Mild alkaline borohydride treatment of glycoproteins—A method for liberating both N- and O-linked carbohydrate chains

ANALYTICAL

BIOCHEMISTRY

119, 351-359 (1982)

Mild Alkaline Borohydride Treatment of Glycoproteins-A Method Liberating Both N and OLinked Carbohydrate Chains’ SHUN-ICHIROOGATAANDKENNETHO. Memorial

Sloan-Keltering

Cancer

Center,

1275

York

Avenue,

for

LLOYD New

York,

New

York

10021

Received July 6, 1981 The generally accepted concept that N-linked oligosaccharides in glycoproteins are considerably more stable to mild alkali than are O-linked chains has been reexamined. A number of ‘H-labeled model glycoproteins (fetuin, transferrin, and glycophorin) were treated with: (i) 0.05 M OH--l M BH; at 50°C for 16 h, (ii) 0.1 M OH--O.8 M BH, at 37°C for 68 h, and (iii) 1 M OH--4 M BH; at 80°C for 24 h. Analysis of the products by gel filtration and paper electrophoresis showed that both N- and O-linked chains were released by all three conditions. A portion (40%) of the asparagine-linked units produced under condition (i) remained as glycopeptides. Although mild alkaline borohydride cannot be used as a diagnostic reagent for distinguishing N- and 0-glycosides, it is a useful general procedure for the analysis of the carbohydrate moieties of glycoproteins. Application of the method to a differentiation antigen (gp 160) of human kidney epithelia is described.

It is generally believed that the N-glycosidic linkages between N-acetylglucosamine and asparagine in glycoproteins are considerably more stable to mild alkaline conditions than are U-glycosidic linkages between serine and threonine and N-acetylgalactosamine (l-5). O-Glycosides are released by an alkali-catalyzed P-elimination reaction and low concentrations of base (0.05-0.2 M) and low temperatures (20-50°C) have been found to be sufficient to carry out the reaction efficiently (6-9). In contrast, the scission of GlcNAc*-asparagine bonds is a hydrolysis reaction and more rigorous conditions (e.g., 1 M OH- at 80- 100°C) have been used for this procedure ( 10,ll). Since the liberated oligosaccharides can

undergo “peeling” reactions when exposed to alkali, a reducing agent (NaBH,) is commonly included in the reaction mixture to minimize or prevent this reaction (reviewed in (12)). The concentration of NaBH, can also influence the rate of scission of oligosaccharide-protein linkages (3). During the course of studies (13) on a common cell surface glycoprotein (gp 110) of human nucleated cells, we encountered difficulties in explaining the results on the basis of the commonly accepted concepts for the alkali stability of N- and O-linked oligosaccharides. This lead us to reexamine the effects of mild alkaline-borohydride treatment on such linkages using model glycoproteins of known structure (i.e., transferrin, fetuin, and glycophorin). The results showed that both N- and O-linked oligosaccharides are released under relatively mild conditions.

’ This study was supported by grants from the National Cancer Institute (CA-08478, CA-21445, and CA19765). A preliminary report of this study was presented at the sixth International Symposium on Glycoconjugates, Tokyo, Japan, September 20-25, 1981, p. 23. * Abbreviations used: GlcN, glucosamine; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; SDS, sodium dodecyl sulfate.

MATERIALS

AND METHODS

Glycoprotein. Fetuin (Type IV) was purchased from Sigma Chemical Company. Human transferrin was obtained from Cal351

0003-2697/82/020351-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

352

OGATA

AND

biochem-Behring Corporation. The major red cell glycophorin was prepared according to the method of Springer et al. ( 14). gp 160, a differentiation antigen of human kidney cells (15) was prepared by immunoprecipitation of [ ‘H]GlcN-labeled renal cancer cell lysate (SK-RC-7) with monoclonal antibody S,. Chemicals and enzymes. Sodium [ ‘Hlborohydride (170 mCi/mmol) was obtained from New England Nuclear (Boston, Mass). It was dissolved in 0.05 M NaOH (12.5 mCi/ml) and stored at -80°C. Neuraminidase (Vibro cholerae, 500 U/ml) was purchased from Calbiochem-Behring Corporation. &Galactosidase (EC 3.2.1.23) and /3-N-acetylglucosaminidase (EC 3.2.1.30) both from jackbean, were purchased from Sigma Chemical Company. Endo-P-N-acetyl-D-glucosaminidase (Diplococcus pneumoniae) was kindly supplied by Dr. Muramatsu (Kagoshima University, Japan). Galactose oxidase (Polyporus circinatus) was obtained from Sigma Chemical Company and purified free of proteases as previously described (16). Protease Type V (from Streptomyces griseus) was purchased from Sigma Chemical Company. Radiolabeling of glycoproteins with galactose oxidase-NaB3H4. Approximately 5

