Anhydrous hydrogen fluoride deglycosylates glycoproteins

ANALYTICAL

BIOCHEMISTRY

Anhydrous

82, 289-309 (1977)

Hydrogen Fluoride Glycoproteinsl

Deglycosylates

ANDREW J. MoRT~,~ AND DEREK T. A. LAMPORT MSUIERDA

Plant Research Laboratory and Department of Biochemistry, State University, East Lansing, Michigan 48824

Michigan

Received September 20, 1976; accepted June 14, 1977 Exposure of glycoproteins to anhydrous hydrogen fluoride cleaves all the linkages of neutral and acidic sugars within 1 hr at 0°C while leaving peptide bonds and glycopeptide linkages of amino sugars intact. More severe treatment with anhydrous hydrogen fluoride (3 hr at 23°C) cleaves the 0-glycosidic linkages of amino sugars, but peptidebonds and the N-glycosidic linkage between asparagine and N-acetylglucosamine still remain intact. Anhydrous hydrogen fluoride, therefore, may be used for the deglycosylation of glycoproteins, thereby assisting in the further purification, proteolysis, and sequencing of the protein component. During the cleavage of glycosidic linkages by anhydrous hydrogen fluoride there is little or no degradation of the sugars themselves, thus allowing their quantitative recovery. Therefore, anhydrous hydrogen fluoride may also be useful in the analysis of complex polysaccharides.

As the role of glycoproteins in eukaryotic form and function becomes more evident, so does the need for their chemical characterization. Unfortunately, glycoproteins do not always respond kindly to the classical methods of protein and carbohydrate chemistry. Glycoproteins, for example, are often resistant to proteolysis (2), especially if they are highly glycosylated. The identification of Edman degradation products is usually not attempted (3) in the region of oligosaccharide attachment, thus necessitating subtractive sequencing techniques (4). Therefore, deglycosylation should help subsequent determination of the amino acid sequence. However, enzymic deglycosylation suffers from the disadvantage that specific enzymes must be obtained while chemical deglycosylation involving periodate oxidation is invariably incomplete and also oxidizes amino 1 This research was supported by the U.S. Energy Research and Development Administration through Contract No. EY-76-C-02-1338 and by the National Science Foundation through Grant PCM76-02549. A preliminary report of this work was presented at the annual meeting of the American Society of Plant Physiologists (1). * Present address: C. F. Kettering Research Laboratory, 150 E South College, Yellow Springs, Ohio 45387. 3 This research was submitted by A.J.M. in partial fulfillment of the requirements of Michigan State University for the degree of Doctor of Philosophy in Biochemistry. 289 CopyrIght 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003.2697

290

MORT AND LAMPORT

acid residues such as cysteine and tyrosine (5), although this oxidation can be ameliorated to some extent (6). We have been acutely aware of these problems during our attempts to determine the amino acid sequence of extensin, a highly glycosylated cell wall protein present in most photosynthetic eukaryotes (7). Because of difficulties with the usual methods for deglycosylation we investigated other possibilities. Basing our approach on the analogy between glycosylated amino acid residues and protected groups in synthetic peptide chemistry we tried several reagents which bring about deprotection. We report here results with anhydrous hydrogen fluoride (HF)4 which we have used both for the solvolysis of polysaccharides and the deglycosylation of glycoproteins and glycopeptides of extensin. MATERIALS

AND METHODS

Materials

Fetuin and crab shells were bought from Sigma Chemical Co., St. Louis, MO., and larch arabinogalactan was from K and K Laboratories Inc., New York. Pig submaxillary mucin, a gift from Joseph Sung (Michigan State University), was prepared according to the method of de Salegui and Polonska (S), but without separating the major and minor fractions. Cell walls from tomato suspension cultures were prepared by homogenization in ice with an Ultra Turex homogenizer for 2 min at maximum speed in the presence of a small amount (2 g/500 ml) of sodium dithionite (to prevent oxidation of phenols), followed by a 2-min sonication of 150ml aliquots at full power in a Bronwill Biosonic III. The wall preparation was then extensively washed with cold water on a coarse fritted glass funnel, followed by 1 M NaCl, water, acetone, and finally was dried overnight in a vacuum desiccator at 60°C. Alcohols used in gas chromatography were dried by refluxing over magnesium turnings and iodine as described by Bhatti et al. (9). All other reagents were used without further purification. Methods

Anhydrous hydrogen fluoride can best be handled in a closed system. The apparatus used for the experiments, purchased from Peninsula Laboratories Inc. (San Carlos, California) was designed for deprotection of synthetic peptides after their chemical polymerization. The entire system consists of an HF cylinder, a reservoir, two reaction chambers, another reservoir, and a trap filled with calcium oxide to neutralize the HF when it is removed from the reaction vessel. These components are 4 Anhydrous hydrogen fluoride is subsequently referred to as HF.

