307

Biochimica et Biophysica Acta, 582 (1979) 3 0 7 - - 3 2 1 © E l sev ie r /Nor th -Hol l and Biomedica l Press

BBA 2 8 7 8 6

THE MOLECULAR SIZE OF GLYCANS LIBERATED BY HYDRAZINOLYSIS FROM SEMLIKI FOREST VIRUS PROTEINS

M A I J A - L I I S A R A S I L O and OSSI R E N K O N E N

Department of Biochemistry, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29 (Finland)

(Received May 18 th , 1978)

Key words: Glycopeptide; Glycoprotein; Hydrazinolysis; Glycan release; (Semliki Forest virus)

Summary

The glycans of well characterized, [6-3H ]galactose-labelled glycopeptides, GC-4 from bovine IgG1 as well as GP-V-2 and GP-V-5 from ~l-acid glycopro- tein, were liberated by hydrazinolysis. Molecular weights close to the expected values were observed by gel filtration. Desialated glycans of Semliki Forest virus proteins were likewise liberated by hydrazinolysis and subjected to gel filtration. Metabolically labelled [ 1-3H]gaiactose-oligosaccharides of the mixed viral proteins revealed an apparent molecular weight of 1800. The bi-antennary glycan liberated from the reference glycopeptide GC-4 was of 1750 daltons. A mixture of [2-3H]mannose-labelled El- and E:-proteins of the virus contained L-type glycans of 1800 daltons (formerly called A-type), and M-type glycans of 1200 daltons (formerly called B-type). A fraction of the E3-glycans isolated by affinity chromatography on Concanavalin A-Sepharose showed an average mo- lecular weight of 2150, a value intermediate between the three- and four-anten- nary glycans liberated from the reference glycopeptides GP-V-5 and GP-V-2. The rest of the E3-glycans were of 1850 daltons, a value close to the bi-anten- nary GC-4 glycan. We suggest that the comparatively large size of the E3-glycans and the exposed position of E3-proteins on the viral surface may be interrelated.

Introduction

Our group is studying the membrane of Semliki Forest virus, an alpha virus of the Togavirus family, as a simple model for cellular plasma membranes [1].

Abbrev iat ions : SDS, sodium dodecyl sulfate; Gal, Galactose; Glc, glucose; GIcNAc, N-acetyl glucosamine; Man, mannose; Fuc, fucose; V0, void volume of gel filtration columns; Ve, elution volume; Vtot, to ta l volume of gel filtration columns.

308

The viral membrane consists of lipids [ 2] and of an equimolar mixture of three glycoproteins El, E2 and E3 [3]. The viral proteins are believed to carry aspara- gine-linked glycans of the N-acetyl lactosamine type (L-type, formerly called A-type) [3,4]. E2 appears to contain additionally glycans of oligomannosyl type (M-type, formerly called B-type) [4].

The size of the viral glycans has been estimated previously by gel filtration of glycopeptides obtained by pronase digestion [4]. However, the peptide moieties of the resulting glycopeptides may not be as small, nor as homogene- ous as would be required for a reliable analysis of the oligosaccharides. More- over, glycopeptides and glycans appear to have different elution rates in gel filtration [5]. Therefore, it is better to use hydrazinolysis which appears to liberate asparagine-linked oligosaccharides in an amino acid-free form [6--10].

In the present report we describe hydrazinolysis experiments with reference glycoproteins and glycopeptides of well-known structure, and having shown that the reaction works properly we describe its application to desialated viral glycoproteins. Finally we present gel filtration experiments for the estimation of the molecular weight of the liberated Semliki Forest virus glycans.

Materials and Methods

Reference glycoproteins and glycopeptides The chymotryptic immunoglobulin IgG1 glycopeptides GC-4 and GC-2 from

bovine colostrum [11] were gifts from Drs. J. Montreuil and A. Cheron. The orosomucoid glycopeptides GP-V-5 and GP-V-2 were gifts from Dr. K. Schmid; they were obtained from the chymotryptic glycopeptide V after pronase diges- tion and chromatography on Dowex-50 W X2 [12--14]. Human serum transfer- rin was purchased from Kabi, Stockholm, and ovalbumin from Sigma, St. Louis.

The glycopeptides GC-4, GP-V-5 and GP-V-2 were labelled with tritium in their galactose residues. The procedure used was essentially that described by Gahmberg [15]. The glycopeptides (100--300 pg) in 0.5 ml of Dulbecco's phosphate buffer, pH 7.4, were incubated with 10 U of galactose oxidase (EC 1.1.3.9) (Kabi, Stockholm) for 30 min at 37°C. The oxidized glycopeptides were reduced by adding NaB3H4 (0.2--0.7 mCi, 7.6 Ci/mmol, Amersham) in 10--30 pl of 0.01 N NaOH. After purification by gel filtration the tritium- labelled glycopeptides were stored at --70°C in the elution buffer, 0.15 M Tris/ acetate, pH 7.8, containing 0.1% sodium dodecyl sulfate (SDS).