mg of fetuin were digested with 6.5 units of neuraminidase at pH 5.5 for 16 h. The digest was then dialyzed extensively against water and freeze-dried. Desialyated fetuin was then dissolved in 1 ml PBS, treated with 10 units of purified galactose oxidase at 37’C for 1 h and reduced by 1.25 mCi of NaB3H4 (300 mCi/mmol) at room temperature for 15 min. After the incubation, the reaction mixture was subjected to Bio-Gel P-60 COIumn chromatography. Radioactive fractions ([ 3H ]Gal-asialofetuin), eluted at the void volume of the column were collected, concentrated, and dialyzed against water. Transferrin and glycophorin were also labeled in the same way. Alkaline borohydride treatment of Iabeled glycoproteins. [ 3H]Gal-asialofetuin

LLOYD

(approx 2 X lo5 cpm) was treated with 0.3 ml of 0.05 M NaOH-1 M NaBHa at 50°C for 15 h according to the procedure of Iyer and Carlson (8). After incubation, the reaction mixture was adjusted with acetic acid to pH 4.5 and the products separated by gel filtration as described below. Other samples of [ 3H]Gal-asialofetuin were treated under the following conditions: (i) 0.1 M NaOH8 M NaBH4 at 37°C for 68 h (9) and (ii) 1.0 M NaOH-4 M NaBH4 at 80°C for 24 h ( 10,ll). [ 3H]Gal-asialotransferrin and [ 3H JGal-asialoglycophorin were treated with 0.05 M NaOH-1 M NaOH4 as described above. Hydrazinolysis

of labeled glycoproteins.

[3H]Gal-asialofetuin (2 X lo5 cpm) was treated with 0.3 ml of anhydrous hydrazine at 100°C for 10 h as described by Yamashita et al. ( 17). The released oligosaccharides were reduced with NaBH4 (17). Protease digestion of labeled glycoproteins. [3H]Gal-asialofetuin (2 X lo5 cpm) was digested at 37°C with protease in 50 mrvr Tris-HCl, pH 8.0, buffer containing 10 mM CaCl,. Enzyme was added at a concentration of 3 mg/ml every 24 h for 3 days. The reaction mixture was adjusted to pH 8.0 with 0.5 N NaOH after each addition of the enzyme. Treatment of glycoproteins with NaOHNaB3H4. Asialofetuin (0.5 mg) was dissolved in 0.2 ml of 0.05 M NaOH containing 1 M NaB3H4 (7 mCi/mmol) and incubated at 50°C for 15 h. The sample was then deionized with Dowex 50 (H+) and by evaporation with methanol. After separating the products into peaks I and II on a Sephadex G-50 column, the labeled, reduced end group in each peak was determined by hydrolyzing the samples in 2 N HCl for 3 h at lOO”C, re-N-acetylating and analyzing for N[ 3H]acetylglucosaminitol and N-[ ‘Hlacetylgalactosaminitol by paper chromatography on borate-impregnated paper ( 18). GelJiltration chromatography. Gel filtration was performed on Sephadex G-50 and G-25 columns (1.5 X 88 cm) in 0.1 M pyr-

ALKALINE

BOROHYDRIDE

TREATMENT

OF PROTEIN-CARBOHYDRATE

idine-acetate buffer, pH 5.7. Fractions (1.5 ml) were collected, and aliquots were counted by liquid scintillation counting. Discrimination between oligosaccharides and glycopeptides by N-acetylation and Radioactive peaks from electrophoresis.