HF DEGLYCOSYLATES

GLYCOPROTEINS

291

connected with Kel-F tubing and stopcocks and a mercury manometer. The outlet from the trap is connected to a vacuum pump. A more complete description can be found in Refs. (lO,ll, 12). HF solvolysis. Add thoroughly dried sample (up to 100 mg) and 1 ml of anisole scavenger5 to the Kel-F reaction vessel. Evacuate the entire HF line except for the HF reservoir. Coo1 the reaction vessel in a dry ice/ acetone bath and then allow ca. 10 ml of HF to distill over from the reservoir (which also contains cobalt trifluoride as a drying agent) stirring both vessels continuously. Allow the reaction vessel to warm up to appropriate temperature (vapor pressure of HF is 364 mm Hg at 0°C and 850 mm Hg at 23”(Z), timing the reaction from the appropriate point. If performing the reaction at 0°C use an ice bath. At the end of the reaction evacuate the reaction vessel via calcium oxide trap. [To prevent excessive frothing of the reaction mixture vacuum must be applied slowly but sufficiently for rapid HF removal to prevent oligomerization (13)]. To ensure complete removal of HF leave the line evacuated for an additional hour after removal of visible HF. After complete HF removal, generally shown by a change from light brown through a transient reddish color to colorless, take the sample up in 0.1 N acetic acid or 50% acetic acid in water if the residue is difficult to solubilize. Immediately chromatograph the product on an appropriate Sephadex column or dialyze. Analytical methods. HF-Treated glycopeptides (obtained by chlorite oxidation of tomato cell walls) and HF-treated chitin were fractionated on a Bio-Gel P-2 column eluted with 0.1 M acetic acid. After HF treatment of the hydroxyproline-rich extracellular glycoprotein it was fractionated on a Sephadex G-100 column eluted with 0.1 M acetic acid. Disc gel electrophoresis was performed according to the methods of Fairbanks et al, (15). Hydroxyproline was determined on an automated hydroxyproline analyzer (7) after prior hydrolysis of the sample in 5 N NaOH at 121°C for 1 hr, followed by neutralization with 5 N HCI. Gas-liquid chromatography was performed on Perkin-Elmer 900 and 910 gas chromatographs fitted with dual columns, with the output connected to a Spectra Physics System IV Autolab integrator. The support, Gas Chrom Q, and stationary phases SE-30 and SP-2100 were bought from Supelco Co., Bellefonte, Pa. Most amino acid analyses were performed on the heptafluorobutyryl isobutyl ester derivatives as described by MacKenzie and Tenaschuk (16); others were performed by liquid chromatography (17). Sugar analyses were performed on the trimethylsilylated methyl 5HF solvolysis generates fluorinatedcompounds, e.g., glycosylfluorides,whichreadily alkylatearomaticaminoacids,the HF actingas a Friedel-Crafts catalyst (14). Addition of a large excess of anisole protects these amino acids by competing for the alkylating compounds. Thus, when studying sugar recovery, the anisole must be omitted because the adducts involve a C-C linkage which does not permit recovery of the original sugar.

292

MORT

AND

LAMPORT

glycosides as described by Bhatti et al. (9) or by the alditol acetate method of Albersheim et al. (18). Glucosamine and N-acetylglucosamine were distinguished by formation of their TMS derivatives in a 1: 1 mixture of bistrimethylsilyltrifluoroacetamide and pyridine at room temperature for at least 2 hr and subsequent chromatography on a 12 ft x l/8 in.-i.d. 3% SP-2100 column programmed from 165 to 200°C at 1.5” min-‘. Weights of polypeptides were calculated from the sum of the residue weights of amino acids released by hydrolysis for 18 hr with constant boiling HCl at 110°C. As histidine, cysteine, and methionine were not determined, this is only used as a basis from which to calculate the relative amounts of sugars left attached to the protein after HF treatment. Weights of sugars and amino acids given in the tables are residue weights. Carbohydrate contents of the glycoproteins fetuin and PSM are expressed as milligrams of sugar residue per 100 milligrams of polypeptide. Thus, the percentage of original sugar remaining attached to the protein = final mg of sugar/100 mg of deglycosylated polypeptide initial mg of sugar/100 mg of untreated polypeptide

x loo

9

or the percentage of deglycosylation = 1 _ final mg of sugar/100 mg of deglycosylated polypeptide initial mg of sugar/100 mg of untreated polypeptide

x loo

,

where weights of polypeptide exclude sugar. Note that while sugar contents are often expressed as a weight percentage of the total, i.e., sugars + polypeptide, we prefer to use the formula given above because a weight loss of sugars expressed on the basis of polypeptide weight is directly proportional to the percentage of deglycosylation. RESULTS

We attempted to characterize the reaction of HF with polysaccharides and glycoproteins by selecting examples containing various linkage types. Thus, larch arabinogalactan is a simple polysaccharide; tomato cell walls can be regarded as a complex polysaccharide of neutral and acidic sugars; tomato cell wall glycopeptides contain neutral sugars 0-glycosidically linked to hydroxyamino acids; the glycoprotein fetuin has both N- and 0-glycopeptide-linked amino sugars; pig submaxillary mucin has 0-glycopeptide-linked amino sugars; chitin consists of O-glycosiditally linked amino sugars (poly-N-acetylglucosamine); while the extracellular glycoprotein from suspension cultures of sycamore maple illustrates

