMBER
SI s2 s3 s4
--,
0 +
FIG. 3. Comparison of Bio-Gel P-6 chromatography and Tris- borate gel electrophoresis using Sindbis virus glycopeptides. Top panel: Sindbis virus glycopeptides were fractionated as described in Fig. 2 for immunoglobulin glycopeptides. Bottom panel: Isolated pools of Sindbis virus gfycopeptides were analyzed by Tris-borate gel electrophoresis and compared to unfractionated Sindbis virus glycopeptides in the first lane (SBV).
444
WEITZMAN,
SCOTT,
carried out using Sindbis virus glycopeptides. In the top panel, Fig. 3, the glycopeptide profile of Sindbis virus is resolved into four peaks, as previously described (14). Again, gel electrophoresis of the pooled fractions yields a definite pattern: Smaller glycopeptides migrate slower in the gel than larger glycopeptides. Each pool from the Bio-Gel P-6 column appears distinctly different on the Trisborate gel, although the S 1 pool has some S2 glycopeptides, probably due to overlap of these pools. The interesting finding is that electrophoresis in Tris-borate gels exposes a heterogeneity in each pool that is not evident during gel filtration. Two explanations for this heterogeneity were investigated: first, that it is caused by peptide heterogeneity in the glycopeptides; and second, that it is caused by fucose heterogeneity. Peptide heterogeneity exists because the Sindbis virus glycopeptides are derived from two glycoproteins, El and E2, and the amino acid composition of the Pronasederived glycopeptides from El are different than those derived from E2 (D. J. Burke, Ph.D. thesis, 1976). In order to determine whether the double band in each glycopeptide peak reflected variation in amino acids, El was separated from E2 as previously described (7). Each was digested separately with Pronase and subjected to electrophoresis in adjacent lanes on a Trisborate slab gel. Both sets of glycopeptides resolved into similar patterns with double bands for glycopeptides S 1, S2, and S3 (data not shown). Previous studies have shown that glycopeptides Sl, S2, and S3 contain less than 1 mol of fucose per mole of glycopeptide (7,13). It seemed possible that one glycopeptide band contained fucose, while the second did not. In order to determine whether this fucose heterogeneity caused the double band, Sindbis virus glycopeptides labeled with [3H]fucose were mixed with glycopeptides labeled with [14C]glu-
AND
KEEGSTRA
cosamine and subjected to electrophoresis on a cylindrical gel. The gel was sliced and the radioactivity determined in each slice. Although the double bands were not clearly resolved on the cylindrical gels, the fucoselabeled glycopeptides exhibited the same pattern as the glucosamine-labeled ones, suggesting that both the glycopeptides of the double band contain fucose. Analysis of Glycosidase Products
Digestion
Glycosidase digestion of glycopeptides with known structures and, therefore, known reaction products were first examined to test the feasibility of monitoring glycosidase digestions on Tris-borate gels. Ovalbumin glycopeptides were prepared and acetylated with [14C]acetic anhydride following published procedures (see Experimental Procedures). Three of the glycopeptide peaks obtained in this manner correspond to peaks C, D, and E of Huang et al. (8). Peak C has been shown to consist of three different glycopeptides, all of which can be cleaved by endo-p-N-acetylglucosaminidase H, but not by endo+-N-acetylglucosaminidase D (14). Peaks D and E have been shown to consist of Asn-GlcNAc,Man, and Asn-GlcNAc,Man,, respectively (9). Peak D is similar to peak C in that it can be cleaved by both enzymes. The patterns obtained after electrophoresis in Tris-borate buffer of the untreated glycopeptides, as well as those treated by endo-@-N-acetylglucosaminidases D and H, are shown in Fig. 4. Lanes 1,4, and 7 show the patterns obtained with untreated glycopeptides C, D, and E, respectively. Other samples of glycopeptides C, D, and E were treated with a partially purified preparation of endo-p-N-acetylglucosaminidase D prior to electrophoresis. The products of these digestions are shown in Lanes 2,5, and 8 for glycopeptides C, D, and E, respectively. Finally, other samples of these glycopeptides were treated with highly purified endo-/3-Nacetylglucosaminidase H and these products
GEL ELECTROPHORESIS
0
TOP -
0+
BOTTOM d
I
2.
3
445
OF GLYCOPEPTIDES
4
5
6
7
8
9
FIG. 4. Analysis of endo-glycosidase digestions of ovalbumin glycopeptides. Ovalbumin glycopeptides were “C-acetylated and fractionated into three groups-C, D, and E-as described under Experimental Procedures. The untreated glycopeptides (-) were applied to a Tris-borate gel, lanes 1, 4, and 7. Glycopeptides after digestion with endo+-N-acetylglucosaminidase D (D) were applied to lanes 2,5, and 8 and after digestion with endo-b-N-acetylglucosaminidase H (H) to lanes 3,6, and 9.
are shown in Lanes 3, 6, and 9 for glycopeptides C, D, and E, respectively. The data in Fig. 4 show the expected results. Peak E is cleaved by both endo+-Nacetylglucosaminidases H and D. Peaks C and D are cleaved by endo+-N-acetylglucosaminidase H but not by D, although peak C migrates slightly faster after incubation with the crude endo-p-N-acetylglucosaminidase D mixture. The 14C-acetylated glycopeptides are cleaved to yield an unlabeled oligosaccharide and a smaller 14C-acetylated product consisting only of Asn-GlcN. This latter product appears as a radioactive band near the ion front. The loss of intensity in this band, compared to the undigested substrate, is probably due to diffusion of this relatively smaller molecule out of the gel during drying. A similar series of experiments using specific glycosidases on Sindbis virus glycopeptides establishes the ease with which this technique can provide partial structural data. A mixture of glycopeptides from
Sindbis virus grown in chick cells was applied to lane A (Fig. 5); the same mixture was treated with neuraminidase and applied to lane B. Neuraminidase treatment caused the disappearance of glycopeptides Sl and S2 with a concomitant increase in glycopeptide S3, as previously described (9). Subsequent treatment of neuraminidasetreated glycopeptides with /3-galactosidase caused no observable change in the glycopeptide pattern (Fig. 5, lane C). The mixutre of neuraminidaseand pgalactosidase-treated Sindbis virus glycopeptides was treated with endog-IV-acetylglucosaminidase D and subjected to electrophoresis (Fig. 5, lane D). This enzyme completely hydrolyzed glycopeptide S3, yielding two radioactive products. One product which migrates near the ion front is poorly labeled and can be seen as a faint band in the original gels but is not visible in Fig. 5. A second product migrates as a diffuse band slightly faster than one of the components of glycopeptide S4. Three dif-
446
WEITZMAN,
A
SCOTT, AND KEEGSTRA
B
C
D
E
FIG. 5. Structural studies of Sindbis virus glycopeptides using specific exo- and endo-glycosidases. Purified Sindbis virus was digested with Pronase and an ahquot removed for electrophoresis as an untreated sample in lane A. The remaining sample was incubated with neuraminidase and an aliquot removed for lane B. The sample was then incubated with purified P-galactosidase and an aliquot removed for lane C. The remaining sample was divided into two equal parts; one was incubated with endoP-N-acetylglucosaminidase D (lane D) and the other with endo-P-N-acetylglucosaminidase H (lane E).
