146

Biochimica et Biophysica Acta, 586 (1979) 146--158 © Elsevier /North-Hol land Biomedical Press

BBA 28976

THE GLYCANS OF p-62, A VIRUS-SPECIFIC GLYCOPROTEIN IN SEMLIKI FOREST VIRUS INFECTED BHK CELLS

H A N N E L E S T E N V A L L and OSSI R E N K O N E N *

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

(Received December 15th , 1978)

Key words: Virus infection; Glycan; Virus-specific glycoprotein; p-62 glycoprotein; (BHK cell, Semliki Forest virus)

Summary

P-62, the precursor of viral envelope glycoproteins E2 and E3, was isolated by polyacrylamide gel electrophoresis from BHK cells infected with Semliki Forest virus. Metabolic labelling with radioactive monosaccharides revealed that p-62 contained mainly type M (high mannose) glycans. Glycopeptides were obtained from the cellular p-62 by pronase hydrolysis, and their structure was elucidated by sequential digestions with exo- and endohydrolases. They resembled the type M glycopeptides isolated from the virus particle and had probably the following average structure: (Man-~)a_5-Man-fl-(1-* 4)-GlcNAc-fl- (1-~ 4)-GlcNAc-peptide. The glycans labelled with [3H]mannose and [14C]- glucosamine were cleaved quantitatively from the peptides by endo-fl-N-acetyl- glucosaminidase H; the absence of unreactive peptides confirmed the absence of type L (lactosamine) glycans in the cellular p-62. Paper chromatography showed the type M glycans to be heterogeneous. The possible conversion of the type M glycans of the cellular p-62 to type L glycans of the viral E2 and E3 is discussed.

Material containing label from [3H]fucose and [~4C]galactose migrated also at the p-62 band in the gel electrophoresis. Most of this label was not carried by the same protein as the type M glycans, as shown by affinity chromatog- raphy on concanavalin A-Sepharose.

Introduction

In addition to three viral envelope glycoproteins E l , E2 and E3, a fourth gly- coprotein, p-62, is present in Semliki Forest virus infected cells [1--3]. P-62 is

* To whom correspondence should be addressed.

147

known to be the precursor of E2 and E3 [4,5], suggesting that the glycans of p-62 may represent an earlier stage of glycosylation than those of E2 and E3 [6].

In the present report it will be shown that the p-62 found in the infected BHK cells carries only type M (high mannose) glycans, whereas its products, the viral proteins E2 and E3, carry additionally type L (lactosamine) glycans (Refs. 7--9 and Pesonen and Renkonen, unpublished results).

Materials and Methods

Isolation of labelled glycopeptides. A monolayer culture of BHK-21 cells in a Falcon flask of 75 cm 2 was infected with a pro to type strain of Semliki Forest virus and labelled from 3 to 10 h post infection in separate experiments with 1 mCi of D-[2-3H]mannose (2 Ci/mmol), 250pCi of D-[1-14C]glucosamine hydrochloride (58 Ci/mol), 1 mCi of L-[1-3H]fucose (2.6 Ci/mmol) or 250 pCi of D-[1-14C]galactose (60.7 Ci/mol). (The Radiochemical Centre, Ltd., U.K.) as described by Mattila et al. [8]. The labelled cells were washed twice with cold phosphate-buffered saline, dissolved in 0.1% sodium dodecyl sulphate (SDS) and fractionated by discontinuous polyacrylamide gel electrophoresis in the presence of SDS according to Laemmli [10]. The p-62 was isolated as described by Mattila et al. [8] and hydrolyzed with 0.13% pronase as described by Peso- nen and Renkonen [11].

Gel chromatography. A column (1 × 60 cm) of Bio.Gel P-6 (100--200 mesh, Bio-Rad), eluted with 0.15 M Tris/acetate buffer (pH 7.8) containing 0.1% SDS, was used for apparent molecular weight determinations. The column was calibrated using the following NaB3H4-reduced reference standards: Gal-/3- (1 -* 4)-GlcNAc~6-(1 -~ 2)-Man~-(1 -* 3)-[Gal-/~-(1 -~ 4)-GlcNAc-/3-(1 -+ 2)-Man- fi-(1 -* 6)]-Man~6-(1 -~ 4)-GlcNAc (Mr = 1434) [9], lacto-N<lifucohexaose (M r = 1004) [8] and Gal-fi-(1 -~ 3)-Gal-fl-(1 ~ 4)-Glc (Mr = 504) [8]; D-[1-14C]man - nose (The Radiochemical Centre Ltd., U .K. )and [14C]acetylated type L glyco- peptides of thyroglobulin (M~ = 4100) [8] were also used. The exact position of each peak was determined by fitting a parabola through the three highest points.