Labelling of the viral glycans The protein-bound glycans of Semliki Forest virus were labelled with chemi-

cal and with metabolic methods. For the chemical labelling the desialated virus proteins were treated with galactose oxidase and NaB3H4 as described by Luuk- konen et al. [16]. The metabolic labelling of the viral glycans was carried out by growing the virus in BHK cells in the presence of radioactive monosaccha- rides as described previously [4,17]. Viral proteins labelled either with [1-3H]- galactose and [1-14C]mannose [4] or with [2-3H]mannose were used. In the former preparation the 14C label was so small that it was ignored.

309

Gel electrophoresis o f the viral proteins The virus was disrupted with SDS in the presence of fl-mercaptoethanol and

the dissociated proteins were separated on polyacrylamide gel electrophoresis in the presence of SDS; either the system of Neville [18] or of Laemmli [19] was used. The proteins were extracted from the sliced gels and digested with pronase (Calbiochem, B grade) as described by Mattila et al. [4].

Fractionation o f the viral glycopeptides by affinity chromatography Fractionation of the viral glycopeptides on Concanavalin A-Sepharose

columns into two fractions of L-type glycopeptides (fraction I and II) and the M-type glycopeptides (fraction III) [20,21] was carried out as described by Mattila, K. and Renkonen, O. (unpublished data).

Hydrazinolysis Hydrazinolysis of glycoproteins and glycopeptides was carried out with the

method used by Montreuil 's group [7,8]. Tracer quantities of labelled mate- rials, and 1--30 mg samples of unlabelled compounds were used. The samples were dried at 0.1 mmHg over P2Os for 48 h and hydrazinolyzed at 105°C for 48 h in 0.2--0.5 ml of dry hydrazine (Pierce, Rockford, Ill.) in Teflon-capped tubes under nitrogen. At the end of the reaction hydrazine was removed by evaporation, either with toluene in a nitrogen flow or in a vacuum desiccator over H2SO4. The liberated glycans were either chromatographed directly or re-N-acetylated by treating the reaction mixture with acetic anhydride under conditions similar to those described by Carlson et al. [22].

Gel filtration All glycopeptides were gel filtrated and their molecular weights were deter-

mined on a Bio-Gel P-6 column in the presence of SDS exactly as described pre- viously [4,17].

The liberated glycans were gel filtrated on a column of Bio-Gel P-6 (Bio-Rad Laboratories, Richmond, Calif.} (200--400 mesh, 1 × 140 cm)equ i l i b r a t ed with 0.2 M pyridine acetate, pH 5.0, containing 0.02% NaNs. The molecular weights of the glycans were estimated by calibrating the column with Blue Dex- tran 2000 (Pharmacia, Uppsala), lacto-N-difucohexaose I (Mr = 1003 in reduced, tritiated form) [23] and with octasaccharide IXD, Gal/31-~ 4 GlcNAc~l -+ 2 Man~l -* 6 [Galfll -~ 4 GlcNAc/~l -~ 2 Man~l -~ 3] Man~l -~ 4 GlcNAc (M r = 1441 in reduced, tritiated form) [24]. The former was a gift from Dr. A. Gauhe, the latter from Drs. J. Montreuil and G. Strecker. All pure oligosaccharides were actually used after NaBSH4 reduction, which was carried out by a procedure similar to that described by Takasaki and Kobata [25]. Lacto-N-difucohexaose I and its reduction product had identical elution rates.

In all gel filtration experiments the columns were eluted beyond the Vtot, which was determined with the aid of various small molecules.

Chemical analyses Hexoses were assayed by a phenol sulphuric acid method [26]. For amino

acid analysis, glycans were hydrolyzed with 6 N HCI for 23 h at l l 0 ° C in sealed glass ampoules in vacuum, and the hydrolysates were analyzed with a Beckman Multichrom analyzer [27].

310

Miscellaneous procedures The digestions with pronase (Calbiochem, B grade) were carried out under

the conditions described by Sefton and Keegstra [28] using three incubations of 24 h at 60°C.

The fucosidase digestion of GC-4-peptide was carried out in 150 pl of sodium citrate buffer pH 4.0 under toluene with a-L-fucosidase (EC 3.2.1.51) (0.4 U/ml) from beef kidney (Boehringer) for 14 h at 37°C. The treatment was repeated 3 times with fresh enzyme.

The galactosidase digestion of [6-3H]galactose-GC-4 peptide was carried out under the conditions previously described [ 17].

Glycopeptides were desialated with acid according to Warren [29]. Radioactivity was determined in a Wallac 8100 scintillation counter using a

Xylene-Triton X-114 cocktail (8 : 3, v/v). Quenching was occasionally checked with an external standard.