HF DEGLYCOSYLATES

the way in which a highly glycosylated deglycosylation. HF Solvolysis

293

GLYCOPROTEINS

glycoprotein

may be purified

via

of Polysaccharides

Some polysaccharides such as cellulose are notoriously difficult to hydrolyze, without considerable destruction of the sugars. Anhydrous hydrogen fluoride readily depolymerizes such polysaccharides, and, therefore, we considered the possibility of using this as a method to obtain good quantitative sugar analyses of “difficult” material. Thus, we compared the sugar recovery after HF solvolysis of (a) larch arabinogalactan and (b) tomato cell walls with the sugar recovery after TFA hydrolysis only. (a) Larch arabinogalactan. Larch arabinogalactan (19.68 mg) was dissolved in 1 ml of water containing 1 mg/ml of mannitol. Aliquots of this solution were hydrolyzed in 2 N TFA and then were analyzed as alditol acetates or methanolyzed and then analyzed as the TMS-O-methyl glycosides. The remaining solution of arabinogalactan was’dried in the HF reaction vessel, treated with HF for 1 hr at 0°C in the absence of scavenger and then was analysed by the two methods mentioned above. The results in Table I show that, within experimental error, HF treatment of the arabinogalactan followed by mild methanolysis gives a 100% yield of sugar residues. (b) Tomato cell walls. Plant cell walls are among the most difficult materials to analyze quantitatively. Cellulose is difficult to hydrolyze, polygalacturonic acid is prone to degradation during hydrolysis, while protein tends to react with sugars and sugar degradation products, for example, via the Maillard reaction, Because of these and other problems, cell wall analyses usually involve at least four separate assays: (i) cellulose, (ii) uranic acids, (iii) neutral sugars, (iv) protein. Anhydrous HF (1 hr at 0°C in the absence of scavenger) renders water-soluble about 90% of a cell wall preparation obtained from tomato TABLE

1

PERCENTAGEOFRECOVERYOFSUGARSFROM LARCHARABINOGALACTAN:ACOMPARISON OF HF SOLVOLYSIS,METHANOLYSIS, AND TFA HYDROLYSIS TFA” No HF solvolysis Initial HF solvolysis o TFA concentration: * Conditions:

2 N. Conditions:

I.5 M HCI in methanol,

90 100

Methanolysis’ 73 96

1 hr, 121°C. Analyzed as alditol acetates. 8 hr, 85°C. Analyzed as TMS-O-methyl glycosides.

294

MORT AND LAMPORT

cell suspension cultures. We determined the sugar recovery by analyzing the water-soluble products as alditol acetates and as TMS-O-methyl glycosides (Table 2). For maximum sugar recovery it was necessary to hydrolyze the water-soluble HF solvolysis products in 2 N TFA. The 10% of the HF-treated cell wall which remained insoluble in water or buffers appeared microscopically as very thin walls and consisted of protein plus an unidentified component in roughly equal proportions. Using HF solvolysis we were able to account for 70-80% of the wall as sugars. The protein content was ca. 5%. The amino acid composition of untreated cell walls was almost the same as that of the insoluble cell wall residue which remained after HF treatment (Table 3). HF Deglycosylation

of Hydroxyproline-Rich

Glycopeptides

Because HF cleaves the glycosidic linkages of neutral sugars we investigated the extent to which HF would deglycosylate some glycopeptides from cell walls of suspension-cultured tomato cells. These peptides were released from the walls by oxidation for 30 min at 75°C in a 1% acetic acid-l% sodium chlorite solution, a slight modification of the TABLE

2

SUGAR RECOVERIES FROM TOMATO CELL WALLS: TFA HYDROLYSIS AND HF SOLVOLYS~S

COMPAREP

TFA hydrolysis

Rha Fuc Ara XYl Man Gal GIG

Total neutral sugars Galacturonic

HF solvolysis followed by TFA hydrolysis

(nmol)

(pg of residue)

(nmol)

39 144 71 16

5.1 19.0 10.2 2.6

35 102 60

100

16.2

86 350

33

5.4 59.1

(Kg of residue) 5.1 13.5 7.9 (2.6)* 13.9 54.7 97.1 41.0

(16)

n.d.

Cell wall recovered as sugars (weight basis) (%) Cell wall recovered as amino acids (weight basis) (%)

72.1 4.2

4 We took 10 mg of cell walls for the TFA hydrolysis (2 N, 1 hr, 121°C) and 100 mg of cell walls for the HF solvolysis (1 hr, 0°C). After each treatment we removed the solvent, then added water, and centrifuged. For gc analyses we took a volume of the supernatant equivalent to 200 pg of cell wall. We analyzed neutral sugars as alditol acetates and galacturonic acid as its TMS-O-methyl glycoside. b Both mannitol and inositol were used as internal standards in this experiment.