A column (1 × 63 cm) of Bio-Gel P-2 {100--200 mesh, Bio-Rad) eluted with 0.02% NaNa was used for 'desalting' purposes.

Glycosidase digestions. Digestions with exoglycosidases were carried out at 37°C as described by Pesonen and Renkonen [11]. N-Acetyl-~-D-hexosamini- dase (EC 3.2.1.52) from jack bean (gift of Dr. Y.-T. Li, New Orleans) was added three times to final concentrat ion of 5.9 or 13.3 U/ml, with an incuba- tion time of 72 or 74 h. a-D-Mannosidase (EC 3.2.1.24) from jack bean (Boeh- ringer) was added three successive times to a final concentrat ion of 24 U/ml, incubation time 72 h. a-D-Mannosidase from jack bean (gift of Dr. Y.-T. Li, New Orleans) was added three times to final concentrat ion of 50 U]ml, with an incubation time of 86 h. Endo-/3-N-acetylglucosaminidase H digestions were carried out essentially as described by Tai et al. [12]. The enzyme was added once to a final concentrat ion of 20 mU/ml. The incubation t ime was 24 h at 37°C.

Paper chromatography. Descending paper chromatography was performed

148

on Whatman 1 paper using the solvent system ethylacetate/pyridine/acetic acid/water (5 : 5 : 1 : 3). The paper was cut into 1 X 3 cm strips and counted for radioactivity in 0.5 ml of water and 5 ml of scintillation liquid.

Affinity chromatography of glycoproteins. A concanavalin A-Sepharose 4B (Pharmacia Uppsala, Sweden) column of 1 X 15 cm was used essentially as described by Zanetta et ,al. [13] and Mattila, K. {unpublished data). The column was equilibrated first with 50 ml of 0.01 M Tris/HC1 buffer (pH 7.5) containing 0.08% SDS. The sample was introduced in 100 t~l of the buffer. Fractions of 1.5 ml were collected with the flow rate of 240 pl/min. The column was eluted first with 22 ml of the sugar free buffer, and with 30 ml of the buffer containing 5 mM a-methyl-D-mannoside {Grade II, Sigma) and finally with 30 ml of the buffer containing 200 mM a-methyl-D-mannoside.

Affinity chromatography of glycopeptides. A concanavalin A-Sepharose 4B (Pharmacia, Uppsala, Sweden) column of 0.9 X 3 cm was used essentially as described by Ogata et al. [14] and by Mattila and Renkonen [15].

Labelling of Semliki Forest virus by galactose oxidase. A pro to type strain of Semliki Forest virus was grown in BHK-21 cells and purified by gradient cen- trifugation [16]. Neuraminidase (Vibrio cholerae) and galactose oxidase (Kabi AB, Stockholm, Sweden) digestions of the virus, followed by reduction with NaB3H4, were performed as described by Luukkonen et al. [17].

Chemical methods. Acid hydrolysis to cleave the glycosidic linkages was carried ou t with 2 or 4 M HC1 for 2 or 16 h at 100°C. HC1 was removed from the samples by evaporating several times with water under nitrogen.

Reduct ion of type M glycans was performed in 200 pl of NaBH4 (5 mg/ml of water). After incubation for 2 h at room temperature a few drops of glacial acetic acid were added 'til neutrality, 2 ml of methanol were added and the sample was evaporated to dryness under nitrogen. This was repeated three times. The sample was desalted on a coupled column of Dowex 1X8 200/400 mesh (HCOO-) /Dowex 50 WX8 200/400 mesh (H ÷) ion exchange resin. Neutral products were eluted by water and charged molecules by 1.0 M pyridine/ace- tate, pH 5.0.