ferent radioactive digestion products would have been expected, [14C]glucosamine, [‘“Clglucosamine-labeled oligosaccharide, and [14C]glucosamine-labeled glycopeptide. It is not clear from the present experiments which products give rise to the observed bands. Treatment of the neuroaminidase- and pgalactosidase-treated Sindbis virus glycopeptides with endo+-N-acetylglucosaminidase H does not affect glycopeptide S3 but results in the disappearance of glycopeptide S4 (Fig. 5, lane E). Again two radioactive products are observed from this reaction, one migrating at the ion front (not visibile in this figure) and one migrating just slightly faster than untreated S4. These experiments suggest that glycopeptide S4 is a simple, high-mannose structure, while glycopeptides Sl, S2, and S3 are similar to other previously reported complex structures. This conclusion is similar to that obtained
from other studies on these glycopeptides (7, 13). DISCUSSION
The experiments reported in this paper illustrate the electrophoretic separation of glycopeptides in a polyacrylamide gel using a Tris-borate buffer. This system offers resolution of glycopeptides equal to or better than conventional gel filtration columns (Figs. 2 and 3). Two additional features of this Tris-borate gel electrophoresis system provide unique advantages in analyzing glycopeptides compared to other available techniques. First, radioautography allows small amounts of radiolabeled glycopeptides to be visualized by prolonging the exposure time. This technique can be used with a wide variety of glycopeptides which can be made radioactive, either by in viva labeling as with the Sindbis virus or immunoglobulin gly-
GEL
ELECTROPHORESIS
copeptides or by in vitro labeling as with the ovalbumin glycopeptides. Second, the potential exists to compare simultaneously up to 24 samples on a single slab gel. This potential can be advantageous in either structural or kinetic studies. For example, untreated glycopeptides and the products of several exo- and endo-glycosidase digestions can be subjected to electrophoresis simultaneously as in Fig. 5 to yield partial structural data. Another use would be in pulse-chase experiments with multiple time points to study the kinetics of the in vivo processing of oligosaccharides after attach-
H3B03+
OH
_
447
OF GLYCOPEPTIDES
ment to proteins. The advantages of a slab gel system to analyze glycopeptides seem clear. However, in order to interpret a pattern after Tris-borate gel electrophoresis, the basis for the resolution of glycopeptides needs to be understood. Borate complexes have been utilized in the electrophoretic separation of a wide variety of different carbohydrates (5). This technique is founded upon the observation that neutral sugars are converted into charged complexes by reaction with borate ions at alkaline pH. The reaction between borate ions and carbohydrates can be represented by the reactions shown below.
-
-
r
l-
- ‘“\ /“\ /J Hd’\O-:L
L The formation of the charged borate- sugar complex is dependent upon a number of variables including pH, with alkaline pH favoring the formation of the charged complexes. Another variable strongly affecting the formation of complexes involves the stereochemical disposition of the hydroxyl groups. For glycosidically linked sugars, where the sugars are locked into a pyranose ring form, adjacent hydroxyls react much more readily in the cis configuration than the tram configuration. Thus, methyl-/3-D-xylopyranoside (all transhydroxyls) does not form a complex with borate ions and does not migrate during electrophoresis in the presence of borate. Methyl-P-D-ribopyranoside, however (all cis-hydroxyls), forms a complex and migrates toward the anode during electrophoresis (5). Borate can form a complex with nonadjacent hydroxyls if the stereochemistry is correct. For example, methyl-
‘R2-I
p-D-glucopyranoside (all trans-hydroxyls) migrates during borate electrophoresis due to formation of a borate complex at the hydroxyl groups on C4 and C6 (5). However, the equilibrium for charged complex formation is unfavorable and this compound shows a very low mobility in borate electrophoresis. Studies examining the electrophoretic separation of carbohydrates as borate complexes have been carried out with monosaccharides, simple oligosaccharides, and in some cases polysaccharides (5). No studies have reported the electrophoretic separation of glycopeptides. Most of the previous work has been carried out using zone electrophoresis, where paper has been used as the support matrix. In this study polyacrylamide gels serve as the support. This choice has the advantage that the equipment and techniques for acrylamide gel electrophoresis
448
WEITZMAN,
SCOTT,
are readily available in most laboratories because of the widespread utilization of polyacrylamide gels for the electrophoretic separation of proteins and nucleic acids. The percentage of acrylamide in the gel appears to be unimportant in determining electrophoretic mobility. The separation of glycopeptides is not a result of filtration of molecules based on their molecular weight, as is the case for uniformly charged polypeptides in the presence of sodium dodecyl sulfate. The results presented in Fig. 1 support the hypothesis that the observed separations are a result of electrophoretic migration of borate complexes of the glycopeptides. If borate ion is omitted from the buffer system, the glycopeptides migrate only poorly and as a single band. In the presence of borate ions the glycopeptides are resolved into a number of distinct bands. In general, larger oligosaccharides will bind more borate than smaller oligosaccharides, acquire a greater negative charge, and migrate further into the gel. This seems to be true for the immunoglobulin and Sindbis virus glycopeptides as illustrated in Figs. 2 and 3. However, the amount of borate bound and the migration into the gel is not simply a function of the number of monosaccharides in a glycopeptide but also the composition and structure of the oligosaccharide. Sialic acid residues not only bind borate but also contribute an additional negative charge because of carboxyl groups. This results in a marked increase in migration of sialated compared to desialated glycopeptides as demonstrated in Fig. 5. In addition, terminal mannose, galactose, and fucose residues would be expected to bind borate ions while terminal glucosamine residues would not. That these characteristics influence the migration of glycopeptides in the borate gel is seen in Fig. 4. The untreated ovalbumin glycopeptide pool C (lane 1) contains two terminal glucosamine residues on an oligomannosyl core. After treatment with the endo-/3-N-acetylglucosaminidase D