Results

Tritium labelling of reference glycopeptides Three reference glycopeptides were labelled with tritium by oxidizing the

galactose residues at non-reducing termini with galactose oxidase, and by reduc- ing the resulting aldehydes with tritiated sodium borohydride [15]. The glyco- peptides labelled in this manner were GC-4, a chymotrypt ic glycopeptide from bovine colostrum IgG [11], as well as GP-V-5 and GP-V-2, pronase peptides of the chymotrypt ic peptide GP-V from a 1-acid glycoprotein [12]. The structures of these compounds are ~hown in Fig. 1 [11--14]. The specific reaction prod- ucts were obtained by gel filtration of the crude reaction mixtures. They appeared to be [6-3H ]galactose-labelled glycopeptides; 81% of the tritium pres- ent in the labelled GC-4 peptide was released as free monosaccharide after digestion with pure fl-D-galactosidase (EC 3.2.1.23) from Jack bean.

The apparent molecular weights of the purified [6-3H]galactose-labelled GC-4, GP-V-5 and GP-V-2 were 3900, 2900 and 3600, respectively. These values are all significantly larger than the formula weights of the peptides which are 3238, 2277 and 2778 (see Fig. 1). This systematic error is probably caused by the use of oligosaccharides instead of glycopeptides in the calibration of the gel filtration columns [4,5].

Liberation of glycans by hydrazinolysis of reference glycopep tides Hydrazinolysis liberated glycans in an amino acid-free form from glycopep-

tides. Amino acid analysis (Fig. 2) revealed that neither asparagine nor the other amino acids were present in the liberated glycan isolated from a hydra- zinolysate of GC-2 [11], a glycopeptide similar to GC-4 but missing the amino acids 1--8. The liberation of amino acid-free glycans was confirmed also by the finding that re-N-acetylated hydrazinolysate of [6-3H]galactose-GP-V-5 was a neutral compound passing in water through a mixed bed of Dowex-1 (HCOO-) and Dowex-50 X8 (H*); before re-N-acetylation only 6% of the label was eluted.

The re-N-acetylated glycan from [6-3H]galactose-GP-V-2 revealed in gel fil-

Gal Gal

]B-1 ,4 `}5-1,4 GIcNAc GIcNAc

`}~-1,2 ,1~-1,2 Man Xan c~-1 ,6"~ ~ ' ~ - 1 , 3

Man ,~5-1,4 GIcNAc

}B-1,4 ~-1,6

GIcNAc .

`}B-1 Thr- Lys-Pro-Arg-Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr

Fuc

GC-4, M : 3238

Gal Gal Gal Gal Gal

GIcNAc GIcNAc GIcNAc GIcNAc GIcNAc

1 ,<5 \~ .}6-1,2 ~5- -1,4 5-1,6-. `}6-1,2 Man Man ~ Man

Man Man .j6-1,4 `}5-I ,4 GIcNAc GIcNAc

~5-1 ,~ `}5-1 ,~ GIcNAc GIcNAc

~-~ `}B-~ Asn-Gly-ThP Asn-Gly-Thr

Gal Gal Fuc

GIcNAc GlcNAc

`}5-1, ~ / 4 - 1 , 4 Man ~ "

,3

Gp-V-5, M : 2277 P

I Gp-V-2, M r : 2778

Fig. 1. Structures of the reference g lycopept ides GC-4 [ 1 1 ] , GP-V-5 and GP-V-2 [ 1 2 - - 1 4 ] .

311

tration a well defined oligosaccharide peak of 2450 daltons, V e / V o = 1.340 (Fig. 3A). As shown in Table I this value agrees well with the formula weight (2506) calculated from the structure proposed for GP-V-2 on the basis of NMR-findings and methylation analysis [13,14] . Both these values are reason- ably close to the molecular weight (2680) calculated by Schmid et al. [12] on the basis of compositional data. The liberated glycan of GP-V-2 was obtained

312

z o (n ua oK:

i.-

^

Asp S e r Glu G l y A l e T h r

n L e u 2 5 0 n M

i:

J J

k. .... j j! t

G I c N NH 3

T I M E

Fig. 2. A m i n o acid analysis of the g lycan l ibe ra ted f r o m GC-2 g lycopep t ide by hydraz ino lys i s . Nor leucine and the e n d o g e n o u s g lucosamine serve as in te rna l s tandards .

with a yield of 85% as estimated from the distribution of radioactivity in Fig. 3A.

The re-N-acetylated glycan from [6-3H]galactose-GP-V-5 revealed also most

8 0 0

4 0 0

A G p - V - 2 GLYCAN o,

l\

o~ oo o°°ooo o o

6 0 0

3 0 0 Vo ,I

Gp-V- 5 GLYCAN

o ;

B

~o.~_^ : ~ _ o - o - ~ ° ° o - o . o o _ ^ " -~ : -': --

GC-4 GLYCA N

3 0 0 °'°~ o / \ 2 0 0 o o

,oo / k . °°"

"°~-o'°" ° ' ° ' ° o o o

30 50 70 FRACTION NUMBER

Fig. 3. Gel f i l txation on a Bin-Gel P-6 c o l u m n (1 X 140 c m ) of re -N-ace ty la ted g lycans l ibera ted f r o m the t r i t i um label led r e f e r ence g lycopep t ides by hydraz ino lys i s . E lu t ion b u f f e r 0 .2 M pyr id ine ace ta te , p H 5.0, con ta in ing 0 .02% NAN3; f r ac t ion size 1.4 ml . The a r rows show pos i t ions of pu re r e fe rence glycans, Blue D e x t r a n (V0) , oc tao l IX D (O) a nd l ac to -N-d i fucohexao l I (H) . A, [ 3 H ] G P - V - 2 g lycan; B, [ 3 H ] G P - V - 5 g lycan; C, [ 3 H ] G C - 4 g lycan.