Mild alkaline t reatment of the glycopeptides was carried out with 0.05 M NaOH/1.0 M NaBH4 at 45°C for 15 h under nitrogen in sealed tubes, and the reaction was stopped by glacial acetic acid as described [18]. After reaction three drops of toluene were added and the mixture was evaporated to dryness under nitrogen. This step was repeated three times.

Lipid extraction was performed by using chloroform/methanol /water (8 : 4 : 3) [19] and 2 mg of bovine serum albumin as carrier.

Scintillation counting. Radioactivity was determined in Wallac 81 000 scin- tillation counter in plastic vials in Triton X-114/xylene (1 : 2.67, v/v) con- taining 2.9% (W/v) Permablend III (Packard). Quenching was checked with an external standard. In double label experiments the data were corrected for the crossover counts.

Results

(1) Glycoproteins of Semliki Forest virus infected BHK cells Semliki Forest virus infected BHK cells were labelled for 7 h in vivo with

149

3200

El- E 2

E3

P62

~o go ~o l

P62

E 1, E 2 E3

1400

SL$CE NUMBER

4o0

=', 300 z

200

:o' ~UM

500

.=

2oc

10C

Z'O

ES

EI*E 2

,62 c ~ L

1 4"0 ~o ~0

d

E1 * E2

C

20 ~() 20 ~ 60 80 SLICE NUMBER

Fig. I . Gel electrophoretic patterns of Semliki Forest virus infected cells labelled with: (a) D-[I-14C]- galactose; (b) L-[1-3H]fueose; (c) D-[2-3H]mannose and (d) D-[1-14C]glucosamine. E l , E2 and E3, enve- lope proteins; p-62, precursor protein; C, nucleocapsid protein; BPB, bromphenol blue.

radioactive monosaccharides. The resulting glycoproteins, which were labelled in their glycans, were separated with discontinuous polyacrylamide gel electro- phoresis in the presence of SDS [10] . In addition to the peaks corresponding to the viral membrane glycoproteins E1 + E2 (which migrate together in this gel electrophoresis system) and E3, the p-62 band also became labelled with radio- active galactose (Fig. la) , fucose (Fig. lb) , mannose (Fig. l c ) and glucosamine (Fig. ld) .

To find out how specific the metabolic labelling had been, the material in the p-62 band was hydrolyzed and the monosaccharides of the hydrolyzate were analyzed by paper chromatography. The results are presented in Table I. [2 )H]mannose labelling had been quite specific, but some label from [1-14C] -

150

T A B L E I

S P E C I F I C I T Y O F T H E R A D I O A C T I V E L A B E L IN T H E p-62 B A N D

R a d i o a c t i v e m o n o s a c c h a r i d e

used in the label l ing o f p -62

D i s t r i b u t i o n o f r a d i o a c t i v i t y (%) in d i f f e r e n t m o n o s a c c h a r i d e s d

GaIN GlcN Gal Glc Man Fuc

D-[ 1-14C] Ga lac tose a _ 2 63 - - 31 4

L-[ 1-3HI F u c o s e a . . . . . 16 84 D-[ 1-14C] G l u c o s a m i n e b,c _ 75 - - - - 25 --

D-[ 2 -3 H] Man n o se b . . . . 100

a The s a m p l e s t u d i ed was p ronase g l y c o p e p t i d e .

b The s a m p l e s t u d i ed was o l i gosaccha r ide c leaved by endo-N- f l - ace ty lg lucosamin idase H f r o m the p ronase

g l y c o p e p t i d e .

c When the s a m p l e was h y d r o l y z e d p-62 p r o t e i n , 93% of 14C label was as g l u c o s a m i n e and 7% as m a n n o s e

d GalN, g a l a c t o s a m i n e ; GlcN, g l u c o s a m i n e ; Gal, ga lac tose ; Glc, g lucose; Man, m a n n o s e ; and Fuc , fucose .

o = ~' '°°° i b

1 ~00 Z50 i ~

M r 6OO0 I t 150

=o ~ ~o ~o 2o ,o 6o ,o

~00

'.00

3(]0

200

~00

C 20oi

FRACTION NUMBER

~so !