AND
KEEGSTRA
mixture which also contains exoglucosaminidase activity (1 l), the glycopeptide migrates further into the gel (lane 2). The removal of the terminal glucosamine residues, which do not bind borate, led to the availability of two mannose residues which do bind borate. The resulting increase in negative charge results in the further migration of the glycopeptides. In addition to the number and sequence of sugars in the glycopeptide, the linkage between the carbohydrate residues can influence the amount of borate bound. For example, 2linked mannose or 3-linked galactose would not be expected to bind borate, while the 6linked derivatives of these sugars should bind borate. A number of questions remain unanswered about the experiments presented in this paper. The cause of the double bands in the Sindbis virus glycopeptides is unclear. This is obviously not an artifact of the gel system since the major immunoglobulin glycopeptide and the ovalbumin glycopeptides appear as single bands (Figs. 2 and 4). The possibility remains that a single peak isolated from a Bio-Gel P-6 column contains a mixture of oligosaccharides which is resolved by Trisborate electrophoresis. In addition, the influence that the peptide portion of the glycopeptide has on the behavior of these molecules in this system has only been partially characterized. The presence of a negative charge on the carboxyl terminal amino acid is most probably neutralized by the positive charge on the amino terminal end of the peptide (except for the ovalbumin glycopeptides where the amino termini are blocked with [14C]acetic anhydride). The Sindbis glycopeptides from El and E2 have some differences in amino acid composition and yet comigrate on Trisborate gels. An additional possibility to consider is that heterogeneity in peptide length exists due to incomplete Pronase digestion, and this results in the double bands of Sindbis virus glycopeptides. While further
GEL ELECTROPHORESIS
studies on this problem need to be done, the accumulated evidence suggests that the peptide moiety has little or no effect on the electrophoretic separation of glycopeptides. In summary, the system described in this report provides a convenient method for the rapid, inexpensive analysis of glycopeptides. The inability to distinguish three species in the ovalbumin pool C (Fig. 4) indicates that this is not a high-resolution technique and the extent to which it can be used as a precise analytical tool remains to be determined. The resolution of this system, however, seems to be better than conventional Bio-Gel P-6 chromatography. The value, then of Tris-borate gel electrophoresis lies in its capacity to compare multiple samples, to screen for differences and similarities in glycopeptides from a variety of biological sources, and to collect preliminary structural data with the use of specific glycosidases. ACKNOWLEDGMENTS We thank Dr. Matthew ScharlT for his original ideas on this project and Margery Grennon and Jack Bruno for technical assistance.
449
OF GLYCOPEPTIDES
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Sci.
USA
74, 560-564.
3. Komfeld, R., and Komfeld, S. (1976) Annu. Biochem.
Rev.
45, 217-238.
4. Reading, C. L., Penhoet, E. E., and Ballou, C. E. (1968) J. Biol. Chem. 253, 5600-5612. 5. Foster, A. B. (1957)Advan. Carbohyd. Chem. 12, 81-115. 6. Weitzman, S., Nathenson, S. G., and Schartf, M. D. (1977) Cell 10, 679-687. 7. Burke, D. J., and Keegstra, K. (1976) J. Viral. 20, 679-686. 8. Huang, C. C., Mayer, H. E., Jr., Montgomery, R. (1970) Carbohyd. Res. 13, 127-137. 9. Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Swashita, S., moue, Y, and Kobata, A. (1975) J. Biol. Chem. 250, 8569-8575. 10. Laskov, R., and Schartf, M. D. (1970) J. Exp. Med. 131, 515-541. 11. Koide, N.,and Muramatsu, T. (1974)5. Biol. Chem. 249, 4897-4904. 12. Robbins, P. W., Hubbard, S. C., Turco, S. J.,and Worth. D. F. (1977) Gel/ 12, 893-900. 13. Sefton, B. M., and Keegstra, K. (1974)5. Viral. 14, 522-530. 14. Tai, T., Yamashita, K., Ito, S., and Kobata, A. (1977) .I. Biol. Chem. 252, 6687-6694.
97, 438-449 (1979)
BIOCHEMISTRY
Analysis
STEPHEN *Department
of Glycopeptides as Borate Complexes Polyacrylamide Gel Electrophoresisl
WEITZMAN,*
VICTOR SCOTT,*
of Microbiology, State University TDepartment of Botany, University
by
AND KENNETH KEEGsTmt
of New York at Stony of Wisconsin-Madison,
Brook, Stony Brook, Madison, Wisconsin
New York 53706
11794;
Received March 8, 1979 A new method using polyacrylamide Tel electrophoresis in a Tris-borate buffer to analyze Pronase-derived glycopeptides is described. Examination of immunoglobulin, Sindbis virus, and ovalbumin-radiolabeled glycopeptides by this system demonstrates a pattern similar to that seen after Bio-Gel P-6 chromatography and, in addition, exposes a heterogeneity in the immunoglobulin and Sindbis virus glycopeptides not apparent after gel filtration. The resolution of glycopeptides by gel electrophoresis depends on the inclusion of borate ions in the sample, the gel, and the electrophoresis buffer. The borate ions react with neutral sugars, converting them to charged complexes which migrate during electrophoresis. The number of borate ions bound to a glycopeptide is a function of the composition, sequence, and linkages of the carbohydrates. Gel electrophoresis of glycopeptides in a borate buffer has several advantages: (1) The method requires no new equipment or special skills beyond those necessary for conventional polyacrylamide gel electrophoresis; (2) when performed on a slab gel, up to 24 samples can be analyzed simultaneously; and (3) since detection is by radioautography, small amounts of radiolabeled glycopeptides can be visualized by prolonging the exposure time. These characteristics are advantageous for studies of glycopeptides based on digestion products resulting from incubations with specific exo- and endo-glycosidases. Untreated glycopeptides have been compared on the same gel with glycopeptides sequentially treated with different glycosidases to gain structural information.