295

HFDEGLYCOSYLATESGLYCOPROTEINS TABLE

3

AMINOACIDCOMPOSITIONOFTOMATOCELLWALLSBEFOREANDAFTER

HYP ASP Thr Ser Glu Pro G]Y Ala Val CYS Met Ile Leu Tyr Phe LYS His A% ” Normalized and Methods.

HF TREATMENTS

Before

After

30 4.4 2.5 10.1 3.0 n.d. 3.4 2.7 4.3 0 0 1.5 2.7 2.6 1.3 8.4 1.9 1.3

30 3.9 2.8 9.5 3.1 3.6 4.0 2.6 4.7 0 0.3 1.7 3.0 2.3 1.6 8.9 1.7 1.3

to 30 Hyp residues. For experimental details, see Table 2 and Materials

procedure described by Mort and Lamport (19). More than half the amino acid residues in these peptides consist of hydroxyproline in which the hydroxyl group is substituted by a short chain of arabinose residues, ranging from one to four, but with the tri- and tetraarabinosides predominating so that the average Ara/Hyp ratio is 3.1 (7). These peptides also contain 0-galactosyl serine (20). Treatment with HF for 1 hr at 0°C deglycosylated cell wall glycopeptides obtained by chlorite oxidation (Table 4). The sugars cleaved from these glycopeptides were monomers judging from their elution in the included position on a Bio-Gel P-2 column. The 2-3% of the sugars remaining linked to the peptide were not cleaved possibly because they were in a part of the reaction vial not in contact with the liquid HF. Kel F is very prone to static electricity which makes it difXcult to ensure that all of the sample is in the bottom of the reaction vessel. HF Deglycosylation

of a Soluble Hydroxyproline-Rich

Glycoprotein

Cell suspension cultures of plants secrete a mixture of soluble polysaccharides, proteins, and glycoproteins into their growth medium. At least one of these components is a hydroxyproline-rich glycoprotein in which

296

MORT AND LAMPORT TABLE SUGAR

COMPOSITION

OF CELL

WALL HF

Ara/Hyp Gal/Ser Galacturonic/Ser RhalSer Glc/Ser

4 GLYCOPEPTIDES

BEFORE

AND

AFTER

SOLVOLYS~S”

Before

After

5.04 4.2 7.0 1.7 0.84

0.14 0.09 0.04 0.02 0.05

sugar remaining (%I 3 2 1.3 1.2 6

o HF solvolysis at 0°C for 1 hr. Data expressed as molar ratios. Sugars were determined as their TMS-O-methyl glycosides, and amino acids as their N-heptafluorobutyryl isobutyl esters.

arabinogalactan side chains glycosylate > 50% of the hydroxyproline residues through an U-galactosyl hydroxyproline linkage (21). Such a highly glycosylated glycoprotein must be deglycosylated before the amino acid sequence can be determined. We partially purified the crude hydroxyproline-rich glycoprotein of sycamore-maple by ultrafiltration of the culture medium through an Amicon XM-1OOA membrane, followed by desalting on Sephadex G-25 and freeze-drying. (The bulk of this crude material would appear in the void volume of a Sephadex G-100 column.) However, after treatment with anhydrous HF (1 hr at O’C) and extraction with 0.1 M acetic acid, soluble hydroxyproline-rich macromolecular material was considerably retarded on a Sephadex G-100 column (Fig. 1) and was largely deglycosylated. For example, before HF treatment, there were ca. 120 sugar residues for each hydroxyproline residue, while, after HF treatment, there were only ca. 3.6 sugar residues for each hydroxyproline residue. A comparison of amino acid analyses before and after deglycosylation (Table 5) shows a twofold enrichment of hydroxyproline compared with other amino acids, indicating a considerable purification of the hydroxyproline-rich polypeptide. HF Deglycosylation

of Other Glycoproteins

Having verified that liquid HF cleaves 0-glycosidic linkages of neutral and acidic sugars we turned our attention to N- and 0-glycosidic linkages of amino sugars. These linkages also occur frequently as glycopeptide linkages and, therefore, are especially important. (a ) Fetuin (N-and-0-glycosidic linkages). Spiro and co-workers (22) showed that the fetuin molecule contains six oligosaccharides; three Nglycosidically linked (type I) and three 0-glycosidically linked (type II).

297

HFDEGLYCOSYLATESGLYCOPROTEINS

I.

ManI GlcNAc I Gal

I

NANA II.