T A B L E I

M O L E C U L A R W E I G H T S OF G L Y C A N S L I B E R A T E D BY" H Y D R A Z I N O L Y S I S

313

Parent glycopeptide Observed M r o f g l y c a n a Calculated M r o f glycan

Chemical NMR

analysis analysis b

R e f e r e n c e g l y c o p e p t i d e s [6 .3 H]ga lac tose -GC-4 1750 ± 50 [6 .3 H] ga lac tose-afuco-GC-4 1600 [6 -3H]ga lac tose -GP-V-5 2000 + 20 [6 -3H]ga lac tose -GP-V-2 2450 ± 50 Serotrans ferr in 2000 e O v a l b u m i n 1500 e

Viral g l y c o p e p t i d e s M i x e d [ 1-3 H] g a l a c t o s e - g l y c o p e p t i d e s 1 8 0 0 [ 2 -3 H] m a n n o s e - E 3 I -g lycopep t ides 2 1 5 0 [ 2 -3 H] m a n n o s e - E 3 I I -g lycopep t ides 1 8 5 0 [ 2 -3 HI m a n n o s e - E 1 -E2 I I - g l y c o p e p t i d e s 1 8 0 0 [ 2 .3 H ] m a n n o s e - E 1 "E2 I I I -g lycopep t ides 1 2 0 0 [ 6-3 H] galactose-E3 I -g lycopep t ides 1900 [ 6-3 H ] galactose-E3 I I -g lycopep t ides 1 7 5 0 [ 6-3 H ] ga lac tose -E2-g lycopep t ides 1700 [6-3 H ] galactose-E l -g lycopep t ides 1600

(n= 2) 1756 c (n = 1) 1639 c (n = 2) 1641 d 2 0 0 5 b (n = 3) 2680 d 2506 b

1 6 4 0 - 2 0 2 3 f 1 0 7 2 - 2 0 0 5 g

a T h e m o l e c u l a r w e i g h t s w e r e m e a s u r e d o n a B i o -G e l Po6 c o l u m n w h i c h w a s ca l ibrated w i t h t w o p u r e r e f e r e n c e glycans , [ 3 H ] l a c t o - N - d i f u c o h e x a o l I [ 2 3 ] , V e / V 0 = 1 .731 _+ 0 .016 ( m e a n + hal f range , n = 5) and [ 3 H ] o c t a o l IX D [ 2 4 ] , Ve/V 0 = 1 .573 -+ 0 .008 ( m e a n ± hal f range , n = 4). T h e observed Mr values are m e a n s ( ± h a l f range) o f n d e t e r m i n a t i o n s .

b F o r m u l a w e i g h t s c a l c u l a t e d f r o m s truc tures w h i c h w e r e d e d u c e d f r o m NMR da ta [ 1 3 , 1 4 ] . c F o r m u l a w e i g h t f r o m ref. 11. d F o r m u l a w e i g h t f r o m ref. 12. e T h e m o l e c u l a r w e i g h t was m e a s u r e d e x c e p t i o n a l l y in the b u f f e r u s e d for the glycopep t ides . f F o r m u l a w e i g h t s f r o m ref . 30. g F o r m u l a w e i g h t s f r o m refs . 31 and 32.

of the radioactivity (90%) in a well defined oligosaccharide peak of Ve/Vo = 1.432 corresponding to a molecular weight of 2000 (Fig. 3B). As shown in Table I the formula weight (2005) of GP-V-5 proposed on the basis of NMR- findings and methylation analysis [13 ,14] , is rather close to our value. But the analytical data of Schmid et al. [12] suggest a much smaller molecular weight (1641).

The re-N-acetylated glycan from GC-4 revealed in gel filtration a major peak of Ve/Vo = 1.486 (Fig. 3C) corresponding to a molecular weight of 1750 (Table I). The formula weight calculated from the structure of GC-4 proposed by Che- ron et al. [11] is 1756. Fig. 3C shows also another tritium containing peak in the hydrazinolysate of GC-4 peptide. This unidentified peak seemed to increase during the storage; in hydrazinolysates prepared from freshly labelled GC-4 there was only 10% of this material, but after a year's storage of [3H]GC-4 the unidentified peak contained 38% of the total label as shown in Fig. 3C.

The glycan from GC-4 defucosylated with a-L-fucosidase revealed an elution rate on Bio-Gel P-6 (Ve/Vo = 1.525) corresponding to a molecular weight of 1600 (Table I).