Polyacrylamide gel electrophoresis has been a valuable analytical and preparative tool for resolving individual components from complex mixtures of biological macromolecules. This technique has been used for years to study proteins from complex mixtures and, more recently, to do peptide mapping of purified proteins using specific proteases (1). Polyacrylamide gels have also been combined with specific chemical and enzymatic reactions to sequence DNA (2). Both peptide mapping and DNA sequencing take advantage of a similar principle-the availability of specific degradative enzymes and the ease with which multiple samples can be compared on a single slab gel. r This work was supported by funds from a National Science Foundation grant award, PCM 760 1975. 0003-2697/79/120438-12$02.00/O Copyright D 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Such techniques have played an important role in the recent advances in molecular biology since they offer an inexpensive, rapid method for protein and nucleic acid analysis which can be undertaken by most laboratories without large investments in specialized equipment or prolonged training. In the field of glycoprotein biochemistry, however, analyses of the oligosaccharide portions of these molecules remain cumbersome. Most of the structural characterizations of oligosaccharides attached to proteins have depended upon the isolation of decigram or gram quantities of purified glycoproteins and have, therefore, tended toward studying a limited number of serum glycoproteins, myeloma immunoglobulins, and animal viruses (3,4). Even techniques which can be used for analysis of radio438
GEL ELECTROPHORESIS
labeled glycopeptides such as gel filtration, ion exchange, and paper chromatography provide limited information and are restricted in the number of samples which can be compared simultaneously. A polyacrylamide gel technique which resolves glycopeptides generated by proteases could provide an important analytical tool for studying multiple samples of these molecules. In addition, partial sequencing of the oligossaccharide portions could be carried out relatively easily in such a system using highly specific endo- and exoglycosidases. Since borate esterification of carbohydrates has proved valuable in separating monosaccharides by paper electrophoresis (5), an attempt was made to exploit this same chemical reaction for a&amide gel analysis. The Pronase-derived glycopeptides from ovalbumin, Sindbis virus, and an IgGzb mouse myeloma immunoglobulin were used as “markers” to determine conditions for optimal resolution of glycopeptides in acrylamide gels. These glycopeptides have been, and are continuing to be, characterized by us and others with regard to heterogeneity and carbohydrate composition, sequence, and linkages (6-9). They offer a test of a system’s ability to resolve and analyze a complex mixture of glycopeptides that are representative of serum and membrane glycopeptides in the molecular weight range of 1800 to 3500. EXPERIMENTAL
PROCEDURES
Acrylamide gel electrophoresis. A stock solution of 1 M Tris was titrated to different pH levels with either H,BO, or HCl. Dilutions were made from these stocks for sample buffer, reservoir buffer, and gel buffer. Three loo-ml stock solutions each with 30 g of acrylamide (Bio-Rad) and either 1.5,0.75, or 0.375 g ofbis(N, N’-methylenebis-acrylamide) (Bio-Rad) were used for gels with 5- 10, 15, or 20% acrylamide (w/v), respectively. Gels were polymerized by the
OF GLYCOPEPTIDES
439
addition of 0.001% N, N, N’, N’-tetramethylethylenediamine (w/v) and 0.1% ammonium persulfate (w/v). For radioautography, acrylamide gels were cast in a standard slab gel apparatus. This resulted in a gel 13 cm wide x 13 cm long x 1 mm thick. After the addition of lo-20 ~1 of a solution of 0.1% bromophenol blue in 60% sucrose to radiolabeled glycopeptide samples, 25-50 ~1 of the sample was applied to a well and subjected to electrophoresis toward the anode at 100 V (approximately 15 mA) until the dye front was 1 cm from the bottom of the gel (approximately 3 h). After electrophoresis, the slab gels were immediately dried and radioautographed. The radiolabel must be one, such as carbon-14, which can be detected by autoradiography. Attempts to detect tritiumlabeled glycopeptides by fluorography were not successful, due to the loss of the glycopeptides by diffusion during treatment of the gel to introduce the fluorescent compounds. The glycopeptides could not be precipitated in the gel, and any attempt to stain or fix them led to their diffusion out of the gel. (Although not used in these experiments, LKB has recently made commercially available sensitive radioautography paper that will develop 3H-labeled isotopes without any pretreatment of the gel.) For experiments using 3H-labeled glycopeptides in this report gels were cast in Pyrex tubing (0.5 mm in diameter x 12 cm long). After electrophoresis, carried out as previously described at 100 V, the gels were removed from the tubes and sliced at l-mm intervals with a Mickle gel slicer. Each slice was shaken for 16-24 h in 0.5 ml of H,O at 37°C and then mixed with 3-5 ml of tolueneTriton X-100 scintillation fluid for determination of radioactivity in a Searle scintillation counter. Radiolabeled glycopeptide preparation.