ManI GlcNAc I Gal

I

NANA

Man-

GlcNAc -

GlcNAc -

Asn

I GlcNAc I Gal

I

NANA

-Ser NANA-Gal-GalNAc I (NANA)M

(or Thr)

Treatment of fetuin with 10 ml of HF for 1 hr at 0°C with 1 ml of anisole as scavenger removed most of the attached neutral sugar residues (Table 6), but substantial amounts of the N-acetylated hexosamines remained. This indicates that the links between GlcNAc and GlcNAc, GlcNAc and Asn, and GalNAc and Ser are stable in HF at 0°C. However, harsher conditions (HF for 3 hr at 23°C) removed all the N-acetylgalactosamine and left only two to three residues of N-acetylglucosamine per mole of fetuin (Table 7). We conclude that HF at 23°C cleaves the GlcNAc-GlcNAc (cf. effect of HF on chitin) and GalNAc-Ser linkages, but not the GlcNAc-Asn linkage. Amino acid analyses of fetuin were similar both before and after treatment with HF at 0°C followed by dialysis (Table 8). Additional analysis by gel electrophoresis on polyacrylamide gels showed that HF-treated fetuin decreased in molecular weight but remained

HYP

FRACTION

FIG. 1. Gel filtration

of a hydroxyproline-rich

NUMBER

glycoprotein

(9.5 ml)

after

HF

solvolysis:

Sephadex

We treated 800 mg of crude glycoprotein with 40 ml of HF for 1 hr at 0°C as described in Materials and Methods, dissolved the residue in 20 ml of 0.1 M acetic acid, and fractionated by two separate applications on a 2.5 x 85-cm Sephadex G-100 column equilibrated with 0.1 M acetic acid. We assayed 500~~1 aliquots of each fraction for hydroxyproline. The major peak contained 827 pg of hydroxyproline which accounted for 51% of the hydroxyproline (1624 pg) in the crude glycoprotein before HF solvolysis.

G-100

elution

profile.

298

MORT AND LAMPORT TABLE

5

HF DEGLYCOSYLATION OF A SOLUBLE HYDROXYPROLINE-RICH GLYCOPROTEIN SECRETED BY SYCAMORE MAPLE CULTURE@ Before HF

After HF and G-100

Galacturonic

1 2 1.2 16.8 6.7 0 13.2 57.1 19.4

I 0.2 0.1 1.4 0.2 0 1.0 0.7 0

HYP Asp Thr Ser Glu Pro GUY Ala Val cyst Met Be Leu ‘br Phe LYS His Arg

6.7 4.9 8.2 9.6 9.8 n.d. 10.4 9.3 7.6 0 1.1 4.9 7.8 1.1 5.1 7.1 3.3 3.1

14.2 11.3 9.0 12.3 7.5 n.d. 7.1 12.3 5.2 0 0 3.3 5.2 0.5 3.3 4.7 2.4 1.9

HYP Rha Fuc Ara XYl Man Gal GlC

a Data expressed as moles of sugar per mole of hydroxyproline, residues as mole% of total amino acids.

and amino acid

essentially as a single band (Fig. 2) which was in fact sharper than the band corresponding to untreated fetuin, indicating perhaps decreased heterogeneity. Thus, HF did not appreciably degrade or alkylate the fetuin polypeptide. (6) Pig submaxillary mucin (0-glycosidic linkages). Pig submaxillary mucin (PSM) consists of about 60% carbohydrate and 40% protein. The carbohydrate is present as many short oligosaccharides 0-glycosidically attached to at least 85% of the threonine and serine residues (25). Carlson (26) characterized a series of PSM oligosaccharides the largest of the series being:

HF

DEGLYCOSYLATES

GalNAc-

299

GLYCOPROTEINS

(or Thr). GalNAc -Ser I N-glycolylneuraminic

GalI Fuc

After treatment of PSM with HF (anisole scavenger) for 1 hr at o”C, followed by dialysis of the aqueous extract, only N-acetylgalactosamine remained as a major sugar component attached to the peptide (Table 9). From the work of Carlson (26) we know that the presence (or absence) of terminal N-acetylgalactosamine in the PSM oligosaccharides determines the presence (or absence) of blood group A type activity. Thus, PSM pooled from a number of glands shows a mixture of blood group types, and one can infer from the data of Payza et al. (25) that about half the PSM from pooled glands contains two GalNAc residues/oligosaccharide. while half contains only one GalNAc residue/oligosaccharide, i.e., pooled PSM averages ca. 1.5 GalNAc residues/oligosaccharide. HF treatment (1 hr at O’C) of pooled PSM actually resulted in the loss of ca. 33% of N-acetylgalactosamine (Table 9), which is consistent with the data above and indicates that, in PSM as in fetuin, HF solvolysis at 0°C cleaves only neutral sugar linkages, leaving intact the GalNAc-Ser and GalNAcThr links. But more rigorous HF treatment of PSM for 3 hr at 23°C (anisole scavenger) almost completely stripped the protein of its sugars, only 6% of the original N-acetylgalactosamine remaining (Table 9). We conclude that these more rigorous conditions of HF solvolysis cleave the GalNAc-Ser and GalNAc-Thr linkages in PSM as in fetuin. The amino acid composition of PSM did not change appreciably after HF treatment for 3 hr at 23”C, followed by dialysis of the aqueous solution (Table 10). TABLE SUGARCOMPOSITION Before (mg of sugar/ 100 mg of fetuin peptide) NANA GlcNAc GalNAc Man Gal

14.4 7.4 1.34 3.82 4.58

D HF solvolysis * As determined ’ Calculated as: residues per mole

OFFETUIN

6

BEFOREANDAFTER

HF solvolysis

HF SOLVOLYSW After

(Sugar residues/mole of fetuin)* 13 I5 3 9 12

for 1 hr at 0°C. by Spiro and Bhoyroo (23). percentage of the remaining of fetuin.