Sephadex G-50 columns were freeze-dried and subjected to high-voltage paper electrophoresis (Savant Instruments) at pH 5.4 in pyridine: acetic acid:water (3:1:287) at a potential of 73 V/cm for 1.5 h. The paper was scanned with a Packard Radiochromatogram scanner (Model 7201). Radioactive material in the neutral fractions was extracted from the paper with water and Nacetylated with acetic anhydride-NaHC03. The N-acetylated sample was refractionated by paper electrophoresis as described above. The paper was scanned to detect neutral material (oligosaccharides) and material migrating towards the anode (glycopeptides). RESULTS Mild Alkaline Borohydride Treatment Radiolabeled Glycoproteins

of

To evaluate the effects of mild alkaline borohydride, three glycoproteins whose carbohydrate structures and carbohydrate-protein linkages have been determined were studied. These glycoproteins were fetuin, transferrin, and glycophorin (Fig. 1). Figure 2A shows the separation on a Sephadex G-50 column of the products resulting from treating asialofetuin, labeled in its galactose residues by galactose-oxidase boro-[3H]hydride ([ ‘HIGal-asialofetuin), with 0.05 M NaOH-1 M NaBH, at 50°C for 15 h as described by Iyer and Carlson (8). Two sharp peaks (I and II), both within the included volume of the column, were observed. No material was eluted in the excluded volume of the column. Peak II was rechromatographed on a Sephadex G-25 column. It was eluted in the disaccharide region and therefore corresponds to the expected

LINKAGES

353

disaccharide (Gal-GalNAc-01) liberated from asialofetuin by /3 elimination. Peak I was eluted from the Sephadex G50 column at a position expected for a compound with a molecular weight of about 2000; it was therefore provisionally assumed to correspond to the complex carbohydrate chains which are known to be triantennary in fetuin (19,20) and therefore would have a molecular weight of 1862. To obtain further information on this point, the elution profiles of Pronase and hydrazine-treated asialofetuin were determined. The elution profile of hydrazinolyzed [ 3H]Gal-asialofetuin (Fig. 2C) was exactly the same as that of alkaline borohydride-treated material. Thus, peak I from alkaline borohydride treatment was eluted at a position corresponding to N-linked complex chains. Although the second peak resulting from hydrazine treatment was eluted in the same fractions as peak II from alkali borohydride treatment, rechromatography on Sephadex G-25 showed that it was smaller than a disaccharide. Other workers (20) have also recognized that hydrazinolysis may result in degradation of 0glycosidically linked chains. The elution profile on Sephadex G-50 of Pronase-digested [ 3H]Gal-asialofetuin (Fig. 2D) is more complex than the other patterns but again the larger glycopeptides were eluted in the same position as peak I in Fig. 2A. Labeled asialotransferrin ([ 3H]Gal-asialotransferrin) and asialoglycophorin ([ 3H]Gal-asialoglycophorin) were also treated with 0.05 M NaOH- 1 M NaBH4 and the products separated by Sephadex G-50 chromatography. Asialotransferrin which is known to contain only asparagine-linked biantennary complex chains (2 I), produced only one major peak (Fig. 3B) which was eluted at a slightly later position than peak I from asialofetuin. The small second peak in the transferrin elution pattern is a radioactive impurity (determined by Sephadex G25 chromatography). This material could not be removed by prior dialysis of the ra-

354

OGATA

AND LLOYD

E-LINKED

E-LINKED

CHAINS

CtlAlNS

B-Gal(l+4)&GlcNAcl,, 4 a-Man

Asialofetuin' B-Gal(l~4)8-GlcNAcl/'

1 1

,

3? B-Mar1(l+4)B-GlcNAc(1+4)GlcNAc-

i

B-Gal(l+4)B-GlcNAc(l'Z)u-Man

1

&Gal(l+4)B-GlcNAc(l+Z)~-Man

1,

3 chains

3 %-Man(l*4)%-GlcNAc(1'4)GlcNAc-

Asialotransferrin2 8-Ga1(1'4)6-GlcNAc(l

Z)Cx-Wan

1,

Asialoglycophorin" B-Gal(l'4)8-GlcNAc(l+Z)ff-Man and 19;

%eference

21;