Radiolabeled immunoglobulin was isolated as previously described (4). An IgGPbsecreting mouse myeloma cell line derived
440
WEITZMAN,
SCOTT,
from the MPC-11 tumor and previously adapted to tissue culture (10) was grown in 5% CO, suspended in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co.) supplemented with 20% horse serum, nonessential amino acids, glutamine, penicillin, and streptomycin. Cells, 5ml at 0.8-1.0 x lo6 cells/ml, were incubated in their usual growth media with 10 &i of [l-14C]glucosamine per milliliter (New England Nuclear Corp., 56.5 mCi/mmol) for 24-48 h. The cells were pelleted at 400g for 10 min, and the supernatant (secretions) was removed. The secreted, radiolabeled immunoglobulin was immunoprecipitated with rabbit antisera to MPC- 11 immunoglobulin. The immunoprecipitate was washed twice in l-2 ml of 0.1 M NH,HCO,. Radiolabeled Sindbis virus was grown in either BHK-21/13 cells or chick embryo cells, as previously described (7). Cells were infected with Sindbis virus at a multiplicity of lo-50 plaque-forming units/cell. After 1 h, they were incubated in medium containing either 2.5 PCi of [ lJ4C]glucosamine per milliliter (New England Nuclear Corp., 56.5 mCi/mmol) or 10 &i of [6-3H]fucose per milliliter (New England Nuclear Corp., 5-15 Wmmol). At 12 h after infection, the labeled virus was harvested and purified by sucrose gradient centrifugation, as previously described (10). The immunoprecipitated immunoglobulin or pelleted Sindbis virus was suspended in 0.5 ml of 0.1 M NH4NC03. Glycopeptides were generated by Pronase digestion for 30 h at 60°C. Pronase (Calbiochem, A grade) was dissolved in Hz0 at a concentration of lOmg/mlandO.l-0.2ml(l-2mg)wasadded at 0, 8, and 20 h. The samples were then placed in a boiling water bath for 5 min to inactivate the Pronase and lyophilized. Ovalbumin (Sigma, grade V) was digested exhaustively with Pronase. The glycopeptides were purified by gel filtration and further fractionated on Dowex 50, as described by Huang et al. (8). The identity of each glycopeptide was confirmed by deter-
AND
KEEGSTRA
mining the sugar composition of each of the pooled peaks. Neutral and amino sugar analyses were performed by gas chromatography using the alditol acetate derivatives, as previously described (3). Pools C, D, and E, using the nomenclature of Huang et al. (8), were acetylated using [14C]acetic anhydride, as described by Tai et al. (9). Gel jiltration chromatography. Pronasederived glycopeptides were suspended in 0.2 ml of 0.1 M NH,HCO, and chromatographed on Bio-Gel P-6 (200-400 mesh, Bio-Rad). The column (0.9 x 125 cm) was equilibrated and eluted with 0.1 M NH4HC03. Fractions (0.5 ml) were collected and SO-p1 aliquots were removed for determination of radioactivity. Glycosidase digestion. Neuraminidase (Vibrio cholera) was purchased from General Biochemicals. p-Galactosidase, pN-acetylglucosaminidase, and endo-p-Nacetylglucosaminidase D were prepared from S. pneumoniae as described by Koide and Muramatsu (11). Endo+-ZV-acetylglucosaminidase H was a generous gift of Dr. Phillip Robbins (Massachusetts Institute of Technology). Glycopeptide samples were dissolved in 0.20.5 ml of 0.1 M NH,HCO, and titrated to pH 5.5-6.0 with acetic acid. For neuraminidase digestion, 0.02 units were added for 3 h at 37°C. For /3-galactosidase and P-Nacetylglucosaminidase digestion 220 and 37.5 units, respectively, were added for 16 h at 37°C. Endo-p-N-acetylglucosaminidase H and D digestions were carried out as described previously (6,12). RESULTS Conditions for Gel Electrophoresis of Glycopeptides
A 1 M Tris solution was titrated to pH 8.3 with boric acid. Radiolabeled glycopeptide samples were dissolved in 100-200 ~1 of 0.01 M Tris-borate buffer, pH 8.3. Bromophenol blue in sucrose was added and the sample subjected to electrophoresis
GEL ELECTROPHORESIS
on a 10% acrylamide gel. The gel and the reservoir buffer both contained 0.1 M Trisborate buffer, pH 8.3. The results of electrophoresis and radioautography on this gel are presented in Fig. 1. The immunoglobulin and Sindbis virus glycopeptides have been resolved into a number of discrete bands. In a parallel experiment where Tris-hydrochloride was substituted for Tris-borate buffer, the glycopeptides were not resolved, giving a single broad band (data not shown). This establishes the importance of the borate ion in obtaining the pattern shown in Fig. 1. Other electrophoretic conditions were examined to determine their effect on the resolution of the glycopeptides. Gels with 5, 10, 15, and 20% acrylamide were comTRIS - BORATE
SBV
Ig
TOP 0 +
OF GLYCOPEPT’IDES
441
pared. The bands seen on a 5% gel were broader and more diffuse than those on the 10% gel seen in Fig. 1. The 15 and 20% gels, however, appeared indistinguishable from the 10% gel by visual inspection (data not shown). Since it was easier to handle and dry, the 10% gel was considered optimal and used in all future experiments. Since borate-ester formation is dependent on pH, 1 M Tris solutions were titrated to different pH values with H,BO,. Five different gels were examined with the Trisborate buffer adjusted to pH 5,6,7,8, and 9 (see Experimental Procedures). The acrylamide gel and the reservoir buffer again contained 0.1 M solution of the appropriate buffer. The effect of changing the concentration of Tris-borate in the sample buffer was also examined in these same experiments. Samples with undiluted Tris-borate buffer and serial dilutions down to 10e4 M were subjected to electrophoresis. The results of these studies (data not shown) indicated that when Tris-borate buffers at pH 5,6, or 7 were used the resolution was inferior to that at pH 8 and 9. In addition, sample buffer concentrations of Tris-borate higher than 0.01 M also decreased resolution. Concentrations lower than 0.01 M offered no improvement. The general pattern seen in Fig. 1 was similar despite variations in acrylamide concentration, pH, and sample buffer concentration. Optimal resolution, however, was obtained with a 10% acrylamidegelinO.1 ~Tris-borate,pH 8.3. Comparison of Borate Gel Electrophoresis and Gel Filtration Chromatography
BOTTOM 0 --j
FIG. 1. Polyacrylamide gel electrophoresis of Sindbis virus and immunoglobulin glycopeptides. Sindbis virus (SBV) and immunoglobulin (Ig) radiolabeled with [14C]glucosamine were purified, digested with Pronase, and subjected to electrophoresis on a Tris-borate gel as described under Experimental Procedures.