(mg of sugar/ 100 mg of fetuin peptide) 1.25 3.0 1.26 0 0.54

sugar

HF solvolysis (Sugar residues/mole of fetuin)’ 1.7 6.0 2.8 0 1.4

multiplied

by its initial

Sugar remaining (c/o) 8.7 41 94 0 12

number

of

300

MORT AND LAMPORT TABLE SLJGARCOMPOSITIONOFFETUIN

7

BEFOREANDAFTERHFSOLVOLYSW

Before HF Solvolysis (mg of sugar/ 100 mg of fetuin peptide) NANA GlcN AC GalNAc Man Gal

14.4 7.4 1.34 3.82 4.58

(Sugar residues/mole of fetuin)

After HF Solvolysis (mg of sugar/ 100 mg of fetuin peptide)

(Sugar residues/mole of fetuin)

Sugar remaining (%o)

tr. I.2 tr. tr. 0.34

2.4 0.89

16 7.4

I3 I5 3 9 I2

a HF solvolysis for 3 hr at 23°C. Calculations as for Table 6.

HF Treatment

of N-Acetylglucosamine

As most hexosaminidases require the amino group of their substrate to be acetylated we thought it important to prove that HF does not deacetylate amino sugars. Thus, treatment of free N-acetylglucosamine with HF at 0°C for 1 hr followed by analysis as the TMS derivative via combined gas chromatography-mass spectrometry, showed only TMS-Nacetylglucosamine. HF Solvolysis

of Chitin

HF at 0°C for 1 hr partially solubilized crab shells a complex of chitin, calcium carbonate, protein, and pigments. The solubilized material contains polymeric N-acetylglucosamine and protein which voids a Bio-Gel P-2 column. The more rigorous HF treatment of the crab shells for 3 hr at 23°C (in the absence of scavenger) solubilized nearly all the Nacetylglucosamine and left a water-insoluble residue considerably enriched in amino acids. The water-soluble material chromatographed on a Bio-Gel P-2 column gave three fractions: a small void peak containing protein plus a little N-acetylglucosamine, another small peak containing N-acetylglucosamine together with an unidentified compound, and a large peak in the included volume containing only monomeric N-acetylglucosamine which, after direct derivatization (see Materials and Methods), cochromatographed as TMS-N-acetylglucosamine. HF Treatment

of Bovine Serum Albumin

Bovine serum albumin, a nonglycosylated protein, was treated with HF plus anisole for 1 hr at 0°C and then was subjected to SDS gel electro-

HF DEGLYCOSYLATES TABLE AMINOACIDCOMPOSITIONOF

FETUIN

301

GLYCOPROTEINS 8 BEFOREANDAFTER

HF SOLVOLYSW

Amino acid residuesb

Ala GlY Val’ Thr Ser Leu Ile Pro HYP Met Asp Phe Glu LYS Tyr A& His CYS

Before HF

After HF

60 44 78 37 44 50 23 65 0 0 61 21 69 31 13 25 15 0

62 44 68 36 47 44 22 61 0 0 60 21 69 31 14 25 18 0

a HF solvolysis for 1 hr at 0°C. b Normalized to 69 Glu residues for comparison with the published analysis (24) and to offset the inflated valine value. c Glucosamine elutes with valine on our amino acid analyzer and, therefore, gives a high value for valine in the untreated fetuin.

phoresis (Fig. 3). New bands did not appear in the gels, an indication that HF did not degrade the protein. DISCUSSION

Anhydrous hydrogen fluoride is an interesting reagent often used in synthetic peptide chemistry because of its selective cleavage proporties (Table ll), although not previously reported as useful for the specific deglycosylation of glycoproteins. Our experiments show that anhydrous HF cleaves the glycosidic linkages of neutral and acidic sugars within 1 hr at 0°C and the 0-glycosidic linkages of amino sugars within 3 hr at 23”C, but leaves intact the peptide bonds and N-glycosidic linkages of amino sugars. Aqueous hydrofluoric acid also has selective cleavage properties although rather different from those of anhydrous HF. For example, 60% aqueous HF at 0°C specifically cleaves the phosphodiesters of teichoic

302

MORT AND LAMPORT

FIG. 2. SDS-Gel electrophoresis offetuin before and after HF treatment: (A) untreated fetuin; (B) fetuin after treatment with HF for 1 hr at 0°C; (C) A + B run together.