J

50

M r 2100

FRACTION NUMBER

Fig. 2. Gel f i l t r a t i o n p a t t e r n s o f p r o n a s e g l y c o p e p t i d e s of the p-62 b a n d label led w i t h (a) D - [ 1 - 1 4 C ] g a l a c - tose ; (b) L - [ 1 - 3 H ] f u c o s e ; (c) D - [ 2 - 3 H ] m a n n o s e ; o r (d) D - [ 1 - 1 4 C ] g l u c o s a m i n e (Mr, a p p a r e n t m o l e c u l a r we igh t , V 0, vo id v o l u m e ) .

151

glucosamine, [1-3H]fucose and [1-1~C]galactose had been converted to man- nose.

Accordingly, the glycans of p-62 band seemed to contain mainly mannose and glucosamine {which is N-acetylated in glycoproteins [20] ) and some fucose and galactose.

(2) Glycopeptides of p-62 The p-62 band was eluted from polyacrylamide gels and digested with pro-

nase. The resulting glycopeptides were analyzed by gel filtration on Bio-Gel P-6 column {Fig. 2a--d). Two different glycopeptides were found. The larger glycopeptides had an apparent molecular weight of 3000--6000 and became labelled by galactose and fucose (Fig. 2a and b). The smaller glycopeptides seemed more homogeneous, having an apparent molecular weight of 2100. They contained label from galactose, mannose and glucosamine (Fig. 2a, c and b). It seems possible that these smaller glycopeptides contained only mannose and N-acetylglucosamine and thus represented type M glycopeptides; most of the [1-'4C]galactose label present in this peak was probably mannose (see Table I).

The pronase digest obtained from p-62 labelled with [1-14C]galactose, [1-3H]fucose or [1-14C]glucosamine contained some radioactivity which was eluting like amino acids or small peptides (Fig. 2a, b, d). This suggests that some of the label was metabolized into amino acids [8]. Pronase hydrolysates obtained from [2-3H]mannose-labelled p-62 did not contain much amino acid radioactivity (Fig. 2c).

(3) Exohydrolase digestions of the type M glycopeptides N-Acetyl-[J-D-hexosaminidase. An N-acetyl-fl-D-hexosaminidase treatment of

the type M glycopeptides labelled with [1-14C]glucosamine did not change their gel filtration pattern (data not shown}. The apparent molecular weight of the gly- copeptide peak had remained unchanged during the digestion, and less than 7% of the radioactivity of the digest eluted at the position of free N-acetylglucos- amine. This suggests that most of the type M glycopeptides of p-62 did not carry distal N-acetylglucosamine residues.

a-D-Mannosidase. The type M glycopeptides labelled with [2-3H]mannose were digested with highly purified a-D-mannosidase from jack bean (Dr. Y.-T. Li). The incubation liberated 79% of the mannose label as free mannose (Fig. 3); 14.5% of the mannose label was found in a heterogeneous glycopeptide peak of 1300 and 6.5% of the label remained in a glycopeptide peak of 2000 daltons resembling the undigested material. This experiment suggested that type M glycopeptides of p-62 carry about five distal a-D-mannose residues. The reduction in the apparent molecular weight of the glycopeptides from 2100 to 1300 is equivalent to 4.9 mol of a-D-mannose, and the ratio of a- and/~-man- nose is 5.4 assuming that the residual glycopeptides of 1300 daltons each carry one fi-D-mannose unit.

~- and [J-D-Mannosidase. The type M glycopeptides labelled with [2-3H]man - nose and [1-14C]glucosamine were mixed and digested with a preparation of a-D-mannosidase from jack bean (Boehringer), which was contaminated with ~-D-mannosidase [11]. The incubation liberated all the 3H-label and 20% of

152

!5C

50

IO(

50

M r 2000 M r 15OO

Vo I

2'O 40 6O FRACTION NUMBER

\

8O

Fig. 3. Pronase glycopept ides of P-62 labe l led w i t h D-[2-3H]mannose in gel f i l t rat ion after t rea tment with c~-mannosidase (Dr. Y.-T. Li, 50 U/ml, 86 h at 37°C).

the ~4C-label as free monosaccharide (Fig. 4a). The liberated ~4Cdabel behaved upon gel filtration like mannose (see also Table I). The demannosylated glyco- peptides had an apparent molecular weight of 1400, a value which was obvi- ously too large (cf. Fig. 3). On the whole this experiment is compatible with the presence of fl-D-mannosyl residues which are located proximally to the a-D-mannose residues.