The basis for the multiple bands seen on Tris- borate acrylamide gel electrophoresis of Sindbis virus and immunoglobulin glycopeptides was investigated using glycopeptides purified by gel filtration chromatography. Immunoglobulin radiolabeled with [14C]glucosamine was digested with Pronase and applied to a Bio-Gel P-6 column (Fig. 2, top panel). Peaks were pooled, lyophilized,
442
WEITZMAN,
SCOTT, AND KEEGSTRA
1
40
f
1
I
I
52
I
I
64
I
76
,&a#!+
00
“0
FRACTION
Ig
A
B
NUMBER
C
FIG. 2. Comparison of Bio-Gel P-6 chromatography and Tris-borate gel electrophoresis using immunoglobulin glycopeptides. Top panel: Purified immunoglobulin radiolabeled with [i4C]glucosamine was digested with Pronase and applied to a Bio-Gel P-6 column (0.9 x 125 cm) equilibrated and eluted with 0.1 M NH,HC03. Fractions (0.5 ml) were collected and 50-4 aliquots were removed for determination of radioactivity. Peaks were pooled as in the figure and lyophilized to dryness. Bottom panel: T&-borate sample buffer was added to each pool, and each sample subjected to electrophoresis as described in the text. An unfractionated sample of immunoglobulin glycopeptides appears in the first lane (Ig).
dissolved in sample buffer, and subjected to electrophoresis on Tris-borate gels (Fig. 2, bottom panel). The major immunoglobulin glycopeptide peak seen after gel
filtration (pool C, top panel, Fig. 2) clearly results in a single band on Tris-borate gels. Likewise, pool B has a corresponding single band on the gel. Pool A, however, resolves
GEL ELECTROPHORESIS
OF GLYCOPEPTIDES
than peak C on the gel. Although one band is most likely the peak between fractions 75-80, not enough information is available on the immunoglobulin glycopeptides to resolve this problem. Since Sindbis virus glycopeptides have been characterized in much more detail (7,13,14) a series of experiments similar to those in Fig. 2 were
into three distinct bands. On the one hand, then, Tris-borate gel electrophoresis yields a glycopeptide pattern comparable to P-6 gel filtration. On the other hand, this gel system seems capable of exposing a heterogeneity in glycopeptides that is not revealed by gel filtration. A few bands can be seen migrating slower
L
1
?
I
I
I
,
64
52
I
76
88
“0
FRACTION
SBV TOP@
BOTTOM
443
NUMBER
SI s2 s3 s4
--,
0 +
FIG. 3. Comparison of Bio-Gel P-6 chromatography and Tris- borate gel electrophoresis using Sindbis virus glycopeptides. Top panel: Sindbis virus glycopeptides were fractionated as described in Fig. 2 for immunoglobulin glycopeptides. Bottom panel: Isolated pools of Sindbis virus gfycopeptides were analyzed by Tris-borate gel electrophoresis and compared to unfractionated Sindbis virus glycopeptides in the first lane (SBV).
444
WEITZMAN,
SCOTT,
carried out using Sindbis virus glycopeptides. In the top panel, Fig. 3, the glycopeptide profile of Sindbis virus is resolved into four peaks, as previously described (14). Again, gel electrophoresis of the pooled fractions yields a definite pattern: Smaller glycopeptides migrate slower in the gel than larger glycopeptides. Each pool from the Bio-Gel P-6 column appears distinctly different on the Trisborate gel, although the S 1 pool has some S2 glycopeptides, probably due to overlap of these pools. The interesting finding is that electrophoresis in Tris-borate gels exposes a heterogeneity in each pool that is not evident during gel filtration. Two explanations for this heterogeneity were investigated: first, that it is caused by peptide heterogeneity in the glycopeptides; and second, that it is caused by fucose heterogeneity. Peptide heterogeneity exists because the Sindbis virus glycopeptides are derived from two glycoproteins, El and E2, and the amino acid composition of the Pronasederived glycopeptides from El are different than those derived from E2 (D. J. Burke, Ph.D. thesis, 1976). In order to determine whether the double band in each glycopeptide peak reflected variation in amino acids, El was separated from E2 as previously described (7). Each was digested separately with Pronase and subjected to electrophoresis in adjacent lanes on a Trisborate slab gel. Both sets of glycopeptides resolved into similar patterns with double bands for glycopeptides S 1, S2, and S3 (data not shown). Previous studies have shown that glycopeptides Sl, S2, and S3 contain less than 1 mol of fucose per mole of glycopeptide (7,13). It seemed possible that one glycopeptide band contained fucose, while the second did not. In order to determine whether this fucose heterogeneity caused the double band, Sindbis virus glycopeptides labeled with [3H]fucose were mixed with glycopeptides labeled with [14C]glu-
AND
KEEGSTRA
cosamine and subjected to electrophoresis on a cylindrical gel. The gel was sliced and the radioactivity determined in each slice. Although the double bands were not clearly resolved on the cylindrical gels, the fucoselabeled glycopeptides exhibited the same pattern as the glucosamine-labeled ones, suggesting that both the glycopeptides of the double band contain fucose. Analysis of Glycosidase Products
Digestion
Glycosidase digestion of glycopeptides with known structures and, therefore, known reaction products were first examined to test the feasibility of monitoring glycosidase digestions on Tris-borate gels. Ovalbumin glycopeptides were prepared and acetylated with [14C]acetic anhydride following published procedures (see Experimental Procedures). Three of the glycopeptide peaks obtained in this manner correspond to peaks C, D, and E of Huang et al. (8). Peak C has been shown to consist of three different glycopeptides, all of which can be cleaved by endo-p-N-acetylglucosaminidase H, but not by endo+-N-acetylglucosaminidase D (14). Peaks D and E have been shown to consist of Asn-GlcNAc,Man, and Asn-GlcNAc,Man,, respectively (9). Peak D is similar to peak C in that it can be cleaved by both enzymes. The patterns obtained after electrophoresis in Tris-borate buffer of the untreated glycopeptides, as well as those treated by endo-@-N-acetylglucosaminidases D and H, are shown in Fig. 4. Lanes 1,4, and 7 show the patterns obtained with untreated glycopeptides C, D, and E, respectively. Other samples of glycopeptides C, D, and E were treated with a partially purified preparation of endo-p-N-acetylglucosaminidase D prior to electrophoresis. The products of these digestions are shown in Lanes 2,5, and 8 for glycopeptides C, D, and E, respectively. Finally, other samples of these glycopeptides were treated with highly purified endo-/3-Nacetylglucosaminidase H and these products
GEL ELECTROPHORESIS
0
TOP -
0+
BOTTOM d
I
2.