Molecules smaller than the GC-4 glycans (1756) were separated more effec- tively than the larger molecules on the Bio-Gel P-6 column. The peaks of the

314

GC-4 glycan (1756) and lacto-N-difucohexaol I (1003) were separated by 7.6 fractions, whereas the glycans of GP-V-2 (2506) and GC-4 (1756) were sepa- rated only by 4.2 fractions, although the differences of their molecular weights are practically the same. A mixture of octaol IXD (1441) and lacto-N-difuco- hexaol I (1003) was eluted as two separate peaks but the glycans of GP-V-2 (2506) and GP-V-5 (2005) did not separate clearly.

Liberation o f glycans by hydrazinolysis o f intact glycoproteins Hydrazinolysis liberated amino acid-free glycans not only from glycopep-

tides but also from intact glycoproteins. After re-N-acetylation the hydrazino- lysates of human serum transferrin showed a well defined glycan peak of about 2000 daltons; the theoretical molecular weights of the two- and three-antennary glycans of serotransferrin are 1640 and 2005, respectively [30].

Hydrazinolysates of ovalbumin revealed a hexose-containing glycan peak and a ninhydrin-positive peak containing presumably amino acyl hydrazides as well as hydrazides of small peptides (Fig. 4). The re-N-acetylated ovalbumin glycans had apparent molecular weight 1500 which is in the expected range of the gly- cans found in ovalbumin [31--33].

Preparation o f viral proteins labelled with radioactive mannose and galactose Metabolically labelled [2-3H]mannose- and [1-3H]galactose-Semliki Forest

viruses were obtained by presenting the radioactive monosaccharides to infected BHK cells. Under the conditions used the mannose-label was rather specific: hydrolysis of the virus and paper chromatography of the liberated monosac- charides showed that mannose contained practically all of the label present (Mattila, K. and Renkonen, O., unpublished data). Even the galactose label is assumed to be quite specific because pure ~-D-galactosidase removed all label from desialated viral glycans labelled in this manner [17]. Chemically labelled [6-3H]galactose-proteins of Semliki Forest virus were prepared by oxidizing detergent disrupted virus with galactose oxidase and by reducing the resulting aldehyde groups with sodium borotritiate [ 16].

E O 0 . 6 0

d Z

nn

c~ 0.30 (.0

"-2

AMINOACYL HYDRAZtDES

OVALBUMIN

° i :

oo., i /

j .'" ~" • ~ e o e e o e° o o •

30 50 70 FRACTION NUMBER

E

o

1 . 0 0

w- Z <~

3.50 o

Fig . 4. G e l f i l t r a t i o n o n a B i o - G e l P-6 c o l u m n ( 0 . 9 X 6 0 c m ) o f a h y d r a z i n o l y s a t e o f o v a l b u m i n b e f o r e

r e - N - a c e t y l a t i o n . T h e e l u t i o n w a s e x c e p t i o n a l l y c a r r i e d o u t w i t h t h e b u f f e r u s e d f o r g l y c o p e p t i d e s . A n a l - y s i s bY t h e p h e n o l - s u l p h u r i c a c i d r e a c t i o n f o r h e x o s e s ( a b s o r b a n c e a t 4 9 0 n m ) a n d b y t h e n i n h y d r i n r e a c -

t i o n f o r a m i n o g r o u p s ( a b s o r b a n c e a t 5 7 0 n m ) .

315

The individual proteins of the labelled Semliki Forest virus were isolated with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Fig. 5A shows the Laemmli separation of E3 and ErE2 of [2-3H] - mannose Semliki Forest virus. The E~-E2-band which contains 81% of the viral mannose [3] gave extracts containing 75% of the mannose label showing that these proteins were extracted from the gel almost as well as the smaller E3-pro- tein. A L aemmli separation of the Semliki Forest virus [6-3H] galactose-proteins is shown in Fig. 5B. El andE2 were separated on a run shown in Fig. 5C, where the system of Neville was used. As shown in Fig. 5B and 5C the galactose oxidase-NaB3H4 procedure labelled preferentially the E3- proteins.

Hydrazinolysis of mixed viral [1-3H]galactose-proteins and gel filtration of the liberated glycans

Metabolically labelled, mixed viral [1)H]galactose-proteins were digested with pronase, desialated, and the resulting glycopeptides were hydrazinolysed,

2 0 0

I00

1600

G- o

I

800

EI-E E 3

? ?