Digestion of the proximal N-acetylglucosamine residues. The demannosy- lated type M glycopeptides were treated further with N-acetyl-fi-D-hexosamini- dase. This t reatment liberated about 65% of ~4C-label as free monosaccharide (Fig. 4b). About 35% of the label was found at a glycopeptide peak of 700-- 750 daltons. This experiment proves the presence of fi-N-acetylglucosaminyl residues located proximally to the mannose residues. The reduction of the apparent molecular weight of the glycopeptides from 1400 to 700--750 is equivalent to about 3 mol of N-acetylglucosamine. Assuming that the residual glycopeptides carry one N-acetylglucosamine residue, and considering that some of the liberated 14Cdabel represented mannose (see Table I), the number of the liberated N-acetylglucosamine units was 1.4--1.9 as judged from the amount of 14C-label.

153

% I

Mr t 400

; 1 'eo t

,' b

20 40

i o

i o / i •

60 2 0

FRACTION NUMBER

Fig. 4. P r o n a s e g l y c o p e p t i d e s o f p - 6 2 l abe l l ed w i t h D - [ 1 - 1 4 C ] g l u c o s a m i n e (e . . . . . . e ) a n d D - [ 2 - 3 H ] m a n - n o s e ( o - --o) in gel f i l t r a t i o n a f t e r t r e a t m e n t w i t h (a) ~ - m a n n o s i d a s e ( B o e h r i n g e r , 2 4 U / m l , 7 2 h a t 3 7 ° C ) a n d (b) a - m a n n o s i d a s e (Boeh r ingex ) a n d N - a c e t y l - ~ - D - h e x o s a m i n i d a s e (Dr . Y.-T. Li , 5 .9 U / m l , 7 2 h a t 3 7 ° C ) .

:50

:00

50

O0

50

0

M r I100 M r 750

60 4O

\

M r I I O0

/ FRAC TON NUMBER

b

6O

25(3

200

150

I00

5 o

B'o

o

o

Fig, 5. Gel f i l t r a t i on o f e n d o - ~ - N - a c e t y l g l u c o s a m i n i d a s e H d iges t s o f p r o n a s e g l y c o p e p t i d e s o f p - 6 2 l abe l l ed w i t h (a) D-[ 1 - 1 4 C ] g l u c o s a m l n e a n d w i t h (b) D-[ 2 - 3 H ] m a n n o s e ,

154

(4) Endo-fl-N-acetylglucosaminidase H digestion o f the type M glycopeptides Intact type M glycopeptides of p-62 labelled with [1-~4C]glucosamine or

with [2-3H]mannose were digested with endo-fl-N-acetylglucosaminidase H. Upon gel filtration, the ~4C-labelled material gave two peaks of apparent molec- ular weight of 1100 and 750, respectively (Fig. 5a). The former represented an oligosaccharide and the latter consisted of glycopeptides. The digestion of the [2)H]mannose-labelled glycopeptide gave only the oligosaccharide peak of 1100 daltons (Fig. 5b). These digestions were complete; all the glycopeptides had reacted with the enzyme. Accordingly, all [2-3H]mannose - and [1-14C]- glucosamine-labelled glycopeptides were really of type M; the L type glycopep- tides do not react with the enzyme [12].

When the [1-~4C]glucosamine-labelled digestion products were subjected to ion-exchange chromatography, about 55% of ~4C-label was eluted like neutral oligosaccharide and the rest was retained like glycopeptide. Affinity chro- matography on a concanavalin A-Sepharose column showed that about 45% of the 14C-labelled digestion products were eluted with sugar-free buffer; about 55% of the label was retarded, eluting poorly even with 200 mM a-methyl-D- mannoside. The former fraction represented charged glycopeptides and the lat- ter neutral oligosaccharides as revealed by ion-exchange chromatography.

The neutral oligosaccharides obtained from endo-~-N-acetylglucosaminidase H digestion were examined by paper chromatography. They proved to be heterogeneous; two major components and two minor components were ob- served (data not shown).