3
445
OF GLYCOPEPTIDES
4
5
6
7
8
9
FIG. 4. Analysis of endo-glycosidase digestions of ovalbumin glycopeptides. Ovalbumin glycopeptides were “C-acetylated and fractionated into three groups-C, D, and E-as described under Experimental Procedures. The untreated glycopeptides (-) were applied to a Tris-borate gel, lanes 1, 4, and 7. Glycopeptides after digestion with endo+-N-acetylglucosaminidase D (D) were applied to lanes 2,5, and 8 and after digestion with endo-b-N-acetylglucosaminidase H (H) to lanes 3,6, and 9.
are shown in Lanes 3, 6, and 9 for glycopeptides C, D, and E, respectively. The data in Fig. 4 show the expected results. Peak E is cleaved by both endo+-Nacetylglucosaminidases H and D. Peaks C and D are cleaved by endo+-N-acetylglucosaminidase H but not by D, although peak C migrates slightly faster after incubation with the crude endo-p-N-acetylglucosaminidase D mixture. The 14C-acetylated glycopeptides are cleaved to yield an unlabeled oligosaccharide and a smaller 14C-acetylated product consisting only of Asn-GlcN. This latter product appears as a radioactive band near the ion front. The loss of intensity in this band, compared to the undigested substrate, is probably due to diffusion of this relatively smaller molecule out of the gel during drying. A similar series of experiments using specific glycosidases on Sindbis virus glycopeptides establishes the ease with which this technique can provide partial structural data. A mixture of glycopeptides from
Sindbis virus grown in chick cells was applied to lane A (Fig. 5); the same mixture was treated with neuraminidase and applied to lane B. Neuraminidase treatment caused the disappearance of glycopeptides Sl and S2 with a concomitant increase in glycopeptide S3, as previously described (9). Subsequent treatment of neuraminidasetreated glycopeptides with /3-galactosidase caused no observable change in the glycopeptide pattern (Fig. 5, lane C). The mixutre of neuraminidaseand pgalactosidase-treated Sindbis virus glycopeptides was treated with endog-IV-acetylglucosaminidase D and subjected to electrophoresis (Fig. 5, lane D). This enzyme completely hydrolyzed glycopeptide S3, yielding two radioactive products. One product which migrates near the ion front is poorly labeled and can be seen as a faint band in the original gels but is not visible in Fig. 5. A second product migrates as a diffuse band slightly faster than one of the components of glycopeptide S4. Three dif-
446
WEITZMAN,
A
SCOTT, AND KEEGSTRA
B
C
D
E
FIG. 5. Structural studies of Sindbis virus glycopeptides using specific exo- and endo-glycosidases. Purified Sindbis virus was digested with Pronase and an ahquot removed for electrophoresis as an untreated sample in lane A. The remaining sample was incubated with neuraminidase and an aliquot removed for lane B. The sample was then incubated with purified P-galactosidase and an aliquot removed for lane C. The remaining sample was divided into two equal parts; one was incubated with endoP-N-acetylglucosaminidase D (lane D) and the other with endo-P-N-acetylglucosaminidase H (lane E).
ferent radioactive digestion products would have been expected, [14C]glucosamine, [‘“Clglucosamine-labeled oligosaccharide, and [14C]glucosamine-labeled glycopeptide. It is not clear from the present experiments which products give rise to the observed bands. Treatment of the neuroaminidase- and pgalactosidase-treated Sindbis virus glycopeptides with endo+-N-acetylglucosaminidase H does not affect glycopeptide S3 but results in the disappearance of glycopeptide S4 (Fig. 5, lane E). Again two radioactive products are observed from this reaction, one migrating at the ion front (not visibile in this figure) and one migrating just slightly faster than untreated S4. These experiments suggest that glycopeptide S4 is a simple, high-mannose structure, while glycopeptides Sl, S2, and S3 are similar to other previously reported complex structures. This conclusion is similar to that obtained
from other studies on these glycopeptides (7, 13). DISCUSSION
The experiments reported in this paper illustrate the electrophoretic separation of glycopeptides in a polyacrylamide gel using a Tris-borate buffer. This system offers resolution of glycopeptides equal to or better than conventional gel filtration columns (Figs. 2 and 3). Two additional features of this Tris-borate gel electrophoresis system provide unique advantages in analyzing glycopeptides compared to other available techniques. First, radioautography allows small amounts of radiolabeled glycopeptides to be visualized by prolonging the exposure time. This technique can be used with a wide variety of glycopeptides which can be made radioactive, either by in viva labeling as with the Sindbis virus or immunoglobulin gly-
GEL
ELECTROPHORESIS
copeptides or by in vitro labeling as with the ovalbumin glycopeptides. Second, the potential exists to compare simultaneously up to 24 samples on a single slab gel. This potential can be advantageous in either structural or kinetic studies. For example, untreated glycopeptides and the products of several exo- and endo-glycosidase digestions can be subjected to electrophoresis simultaneously as in Fig. 5 to yield partial structural data. Another use would be in pulse-chase experiments with multiple time points to study the kinetics of the in vivo processing of oligosaccharides after attach-
H3B03+
OH
_
447
OF GLYCOPEPTIDES
ment to proteins. The advantages of a slab gel system to analyze glycopeptides seem clear. However, in order to interpret a pattern after Tris-borate gel electrophoresis, the basis for the resolution of glycopeptides needs to be understood. Borate complexes have been utilized in the electrophoretic separation of a wide variety of different carbohydrates (5). This technique is founded upon the observation that neutral sugars are converted into charged complexes by reaction with borate ions at alkaline pH. The reaction between borate ions and carbohydrates can be represented by the reactions shown below.