1

a-Fuc 1 3 4 a-Man(l+4)6-GlcNAc(l+4)GlcNAc6 1' 1 chain

8-Ga1(1+4)8-GlcNAc(1+2)a-Man

18

2 chains

lH6

B-GLCNAC

'References

bGal(l+3)GalNAc-

3 chains

'Reference

1 c 6 6-Ga1(1'3)GalNAc15 chains

22

FIG. 1. Proposed structures for the carbohydrate moieties of the model glycoproteins used in this study.

diolabeled glycoprotein and probably contributes to a small extent to the second peak in the fetuin profile also. The elution profile of [ ‘HIGal-asialoglycophorin treated with mild alkaline borohydride (Fig. 3A) showed one large peak in the disaccharide region and a smaller peak with a larger molecular weight. Since glycophorin is known to contain 1 N-glycosidically linked complex chain and 15 0-glycosidically linked disaccharides (22), these results are consistent with the release of both chains by NaOH-NaBH, Are the IV-Asparagine-Linked Chains Released as Oligosaccharides or Glycopeptides? The question arose as to whether peak I released from the glycoproteins represented oligosaccharide or glycopeptide material. To answer this question peak I and peak II material from [ 3H]Gal-asialofetuin was Nacetylated and the resulting charge determined by paper electrophoresis at pH 5.4.

The rationale for this procedure is that glycopeptides will be negatively charged at this pH because of their carboxyl groups whereas oligosaccharides will be neutral. Figure 4 shows that whereas all the radioactivity in N-acetylated peak II migrated in the neutral position, about 40% of peak I radioactivity migrated towards the anode. These results indicate that the 0-glycosidic chains of fetuin were completely released from the peptide by mild alkaline borohydride treatment. About 60% of the N-glycosidically linked chains were similarly released with remaining material having at least one amino acid still attached. Release of Carbohydrate Chains from Unlabeled Glycoproteins by Mild NaOH-NaB’H, Treatment Further information on the release of carbohydrate chains from glycoproteins by mild alkali treatment was obtained by treating asialofetuin with NaOH in the presence of

ALKALINE

BOROHYDRIDE

TREATMENT

NaB3H4. After separation on Sephadex G50 (Fig. 5) two radioactive peaks were obtained which corresponded to the two peaks shown in Fig. 2A. In contrast to the result obtained with prelabeled asialofetuin (Fig. 2A), the first peak was smaller than the second peak. This is probably due to release of only a portion of the N-glycoside chains as oligosaccharides. Further confirmation of the identity of peak I with the complex chains of fetuin was obtained by digestion with exo- and endoglycosidases. Treatment of peak I with P-galactosidase and &N-acetylhexosaminidase reduced the apparent molecular weight of the component and the resulting product became susceptible to endo&N-acetyl-D-glucosaminidase. In addition, hydrolysis and separation of the re-

o-

OF PROTEIN-CARBOHYDRATE

FRACTION

LINKAGES

355

NUMBER

A + I II

FIG. 3. Gel filtration (Sephadex G-SO) profiles of (A) [‘HIGal-asialoglycophorin and (B) [‘HIGal-asialotransferrin after treatment with 0.05 M NaBH4-I M NaBH, at 50°C for 16 h. Fraction size: 1.5 ml.

A 0,

sulting labeled sugars showed that peak I contained N-[3H]acetylglucosaminitol whereas peak II contained N-[3H]ace-

5-

oo-

II

I -&=Lb!L

5-

o-

,c

n

o-

I 5.

O[ o-

A ’

I

I

n

s-

3-

.;. 30

40

FRACTION

50

60

70

80

NUMBER

FIG. 2. Gel filtration (Sephadex G-SO) profiles of [ ‘HIGal-asialofetuin after treatment with various reagents. (A) 0.05 M NaOH-1 M NaBH4 at 50°C for 16 h; (B) 1.0 M NaOH-4 M NaBH., at 80°C for 24h; (C) hydrazinolysis; (D) Pronase digestion. Fraction size: 1.5 ml.

FIG. 4. Paper electrophoresis of N-acetylated components derived from [‘HIGal-asialofetuin. (A) Peak I produced by mild alkaline borohydride treatment (Fig. 2A); (B) peak II produced by mild alkaline borohydride treatment (Fig. 2A); (C) peak I produced by Pronase digestion (Fig. 2D); (D) peak II produced by Pronase digestion (Fig. 2D); (E) peak I produced by hydrazinolysis.