When the neutral 14C-labelled oligosaccharides were reduced with NaBH4 and subjected to acid hydrolysis (4 M HC1 for 16 h at 100°C) about 83% of 14C- label was found as glucosaminitol and 17% as glucosamine (~4C-label metabo- lized into mannose seemed to have been destroyed under these conditions). This result showed that the liberated oligosaccharides contained N-acetylglucos- amine at the reducing end.

Endo fl-N-acetylglucosaminidase H cleaves type M glycopeptides between the two proximally located N-acetylglucosamine residues [21]. It appears that all reactive substrates found so far contain a core of Man-fl-(1 -~ 4)-GlcNAc-fl- (1-~ 4)-GlcNAc [22]. All our observations with endo-fl-N-acetylglucosamini- dase H are compatible with a similar structure in the type M glycans of p-62.

(5) Nature o f the material labelled with [1-14C]galactose and [1-3H]fucose at the p-62 band in polyacrylamide gel electrophoresis

Lipid extraction. About 60% of the label of the pronase digest of the [1-~4C]galactose-labelled p-62 band eluted in the void volume upon gel filtra- tion (Fig. 2a). In addition to nondegraded glycoproteins, micellar sphingo- lipids are believed to elute in this area [8]. Material in the p-62 band labelled by [1-14C]galactose was therefore subjected to lipid extraction [19]. The 14C-label was distributed among the different phases as follows: methanol/ water phase, 27%; chloroform phase, 2%; and insoluble fraction, 71%. Thus, most of the material labelled by [1-~4C]galactose seemed not to be sphingo- lipid.

Mild alkali treatment. Treatment of pronase glycopeptides with 0.05 M NaOH/1 M NaBH4 did not release small or medium sized oligosaccharides from

1 5 5

T A B L E II

T H E B E H A V I O U R O F M A T E R I A L IN T H E p - 6 2 B A N D IN C O N C A N A V A L I N A - S E P H A R O S E A F F I N - ITY C H R O M A T O G R A P H Y

Radioactive m o n o - s a c c h a r i d e used in labelling o f p - 6 2

Radioactivity (%) eluting from concanaval in A-Sepharose c o l u m n with

Suga r - f r ee ~ - M e t h y l - D - m a n n o s i d e ~ - M e t h y l - D - m a n n o s i d e b u f f e r (5 m M ) ( 2 0 0 m M )

D-[ 1 -I 4 C] G l u c o s a m i n e 10 - - 9 0 D-[ 1 -I 4 C] G a l a c t o s e 4 9 1 8 3 3 L-[ I -3 H] F u c o s e 3 8 4 7 1 5

glycopeptides labelled with [1-t4C]galactose or [1-3H]fucose (data not shown). Accordingly, these glycopeptides do not carry small oligosaccharides linked to serine or threonine via N-acetylgalactosamine. Also, the type M glycopeptides (Mr = 2000), which can be seen (Fig. 2a) in the pronase digest of [1-14C]galac - tose labelled p-62 band (about 30% of 14C-label was mannose, cf. Table I), seemed to be stable in this treatment.

El+ E 2

P62 / /

E 3

[ i 60 80

SLICE NUMBER

Fig . 6 . Gel electrophoret ic pattern o f Seml ik i F o r e s t v i rus labelled with galactose ox idase /NaB3H4 treat- ment .

1 5 6

Affinity chromatography of p-62 protein on concanavalin A-Sepharose. The behaviour of [1-14C]galactose-, [1-3H]fucose- and [1J4C]glucosamine-labelled material present in the p-62 band was examined in a concanavalin A-Sepharose column in the presence of SDS (Ref. 13 and Mattila, K., unpublished results). Table II shows that 90% of p.62 labelled with [1-14C]glucosamine was not eluted from the column until 200 mM ~-methyl-D-mannoside was used as the eluant. This was expected, because the type M glycans retard the membrane proteins (Ref. 13 and Mattila, K., unpublished results}. In striking contrast, only 33% of the material labelled with [1-14C]galactose, and only 15% of the material labelled with [1-3H]fucose behaved in this manner (Table II). The actual difference between the p-62 protein carrying the glucosamine-containing M-glycans and the galactose- and fucose~ontaining materials was probably even larger than shown by the data of Table II, because some of the [1-14C]galactose and [1-3H]fucose label had been converted into mannose of the M-glycans (see Table I). This experiment suggested that most of the [1J4C]galactose- and [1-3H]fucose-containing materials of the p-62 band do not reside in the same protein as the type M glycans labelled with [1-14C]glucosamine.