-
-
r
l-
- ‘“\ /“\ /J Hd’\O-:L
L The formation of the charged borate- sugar complex is dependent upon a number of variables including pH, with alkaline pH favoring the formation of the charged complexes. Another variable strongly affecting the formation of complexes involves the stereochemical disposition of the hydroxyl groups. For glycosidically linked sugars, where the sugars are locked into a pyranose ring form, adjacent hydroxyls react much more readily in the cis configuration than the tram configuration. Thus, methyl-/3-D-xylopyranoside (all transhydroxyls) does not form a complex with borate ions and does not migrate during electrophoresis in the presence of borate. Methyl-P-D-ribopyranoside, however (all cis-hydroxyls), forms a complex and migrates toward the anode during electrophoresis (5). Borate can form a complex with nonadjacent hydroxyls if the stereochemistry is correct. For example, methyl-
‘R2-I
p-D-glucopyranoside (all trans-hydroxyls) migrates during borate electrophoresis due to formation of a borate complex at the hydroxyl groups on C4 and C6 (5). However, the equilibrium for charged complex formation is unfavorable and this compound shows a very low mobility in borate electrophoresis. Studies examining the electrophoretic separation of carbohydrates as borate complexes have been carried out with monosaccharides, simple oligosaccharides, and in some cases polysaccharides (5). No studies have reported the electrophoretic separation of glycopeptides. Most of the previous work has been carried out using zone electrophoresis, where paper has been used as the support matrix. In this study polyacrylamide gels serve as the support. This choice has the advantage that the equipment and techniques for acrylamide gel electrophoresis
448
WEITZMAN,
SCOTT,
are readily available in most laboratories because of the widespread utilization of polyacrylamide gels for the electrophoretic separation of proteins and nucleic acids. The percentage of acrylamide in the gel appears to be unimportant in determining electrophoretic mobility. The separation of glycopeptides is not a result of filtration of molecules based on their molecular weight, as is the case for uniformly charged polypeptides in the presence of sodium dodecyl sulfate. The results presented in Fig. 1 support the hypothesis that the observed separations are a result of electrophoretic migration of borate complexes of the glycopeptides. If borate ion is omitted from the buffer system, the glycopeptides migrate only poorly and as a single band. In the presence of borate ions the glycopeptides are resolved into a number of distinct bands. In general, larger oligosaccharides will bind more borate than smaller oligosaccharides, acquire a greater negative charge, and migrate further into the gel. This seems to be true for the immunoglobulin and Sindbis virus glycopeptides as illustrated in Figs. 2 and 3. However, the amount of borate bound and the migration into the gel is not simply a function of the number of monosaccharides in a glycopeptide but also the composition and structure of the oligosaccharide. Sialic acid residues not only bind borate but also contribute an additional negative charge because of carboxyl groups. This results in a marked increase in migration of sialated compared to desialated glycopeptides as demonstrated in Fig. 5. In addition, terminal mannose, galactose, and fucose residues would be expected to bind borate ions while terminal glucosamine residues would not. That these characteristics influence the migration of glycopeptides in the borate gel is seen in Fig. 4. The untreated ovalbumin glycopeptide pool C (lane 1) contains two terminal glucosamine residues on an oligomannosyl core. After treatment with the endo-/3-N-acetylglucosaminidase D
AND
KEEGSTRA
mixture which also contains exoglucosaminidase activity (1 l), the glycopeptide migrates further into the gel (lane 2). The removal of the terminal glucosamine residues, which do not bind borate, led to the availability of two mannose residues which do bind borate. The resulting increase in negative charge results in the further migration of the glycopeptides. In addition to the number and sequence of sugars in the glycopeptide, the linkage between the carbohydrate residues can influence the amount of borate bound. For example, 2linked mannose or 3-linked galactose would not be expected to bind borate, while the 6linked derivatives of these sugars should bind borate. A number of questions remain unanswered about the experiments presented in this paper. The cause of the double bands in the Sindbis virus glycopeptides is unclear. This is obviously not an artifact of the gel system since the major immunoglobulin glycopeptide and the ovalbumin glycopeptides appear as single bands (Figs. 2 and 4). The possibility remains that a single peak isolated from a Bio-Gel P-6 column contains a mixture of oligosaccharides which is resolved by Trisborate electrophoresis. In addition, the influence that the peptide portion of the glycopeptide has on the behavior of these molecules in this system has only been partially characterized. The presence of a negative charge on the carboxyl terminal amino acid is most probably neutralized by the positive charge on the amino terminal end of the peptide (except for the ovalbumin glycopeptides where the amino termini are blocked with [14C]acetic anhydride). The Sindbis glycopeptides from El and E2 have some differences in amino acid composition and yet comigrate on Trisborate gels. An additional possibility to consider is that heterogeneity in peptide length exists due to incomplete Pronase digestion, and this results in the double bands of Sindbis virus glycopeptides. While further
GEL ELECTROPHORESIS
studies on this problem need to be done, the accumulated evidence suggests that the peptide moiety has little or no effect on the electrophoretic separation of glycopeptides. In summary, the system described in this report provides a convenient method for the rapid, inexpensive analysis of glycopeptides. The inability to distinguish three species in the ovalbumin pool C (Fig. 4) indicates that this is not a high-resolution technique and the extent to which it can be used as a precise analytical tool remains to be determined. The resolution of this system, however, seems to be better than conventional Bio-Gel P-6 chromatography. The value, then of Tris-borate gel electrophoresis lies in its capacity to compare multiple samples, to screen for differences and similarities in glycopeptides from a variety of biological sources, and to collect preliminary structural data with the use of specific glycosidases. ACKNOWLEDGMENTS We thank Dr. Matthew ScharlT for his original ideas on this project and Margery Grennon and Jack Bruno for technical assistance.
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OF GLYCOPEPTIDES
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