\

E 5

I

E I - E 2

E 3

4 0 0 E I

i // ~ o

/ I, I 2 0 0 E 2o

f,.,~/\o °~ ,? '1 BPB o

.~_oOo,~, o.1 °° ~ o ,o° ~/'~ooo~,#~Ooo% ' ~ % o ¢ ( % . n~.°o o , , o %

20 40 6O SLICE N U M B E R

Fig. 5. Polyac~, la~ide gel electrophoresis of Semliki Forest virus proteins in the presence of sodium dode- cy l su l fate . A, Laeramli run o f [ 2 - 3 H ] m a n n o s e - p r o t e i n s w h i c h w e r e labe l led m e t a b o l i c a l l y . B. L a e m m l i run o f [ 6 - 3 H ] g a l a c t o s e - p r o t e i n s labe l led c h e m i c a l l y af ter de t erg ent t r e a t m e n t (stage I) o f the virus [ 1 6 ] . C, Nevi l le run o f [6 -3 H ] g a l a c t o s e - p r o t e i n s labe l led c h e m i c a l l y a f ter d e t e r g e n t t r e a t m e n t (stage III) o f the virus [ 1 6 ] ( the bars s h o w h o w the fract ions w e r e p o o l e d to obta in the individual pro te ins ) . Analys i s by sl icing the w e t gels and by ex trac t ing the pro te ins for the assay o f radioact iv i ty .

3 1 6

re-N-acetylated and subjected to gel filtration on Bio-Gel P-6. As Fig. 6 shows, essentially all radioactivity was recovered as one peak of an apparent molecular weight of 1800 (Table I).

Most of the viral glycans were thus of about the same size as the bi-anten- nary GC-4 glycan. This finding has implications concerning the number of the glycans. The early data suggested that one three-antennary glycan is bound to each of Semliki Forest virus membrane proteins [17]. As the present results show that the glycans are in fact only biantennary, the proteins must carry more than one L-type o!igosaccharide.

The glycans could be liberated by hydrazinolysis also from whole [1-3H] - galactose-labelled virus; the molecular weight obtained was the same as for glycans from pronase glycopeptides.

Fractionation of viral glycopeptides by affinity chromatography To see whether the viral glycans are homogeneous or not, the pronase pep-

tides of the individual viral glycoproteins were fractionated by affinity chro- matography on Concanavalin A-Sepharose [ 20,21,34] prior to liberation of the glycans.

Our reference compounds behaved in the expected manner on a Concanava- lin A-Sepharose column: the fou r - and three-antennary glycopeptides [6-3H] - galactose-GP-V-2 and [6-3H]galactose-GP-V-5 were not retained from sugar- free buffer solution, but the two-antennary glycopeptide [6-3H]galactose GC-4 was retained, and could be eluted with 20 mM a-methyl-mannoside (Table II).

Concanavalin A-Sepharose fractionation of desialated [2-3H]mannose glyco- peptides of E3 gave a large fraction (29% of the label) of non-retained material (fraction I), a still larger amount (71%) of retained material, which could be eluted with 20 mM a-methyl mannoside (fraction II), but not at all strongly retained material (fraction III) (Table II). A similar result was obtained from

SFV L-TYPE G LYCAN S

o

O_ u 200 -l-

re)

I O0 Vo

e . ¢ : - ^ , o , ° o o e ° ° ° a ~ ^ . . _ _ J 30 50 70

FRACTION NUMBER

F i g , 6. G e l f i l t r a t i o n o n a B i o - G e l P-6 c o l u m n (1 X 1 4 0 c m ) o f re-N-acetylated L-type glycans l iberated by hydraz ino lys i s o f m i x e d S e m l i k i F o r e s t v i r u s prote ins labelled metabol ica l ly with [ 1- 3 H ] galactose. Condi- t ions as in F i g . 3. N o t e that labelled products o f l o w m o l e c u l a r w e i g h t w e r e n o t present.

317

T A B L E II

F R A C T I O N A T I O N OF P R O N A S E G L Y C O P E P T I D E S ON C O N C A N A V A L I N A - S E P H A R O S E C O L U M N

G l y c o p e p t i d e Per cent of to ta l label

F rac t i on I a F r a c t i o n I I b F rac t i on I I I c

Model c o m p o u n d s [ 3 H ] G C - 4 5 95 0 [ 3 H ] G P - V - 5 100 0 0 [3 HI GP.V_2 d 100 0 0

Viral g lycopep t ides [ 2 _ 3 H ] m a n n o s e E3 e 29 -+ 1 f 71 -+ 1 f 0 [ 2 _ 3 H ] m a n n o s e E l . E 2 e 6 34 59 [1 -3H]ga lac tose E 3 g 41 59 0 [1 -3H]ga lac tose E I - E 2 g 8 92 t race [6 .3H ]ga l ac t o se E3 e 47 +- 1 f 53 +- 1 f 0 [ 6 . 3 H ] g a l a c t o s e E2 e 20 80 t race [6_3H]galac tose E1 e 5 95 0

a F rac t i on I is no t r e ta ined by the c o l u m n ; it is e lu ted wi th sugar- tree buf fe r . b F rac t i on II is r e t a ined by the c o l u m n ; it is e lu ted wi th 20 m M s - m e t h y l mannos ide . c F rac t i on III is r e t a ined b y the c o l u m n ; it is e lu ted w i th 200 mM s - m e t h y l mannos ide . d G l y c a n l ibera ted f r o m GP-V-2 g lycopep t ide b y hydraz ino lys i s and subsequen t ly re -N-ace ty la ted . e Des ia la ted g lycopep t ides . f Mean +_ half range of two analyses. g These s ia log lycopep t ides were ac tua l ly doub le labelled wi th [ 3 H ] g a l a c t o s e and [ 1 4 C ] m a n n o s e [4 ] .