acids from Bacill~~s subtilis, yielding mainly glucosyl glycerol (27), while 40% HF results in the quantitative hydrolysis of sphingomyelin to ceramide (28) after 72 hr at 40°C. Table 11 shows that, depending on reaction conditions, HF shows a surprisingly wide range of specificities for cleaving various linkages in biologically important molecules. One should note, however, that, under some conditions, complete cleavage of glycosidic linkages, especially in the absence of scavenger, may be followed by oligomer formation. For example, HF solvolysis of cellulose gave glucosyl fluoride quantitatively which, however, on slow evaporation yielded short chain glucans (13). Complications may also arise from the action of HF as a very strong hydrogen bonding agent which may denature the protein causing it to be less soluble in aqueous

HF DEGLYCOSYLATES TABLE SUGAR

COMPOSITION

SUBMAXILLARY

9 MUCIN

BEFORE

AND

HF

AFTER

Before (mg of sugar/ 100 m g of PSM polypeptide)

After 1 hr at 0°C tmg of sugar/ 100 m g of PSM polypeptrde)

Sugar remaining after I hr at 0°C (%I

After 3 hr at 23°C (mg of sugar/ IO0 m g of PSM polypeptide)

Sl 32 27 42

54 2.4 0 0

67 7 0 0

5 trace II 0

Gal NAc Gal FUC

NAGA D Calculation

OF PIG

303

GLYCOPROTEINS

SOLVOLYSIS”

Sugar remaining after 3 hr at 23°C (5%) 6 0 0 0

as for Table 6.

solvents or even heterogeneous in size as reported for lysozyme (33). This heterogeneity is not due to peptide bond cleavage; the work of Koch et al. (34), Lenard and Hess (39, and others shows that peptide bonds are stable in anhydrous HF at 0°C and, with the exception of slow cleavage of methionyl peptide bonds (36) and the easily reversed N to 0 acyl shift of serine and threonine (39, are stable at room temperature for many hours. Our own results showing no increase in the heterogeneity of fetuin after HF treatment (Fig. 2) are in accord with the above conclusions. An additional possibility of peptide bond cleavage arising under TABLE AMINO

ACID

COMPOSITION

OF

10

PSM

BEFORE

AND

Amino

acid residues

Before Ala GUY Val Thr Ser Leu Ile Pro Met ASP Phe GIU Lys Tyr Arg His CYS R HF solvolysis

13 18 7 11 18 3 3 6 0 4 2 8 3 1 3 1 0 for 3 hr at 23°C.

HF

AFTER

HF

SOLVOLYSIS”

(mole%) After

HF 13 18 7 I1 18 3 3 6 0 4 2 6 3 1 3 I 0

304

MORT AND LAMPORT

FIG. 3. SDS-Gel electrophoresis of bovine serum albumin before and after HF treatment: (A) untreated BSA; (B) BSA after treatment with HF for 1 hr at 0°C; (C) A + B run together.

aqueous conditions due to incomplete removal of HF also seems unlikely considering that aqueous HF is only a very weak acid (Kdiss = 7 x 10e4). Anhydrous HF is a highly versatile reagent.6 We have just begun to exploit its usefulness in glycoprotein chemistry. Therefore, we list the following possibilities as worthy of further study: Complete Sugar Analysis of Polysaccharides

This should be especially useful for those polysaccharides difficult to hydrolyze by conventional methods.

which are

6 The recent demonstration that HF-pyridine can be used as a safer form of anhydrous HF for the deprotection of peptides (40) suggests a possible extension to deglycosylation, at least for some sugars.

305

HFDEGLYCOSYLATESGLYCOPROTEINS TABLE HF

CLEAVAGE

OF VARIOUS

LINKAGES SEVERE

II LISTED

IN ORDER

OF INCREASINGLY

CONDITIONS” Conditions:

60%

Example

Type of linkage Pyrophosphates

5-Iodo-UTP

Phosphate esters and diesters

5-Iodo-UMP

aqueous

HF

(hr)

---f 5-lodo-UMP

(29)

-50

I

+

(29)

0

(a

0

3-5

Phosphatidyl diglucosyl diglyceride -+ diglyceride + diglucosyl diglyceride (30)

0

24

Acyl carrier protein -O-P-O-pantetheine (31)

2

24

OH OH

5-Iodouridine

Glucosylpolyglycerolphosphate teichoic acid) + Glucosyl glycerol (27)

Conditions: Anhydrous HF Type of linkage Neutral sugar glycosides

Example Arabinogalactan,

cellulose (13)

CT)

(hr)

0

1

0

1

Man-O-GlcNAc Hyp-O-Ara Acidic sugar glycosides

Ser-O-Gal Cell wall pectin (rhamnogalacturonan)

MORT AND LAMPORT

306

TABLE

11 (Continued) Conditions: Anhydrous HF

Type of linkage

Example

N-Glycosides to purine bases

(“C)

3’5-Cyclic AMP (32)

Room temperature

Chitin . (GlcNAcGlcNAc. .)

Room temperature

(hr)

Short time

ADENINE

0=7----O

OH

dH

Amino sugar O-glycosides CH,OH

Serine or threonine-O-N-acetylgalactosamine Amino sugar N-glycosides

Asparaginyl N-acetylglucosamine

Peptide

Methionyl glycine (8)

Cleaved after 36 hr at 23°C

H

s

OH

NH/ctc 2

Stable for at least 3 hr at 23°C

-

1

,A,/

H

8

u Conditions for cleavage of many of the protecting groups used in chemical peptide synthesis are listed in Ref. (10).