356

OGATA

AND LLOYD

1

derived from N-asparagine-linked chains.

complex

Mechanism and Optimal Conditions for Liberating N-Asparagine-Linked Chains from Glycoproteins with Mild Alkaline Borohydride

0M

30 40 50 60 70 80 FRACTION NUMBER

FIG. 5. Gel filtration (Sephadex G-50) of asialofetuin after treatment with 0.05 M NaOH-1 M NaB’H, at 50°C for 16 h. Fraction size: 1.5 ml.

tylgalactosaminitol (Fig. 6). These data show that the larger molecular weight peak released from fetuin by mild alkaline borohydride treatment corresponds to material

While the mechanism for the release of 0-glycosidically linked oligosaccharides from glycoproteins by alkali is known to be by @ elimination, the mechanism for the release of asparagine-linked chains is unclear. Some information on this point was obtained by studying the time course for the release of carbohydrate chains from t3H JGal-asialofetuin by alkaline borohydride. The model compound was treated with 0.05 M NaOH1 M NaBH4 at 50°C for periods varying from

01234567a?

IO DISTANCE

FROM

ORIGIN

II

l-2

13

(CM)

FIG. 6. Identification of ‘H-alditols in reduced oligosaccharides liberated from asialofetuin by mild alkaline B’H,. The oligosaccharides were hydrolyzed and the sugars re-N-acetylated as described in the test. The sugars were then separated by chromatography on Whatman No. 1 paper (previously impregnated with 0.57% Na2B20,. 10HrO solution containing 0.01 M NaCI) in ethyl:acetate:2-propanol:pyridine:water (7:3:2:2) for 48 h. The chromatogram was cut into 0.5-cm strips which were counted for radioactivity. The migration positions of standard N-acetylglucosaminitol (GlcNA& and N-acetylgalactosamintol (GalNAco,) are indicated by arrows. (A) Peak I, (B) peak II.

ALKALINE

BOROHYDRIDE

TREATMENT

5-

A

O* F

8r.

,

IO -

5-

0

30

40

FRACTION

50

60 70

80

NUMBER

FIG. 7. Time course for the degradation of [‘HIGalasialofetuin with 0.05 M NaOH-1 M NaBH4 at 5O“C. Products separated by Sephadex G-50 chromatography. Fraction size: 1.5 ml.

15 min to 72 h and the products separated by Sephadex G-50 chromatography (Fig. 7). After 15 min treatment, a large amount of reduced disaccharide (peak II) had been released but only a small amount of material

OF PROTEIN-CARBOHYDRATE

LINKAGES

357

was present in the peak 1 region. Most of the radioactivity was eluted in the high-molecular-weight region of the column with material being present in both the excluded and included regions. With more prolonged treatment these high-molecular-weight components disappeared and were replaced by peak I material. Peak I progressively increased in height and became narrower during the first 8 h of treatment (Fig. 7). These results can be explained by assuming that the 0-glycosidically linked chains are released directly and fairly rapidly from the peptide. Peak I, on the other hand, arises by the initial formation of glycopeptides which are mostly (60%) hydrolyzed to oligosaccharides over a period of about 8 h. Interestingly, increasing the reaction time to 72 h does not lead to an increase in the proportion of oligosaccharide formed. Other alkaline borohydride conditions were also examined to determine whether the selective release of either 0- or N-linked chains could be obtained. Treatment with 0.1 M NaOH-0.8 M NaBH, at 37°C for 65 h as suggested by Spiro and Bhoyroo (9) gave essentially the same results as those reported above. Lowering the temperature of the reaction to 4°C (using 0.05 M NaOH-1 M NaBH,) slowed the rate of the reaction but also led to the release of both 0- and Nlinked chains. Hot alkaline borohydride (1 M NaOH-4 M NaBH, at 80°C) treatment resulted in the formation of two peaks (Fig. 2B) as would be expected from the results of Lee and Scocca (10). The first peak, however, was much smaller than that obtained using the milder conditions indicating that these more rigorous conditions may partly degrade the complex chains. Application of the Mild Alkaline Borohydride Procedure to the Study of Novel Cell Surface Glycoproteins