H o w e v e r , only the galactose label, wh ich was m u c h s t ronger t h a n the m a n n o s e label, is cons idered here .

metabolically labelled [ 1-3H] galactose-E3-glycopeptides (Table II); non-retained fraction (41%) was even larger than in the mannose-labelled glycopeptides. However, the two observations are probably equivalent because the large gly- cans of fraction I reveal a higher galactose to mannose ratio than the small glycans of fraction II. Also the chemically labelled [6-3H]galactose-E3-glyco - peptides were rich in fraction I material (Table II).

In contrast to E3 glycopeptides the [2-3H]mannose glycopeptides from El- E2 revealed very little fraction I material, but they were rich in material of both fraction II and fraction III (Table II). Fraction III contains the oligomannosyl type gloycopeptides [20,21,34]. Also [1-3H]galactose-glycopeptides of El-E2 revealed very little of fraction I, but much of fraction II (Table II).

These observations confirmed that the bulk of the viral L-type glycans behave like bi-antennary oligosaccharides. These findings also suggested that E3-glycopeptides may contain more than the others of large three- or four- antennary glycans.

Estimation of the molecular size of the glycans liberated by hydrazinolysis of the fractionated viral glycopeptides

Desialated glycans were liberated by hydrazinolysis from the viral glyco- peptides and their molecular weights were estimated by gel filtration on Bio-Gel P-6 after re-N-acetylation (Table I).

The glycans liberated from [2-3H ]mannose-E3I-glycopeptides gave only one major peak of 2150 daltons (Ve/Vo = 1.396) (Fig. 7A). This value is larger than that of the glycan liberated from the three-antennary orosomucoid glycopro-

3 1 8

tein GP-V-5 (2000), but smaller than the glycan liberated from the four-anten- nary orosomucoid glycopeptide GP-V-2 (2450) (Table I). This finding suggests that the E3I-glycopeptides carried indeed large glycans, possibly both three- and four-antennary ones.

The glycans liberated from [2-3H]mannose-E3II-glycopeptides (Fig. 7B) revealed about the same molecular weight, 1850 ( V e / V o = 1.466), as the bi-antennary GC-4 glycan (Table I). The glycans liberated from the [2-3H]man - nose-E1-E2II-glycopeptides (Fig. 7C) revealed a molecular weight of 1800 ( V e / V o = 1.470); they, too, were of about the same size as the bi-antennary glycan of GC-4 (Table I).

The [2-3H]mannose-labelled oligomannosyl glycopeptides E1-E2III released glycans (Fig. 7D) of V e / V o = 1.647 corresponding to an apparent molecular

I 0 0

5 0

E 3 I - G L Y C A N S A

, / ! o O o o

o c - , ~ . : . - , - . : . - . o _ _ _ _ i \ o o 9 9 9 : - - - , ^ , : , o o ° ° 0o^..~:~ ....

:E O_

L ) i

T

r ~

t E3 II-GLYCANS B

2001 -

,00t;° 4 0 0

200 v0 J

E I - E 2 l l - G L Y C A N S

/'i

2 %0. ° -Oo o o ^ ~ ooo~ ~ ~ , A ~ ~v . . . .

D 400 E I - E 2 I I I - G L Y C A N S /°~ °

; \ 2 O0 ~ \o

Vo / \ J o, ? (,

_ ~ . . . . . . . o ° ° ° ° ° ° ° o o ~ ~ ~ - o

0 5 0 7 0

F R A C T I O N N U M B E R

Fig. 7. Gel f i ltration on a Bio-Gel P-6 c o l u m n (1 X 140 cm) o f re-N-acetylated glycans l iberated by hydra- z ino lys i s of individual [ 2 - 3 H ] m a n n o s e g lycopept ides of Semliki Forest virus. A, E3I glycans; B, E3II gly- cans; C, E l -E2II glycans; D, E1-E2III glycans. Condi t ions as in Fig. 3.

319

weight of 1200 (Table I), a value between our reference hexa- and octasaccha- ride.

The glycans released from the chemically labelled [6-3H]galactose-glycopep - tides confirmed that E3I-glycans are larger in size than the others (Table I). However, all [6-3H]galactose-labelled glycans were distinctly smaller than their [2-3H]mannose-labelled counterparts. We do not know whether this reflects differences in the glycans of the two virus batches, or differences caused by selectivity of the labelling procedures. The viral [6-3H ] galactose-glycopeptides revealed also low molecular weight products in their hydrazinolysates which are not regarded as viral compounds because the hydrazinolysates of metaboli- cally labelled [1-3H]galactose-glycopeptides did not reveal these compounds (Fig. 6).