Identification

of Glycopeptide Linkages

(a) 0-Glycosidic linkages such as GalNAc-Ser. For example, after HF treatment of pig submaxillary mucin at 072, only N-acetylgalactosamine

307

HFDEGLYCOSYLATESGLYCOPROTEINS

remained peptide bound. The amino acid involved in the glycopeptide linkage could have been identified by p-elimination in the presence of borohydride. (b) N-Glycosidic linkages: GfcNAc -Asn . Because treatment of glycoproteins with HF for 3 hr at 23°C cleaves all except N-glycosidic linkages, HF can be used to confirm the presence of the GlcNAc-Asn linkage. Structural

Determination

of Glycoprotein

Oligosaccharides

As a method has already been described to cleave oligosaccharides at amino sugars by deamination (37), it should be possible to assign complete sequences to oligosaccharides by a combination of the two techniques, the HF providing the overlapping sequences to piece together the longer ones obtained by the deaminative cleavage. Facile Generation

of Substrates for Glycoprotein

Glycosyl Transferases

After removal of all neutral sugars from a glycoprotein the product should be similar to the natural substrate for the appropriate glycosyl transferase. This should enable one to distinguish between biosynthetic pathways which involve either stepwise addition of sugars or block transfer of preformed oligosaccharides. The Role of Sugars in Glycoproteins The biological activity of some glycoproteins (38) is absolutely dependent on the sugar components which often seem to act as a recognition code for “topographical location” (39) of the glycoprotein. In other glycoproteins the role of sugar components is less clear, perhaps because it is more subtle. For example, after HF treatment for 1 hr at 0°C ovomucoid largely retained its activity as a trypsin inhibitor (unpublished experiments). This is consistent with the observation (10,l 1,12) that many nonglycosylated proteins such as lysozyme and RNAase retain their biological activity after HF treatment at o”C, although not after treatment at 30°C. Solubilization

of Proteoglycan

Networks

This was our original interest because we expected anhydrous HF to render soluble the protein of plant cell walls. However, while HF solubilized the bulk of the cell wall (ca. 90%), the residue which contained the protein was quite insoluble in various protein solvents such as SDS and urea, suggesting the presence of as yet unidentified cross-links between the protein. In the absence of protein-protein cross-links we predict that anhydrous HF will solubilize the cell wall proteins of yeasts, bacteria, etc.

308

MORT AND LAMPORT

As a Simple Method of Purifying the Protein Component of Some Glycoproteins

Microheterogeneity due to varying degrees of glycosylation contributes to the difficulties inherent in the purification of highly glycosylated glycoproteins, making it especially difficult to establish the presence of one unique peptide species. HF deglycosylation removes the microheterogeneity and reduces the molecular weight of the (glyco)protein. Thus, fractionation of a glycoprotein by gel filtration or ultrafiltration before HF treatment and then again after HF treatment may yield a virtually pure apoprotein, provided of course that the apoprotein is soluble. In our limited experience with the proteins mentioned here there is a distinct tendency to become less soluble after deglycosylation, indicating a rather prosaic role for the sugars of some glycoproteins! To Assist Amino Acid Sequencing of Glycoproteins

The oligosaccharide units of highly glycosylated glycoproteins present two obstacles to sequence determination: first, steric hindrance to proteolytic attack and, second, the absence of an identifiable product after Edman degradation of a glycosylated amino acid residue. Except for the GlcNAc-Asn linkage, anhydrous HF removes these obstacles. It should now be possible to sequence previously intractable proteins such as the soluble hydroxyproline-rich glycoprotein which we described briefly in the Results section. These possible uses for anhydrous HF underline the view that, as the role of glycoproteins in eukaryotic organization and morphogenesis bestructural determination becomes incomes increasingly evident, creasingly desirable. HF deglycosylation is a new technique which shows promise of being useful to those who seek to relate structure and function. ACKNOWLEDGMENTS We thank Mr. Mike Caughey for expert technical assistance, especially for amino acid analyses; Mr. Jack Harten for assistance with the gas chromatography/mass spectrometry; Dr. Joyce Clarke for help with the sycamore-maple extracellular glycoprotein; Dr. Shirley Rodaway for help with the SDS gels; and Ms. Joann Lamport for much help with the manuscript. The work was supported by the U.S. Energy Research and Development Administration through Contact E(ll-l)-1338 and the National Science Foundation through Grant PCM76-02549.

REFERENCES 1. Mort, A. J., and Lamport, D. T. A. (1976) Pfanr Physiol. 57, Annual Meeting Supplement Abstract 297, p. 57. 2. Vegarud, G., and Christensen, T. (1975) AC&Z. Chem. Scam’. Ser B B29,887-888.

HF DEGLYCOSYLATES 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

GLYCOPROTEINS

309

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