[ 3H]Glucosamine-labeled gp 160 was isolated from a NP-40 lysate of human renal cancer cells by immunoprecipitation with a

OGATA

358

AND LLOYD

FIG. 8. Gel filtration (Sephadex G-50) of [3H]glucosamine-labeled gp 160 glycoprotein from human kidney cancer cells after treatment with 0.05 M NaOH-I M NaBH, at 50°C for 16 h. Fraction size: 1.0 ml.

specific monoclonal antibody (15). The immunoprecipitate was dissolved in lysis buffer and separated on a thin ( 1.5 mm) polyacrylamide slab gel by SDS-polyacrylamide gel electrophoresis and the radiolabeled component was detected by fluorography (15). The gel band was excised and treated with 0.5 ml 0.05 M NaOH-1 M NaBH4 at 50°C for 15 h. The solution was then neutralized and applied to a Sephadex G-50 column as described above (Fig. 8). One major peak, which was eluted out in the same fractions as the N-linked complex chains from fetuin, was observed. DISCUSSION

Our results are not in accordance with the widely accepted idea that the N-glycosidic linkages between N-acetylglucosamine and asparagine in glycoproteins are relatively stable to the various mild alkaline conditions that can be used to liberate O-glycosidically linked oligosaccharides from serine and threonin residues. Although it is agreed that GlcNAc-Asn linkages are cleaved by hot alkali, most review articles emphasize the selective cleavage of 0-glycosidic linkages under mild alkaline conditions. Recently, however, Austen and Marshall (23) showed that 10% of N-glycosidic linkages are cleaved under the conditions used by Carlson (8).

Our own results demonstrate a much greater degree of scission and in fact show that mild alkali cannot be used to discriminate between N- and 0-glycosides. An analysis of the time course for the degradation of asialofetuin by mild NaOHNaBHa treatment showed that although Oglycosidically linked chains are released rapidly (cf. 24) simultaneous hydrolysis of the peptide occurs with the initial formation of glycopeptides. These glycopeptides are largely converted to reduced oligosaccharides over a period of 8 h. No conditions were found whereby maximum release of O-linked chains could be obtained without concomitant glycopeptide formation. It is important to note that the release of ‘H-labeled reduced oligosaccharides by alkaline treatment in the presence of NaB3H4 cannot, therefore, be used to determine whether a glycoprotein has O-linked as opposed to Nlinked chains. The results presented in Fig. 5 and those derived using the N-acetylation electrophoresis procedure demonstrate that both N- and O-linked chains can be released as reduced oligosaccharides even by mild alkaline treatment. Mild alkaline borohydride treatment has several advantages as a general method for the analysis of the N- and O-linked carbohydrate moieties of glycoproteins. Protease digestion is a valuable and widely used method in this field. This procedure, however, has the disadvantage that some glycoproteins have clustered oligosaccharide chains such that complete digestion is difficult. Hydrazinolysis is an efficient method for the release of N-asparagine-linked chains but may lead to the degradation of O-linked chains. Mild alkaline borohydride, on the other hand, results in the efficient release of both O- and N-linked oligosaccharides (together with a small proportion of N-glycopeptides with only a small number of amino acids attached) even from glycoproteins with clustered oligosaccharides (e.g., glycophorin). To examine the usefulness of the procedure it was applied to a t3H1-

ALKALINE

BOROHYDRIDE

TREATMENT

glucosamine-labeled cell surface glycoprotein. A differentiation antigen of human kidney cells (gp 160) was isolated by precipitation with specific monoclonal antibody (15) and analytical SDS-gel electrophoresis. The radioactive component was detected by fluorography, cut out from the gel, treated with alkaline borohydride, and the profile of labeled carbohydrate chains determined by Sephadex chromatography (Fig. 8). The method is simple, rapid, and sensitive. In conjunction with the use of monoclonal antibodies which enables one to study specific glycoproteins (as opposed to whole cell trypsinates) this procedure is an effective method for analyzing the carbohydrate moieties of cell surface glycoproteins which are often available only in minute amounts. ACKNOWLEDGMENT We thank Jennifer Ng for skillful technical assistance.

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Biophys.

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