¢J

- t -

30

5C

0(:

5C

Vo

i 2 O

M r 3600

f [

40 60 80 FRACTION NUMBER

Fig. 7. Gel f i l tration of a pronase digest o f the p-62 band iso lated from galactose oxidase /NaB3H4-1abei led Semliki Fores t virus.

157

(6) Glycans of p-62 isolated from Semliki Forest virus Semliki Forest virus swollen by treatment with Triton X-100 was treated

with neuraminidase and galactose oxidase and subsequently reduced with NaB3H4. This treatment led to labelling of the viral envelope glycoproteins E1 + E2 and E3 as shown earlier [9,17]. In addition, p-62 band contained some label as revealed by polyacrylamide gel electrophoresis (Fig. 6). This p-62 band was eluted from the gel and treated with pronase, whereby the label was converted into material behaving like type L glycopeptides in gel filtration (Fig. 7).

Discussion

Our results suggest that the precursor protein p-62 isolated from Semliki Forest virus infected BHK cells carries only type M glycans. The average struc- ture of the type M glycopeptides was probably (Man~)4_5-Man~-(1-* 4)- GlcNAc~-(1 ~ 4)-GlcNAc-peptide. They behaved upon gel filtration and treat- ment with mannosidases and endo~-N-acetylglucosaminidase H like the type M glycopeptides of E2 protein of the virus particle [15]. The reactivity of the type M glycopeptides with endo-~-N-acetylglucosaminidase H suggested that they carried two proximal N-acetylglucosamine residues. This notion is sup- ported also by the distribution of glucosamine label of the digest: both the oligosaccharide fraction and the glycopeptide fraction appeared to contain about half of the total radioactivity as determined by ion~xchange chromatog- raphy and by affinity chromatography on concanavalin A-Sepharose. However, the digestion of the mannose-free glycopeptides with ~-N-acetylglucosaminidase suggested the presence of more than two proximal N-acetylglucosamine resi- dues.

The final biological products of p-62, the virus proteins E2 and E3, carry type L giycans (Refs. 8 and 9 and Pesonen, M. and Renkonen, O., unpublished results). Also, E3 protein isolated from infected cells carries exclusively type L glycans (Haahtela, K. and Renkonen, O., unpublished results}. As there were no L-glycans in the p-62, it would seem that the processing of the precursor pro- tein p-62 into E2 and E3 is accompanied either by elimination of the type M glycans and insertion of new type L glycans, or alternatively by conversion of the type M glycans into type L glycans. The latter conclusion is supported by recent observations of some other laboratories: the type L glycan in G protein of vesicular stomatitis virus envelope appears in pulse chase experiments to be derived from type M glycan [23--25] ; similar observations have been made on Sindbis virus [26]. If a relationship between the cleavage of p-62 and the con- version of M-glycans exists, its nature remains quite unclear.

The cellular p-62 band contained some label also from [1-14C]galactose. However, affinity chromatography with concanavalin A-Sepharose separated most of the galactose labelled material from the protein carrying the type M glycans. The notion that the type M glycans, rather than the galactose label, were associated with the virus specific p-62, is based on the study of the pro- nase digests: the protein carrying the type M glycans gave quantitative yields of regular glycopeptides of the type that is found in the virus. In contrast, a large part of the galactose label was not converted into glycopeptides of this type. Some of the galactose label of the cellular p-62 band may have been associated

158

with host-specific mucopolysaccharide [28--30]. The cellular p-62 band contained some label also from [1-3H]fucose. Some

of this label represented quite likely type L glycans (see Fig. 2b, Table II). However, these glycans were not labelled significantly by [2-3H]mannose or [1-14C]glucosamine. Accordingly, the fucose label may have represented cell- specific glycans which had received the mannose and N-acetylglucosamine residues prior to the infection and were completed with the fucose residues during the infection.

Acknowledgements

We thank Dr. Y.-T. Li for supplying us with the pure a-mannosidase and fi-N-acetylhexosaminidase and Dr. T. Muramatsu for the gift of endo-fi-N- acetylglucosaminidase H. This work was supported by grants from the Finnish Academy of Science.

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