Discussion

The present data confirm the findings of Schmid's [6], Montreuil 's [7,8] and Osawa's [9,10] groups on hydrazinolysis, showing that asialoglycans of L- and M-types are liberated from asparagine glycopeptides in amino acid-free form and wi thout destruction of the O-glycosidic linkages of the oligosaccha- rides. This conclusion is based on the use of reference glycopeptides GC-4 from IgG [11] as well as GP-V-2 and GP-V-5 from ~l-acid glycoprotein [12]. These glycopeptides have been previously characterized quite thoroughly by chemical analysis [11,12] , and with high resolution NMR-techniques [13,14] yielding the structures shown in Fig. 1. Their glycans resembled the L-type oligosaccha- rides of Semliki Forest virus in size and composit ion being suitable for the evaluation of the analytical procedures which were to be applied to the viral glycopeptides. Gel filtration of the hydrazinolyzed reference glycopeptides indicated molecular weights quite close to the calculated ones for the three liberated glycans.

Most of the L-type asialo glycans liberated by hydrazinolysis from Semliki Forest virus proteins were of 1800 daltons, a value close to the molecular

w e i g h t of the bi-antennary glycan from GC-4. Also the L-type glycans of Sindbis virus, a close relative of Semliki Forest virus, are bi-antennary according to a structure proposed by Burke [35]. Curiously enough the L-type glycans of Vesicular Stomatitis virus are three-antennary, although even this virus was grown in BHK cells [36]. Even the glycans of Semliki Forest virus proteins were previously regarded as three- or four-antennary [17]. This erroneous notion was based, at least partly, on too high molecular weights of the viral pronase glycopeptides obtained with our old gel filtration technique [4] ; in the present experiments the old technique gave too high values also for the refer- ence glycopeptides. The early idea of relatively large viral oligosaccharides seemed to imply that E, and E3 proteins contain about one mole of L-type gly- cans [4,17]. However, the present findings showing that most of viral L-glycans are small bi-antennary ones, suggest that the carbohydrate content of these Semliki Forest virus glycoproteins is equivalent to more than one oligosaccha- ride molecule per glycoprotein.

Gel filtration of E2-glycans (Table I) and Concanavalin A-Sepharose chroma- tography of E2-glycopeptides (Table II), together with the early composit ional

3 2 0

data [3], suggest that E~, like E1 and E3, may contain true L-type glycans instead of the unidentified X-type oligosaccharides [ 1,4,17].

The M-type glycans liberated from the mixed E1-E2-glycopeptides of Semliki Forest virus probably originated from E~-protein [4]. These glycans revealed an apparent molecular weight of 1200; a value between the reference octa- and hexasaccharides, which are structurally unrelated to the M-glycans, how- ever. The M-type glycans of Sindbis virus are nonasaccharides according to a structure proposed by Burke [35].

Among the Semliki Forest virus proteins only E3 contained a significant amount (30%) of larger than bi-antennary glycans. These large E3-glycans revealed an average molecular weight 2150 falling between the three- and four- antennary reference oligosaccharides. Also our earlier findings [3] showing higher ratios of galactose to mannose, of fucose to mannose and of sialic acid to mannose in E3 than in E~, suggest that E3-glycans may be larger than El-gly- C a n S .

The large size of E3-glycans is possibly related to their peripheral and exposed position on the viral surface. E3 does not appear to have a hydropho- bic part; in contrast E, and E: are believed to have such a structure which serves to anchor the proteins to the lipid bilayer of the viral membrane (ref. 37, and Garoff, H. and S'bderlund, H., submitted for publication). As E3 is not linked to the lipid bilayer it seems to be anchored into the viral membrane in a more peripheral way than the two other proteins. The E3-glycans themselves appear to be more peripheral and more exposed than the other glycans on the intact virion surface, because they react more extensively than the glycans of E: and E~ with sialidase and galactose oxidase [16]. The present findings of Fig. 5A and 5B confirm and extend the previous data [16] showing that E3 is indeed preferentially labelled, not only preferentially extracted from the gel slices obtained after electrophoresis.

It seems possible that the exposed E3 glycans grow larger than the others just because of this high degree of exposure, leading to frequent interactions with glycosyl transferases and sugar donors. Similar steric factors may lie behind the different sizes of L-type glycans observed on separate locations of certain poly- peptides, e.g. in human serotransferrin [ 30] and in ~ ~-acid glycoprotein [ 12 ].

The regulation of the size of the glycans is of considerable interest; for instance malignant cells carry often larger glycans than normal cells [38].

Acknowledgements

We are thankful for the generous gifts of pure glycans and glycopeptides ob- tained from Drs. A. Cheron, A. Gauhe, J. Montreuil, K. Schmid and G. Strecker, which made this s tudy possible. The amino acid analyses were carried out during a highly appreciated visit of M-L. Rasilo in the laboratory of Prof. J. Montreuil. The work was supported by grants from Suomen Kulttuurirahasto, The Finnish Academy and the Finnish Ministry of Education.

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