HAL Id: hal-00901043

HAL Id: hal-00901043

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Submitted on 1 Jan 1978

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SUBUNIT AND FINE STRUCTURE OF THE GLYCOPROTEIN OF BOVINE LEUKEMIA VIRUS

B. Dietzschold, O.R. Kaaden, B. Frenzel

To cite this version:

B. Dietzschold, O.R. Kaaden, B. Frenzel. SUBUNIT AND FINE STRUCTURE OF THE GLYCO-

PROTEIN OF BOVINE LEUKEMIA VIRUS. Annales de Recherches Vétérinaires, INRA Editions,

1978, 9 (4), pp.613-617. �hal-00901043�

SUBUNIT AND FINE STRUCTURE OF THE GLYCOPROTEIN OF BOVINE LEUKEMIA VIRUS

B.

DIETZSCHOLD O.R. KAADEN

B. FRENZEL

Federal Research Institute for Animal Virus Diseases,

Tübingen,

^Federal Republic of Germany

Résumé

SOUS UNITES ET STRUCTURE FINE DE LA GLYCOPROTEINE DU VIRUS DE LA LEUCOSE BOVINE. ― La structure de la

glycoprotéine

du virus de la leucose bovine (BLV) a fait

l’objet

^de

nos recherches.

L’analyse

dans des conditions non réductrices révèle la

présence

^{de deux}

glyco- protéines

virales de

poids

moléculaire apparent ^162.000 (VGP 1) et 85.000 (VGP 11).

Après

^réduc-

tion et

alkylation,

^{on a} pu démontrer la

présence

de deux chaînes

polypeptidiques glycosylées

^de

poids

moléculaire apparent 60.000

(gp

60) ^et 30.000

(gp

^{30) dans VGP 1 et VGP}

Il ;

^{VGP 1 semble}

contenir deux molécules de _gp 60 et deux _{de gp}

30,

et VGP Il est

simplement

constitué d’une molécule de gp 60 ^et d’une de gp

30,

^reliées par des ponts disulfure. La

comparaison après hydrolyse trypsique

^des

peptides

de gp 60 ^et gp 30 ^montre l’existence d’une

séquence partielle

commune entre gp 60 et gp 30.

La similarité structurale de la molécule de

glycoprotéine

^{de BLV avec les}

immunoglobulines

^est

discutée _par rapport ^{aux fonctions}

possibles

^{de cette}

protéine

^{dans la}

pathogénie

de la leucose bovine.

Introduction

The

envelope glycoproteins

^{of RNA tumor}

viruses are a

complex

group of

proteins.

^Inter-

chain disulfide bonds

play a major

^{role in main-}

tening

the structural

integrity

of the viral

glycoprotein

^{(Leamnson and}

Halpern,

^1976).

Recently,

^a

major

^{and a minor}

glycoprotein

of bovine leukemia virus (BLV) have been iso- lated

by

^{treatment of}

purified

virions with

* Present address : Institut fur

Virologie

^der

Tier5rztlichen Hochschule Hannover

reducing

^and

dissociating

agents (Rohde ei aL 1978). Since

nothing

is known about the arrangement ^{of these two}

polypeptide

^chains

in the viral

glycoprotein

^of

^BLV, experiments

were undertaken ^to resolve the

complexity

^of

the BLV

glycoprotein.

^{In this} communication

we present

experiments indicating

^{that the}

BLV

glycoprotein

has the basic structure of two

heavy (gp

^{60) and two}

light (gp

30) ^chains

or one

heavy

^{and one}

light chain, respectively.

Furthermore,

^we demonstrate that

peptide

sequences of the _gp 30 are

present in

^the gp 60

C

uggesting

^a

partial homology

^{of the}

light

chain to the

heavy

chain of the BLV-

glycoprotein complex.

Resu!ts

Arrangement

^{of the}

polypeptide

^{chains in the}

BL V

glycoproteins.

Fig.

1a shows the SDS PAGE

profiles

^of radiolabeled

polypeptides

from BLV grown in the presence of either 14C-leucine or 3H- fucose. The BLV is

disrupted

with SDS in the presence of iodoacetamide and

electrophore-

sed on SDS

gel.

^{Two main}

peaks

^{of 3H-} fucose label are resolved.

According

^{to the ter-}

minology of

Leamnson and

Halpern

^{11976) the}

first peak

^{will be}

designated

^{as viral}

glycopro-

tein ! I (VGP 1) and the second

major peak

^of

JH-label as viral

glycoprotein

^{II (VGP II).}

Using

^the structural

proteins

of rabies virus f =

190.000,

^G

80.000, M, 40.000, M 2 20.ooo

Dalton) as markers (Madore and

England,

1977) the mol. wt. of these two

glycoproteins

were calculated to be 162.000 Dalton and 85.000 Dalton,

respectively.

^{To determine the}

subunit structure of the two viral

glycopro-

teins of

BLV,

^{VGP I and} VGP II were eluted from SDS

gel,

^and the eluates were reelectro-

phoresed

under

reducing

conditions. Two

new

major peaks

of ³H-fucose and 14C- leucine label were obtained after reduction and

alkylation

^{of VGP}

I (Fig.

^{lb). The}

apparent

molecular

weights

^{of these two}

peaks

^were estimated to be 60.000 Dalton

(gp

^{60) and}

30.000 Dalton

(gp

^30).

Dithiothreitol treatment of the VGP II follo- wed

by alkylation

^with iodoacetamide also

produced

gp 60 _{and gp} 30

(Fig.

^1c).

In contrast,

reelectrophoresis

^{of VGP I and}

VGP II in the _presence of iodoacetamide and without

prior

reduction with dithiothreitol did not alter the

mobility

^{of these two}

glycopro-

teins (data not

shown), indicating

^{that VGP I}

and VGP II consist of gp 60 and gp

30,

which

are linked

by

disulfide bonds. The ratio of t4C- leucine labeled _gp 60 to t4C labeled gp 30 is 1 : 0.52 in VGP I and 1 : 0.53 ⁱⁿ VGP

II,

^res-

pectively.

^{For 3}H-fucose labeled Broteins a

ratio of 1 : 0.16 in VGP I and 1 : 0.16 in VGP

II, respectively,

^was calculated. With

regard

^to

the mol. wt. of VGP I and VGP II and the ratios of gp 60 _{and gp} 30 in VGP I and VGP II, ^we conclude that VGP I contains two molecules

of gp 60 and two molecules of _gp 30. VGP 11 appears ^to contain

only

^one molecule of gp 60 and one molecule of gp 30.

Analysis

^of

tryptic peptides

^{of the two}

poly- peptide

chains of the BL V

glycoprotein

^com-

plex.

In order to determine the

relationship

^bet-

ween the

major

^{and the minor}

polypeptide

^of

the viral

glycoprotein,

^the

tryptic peptides

^of

the gp 60 _{and gp} 30 were

analyzed by

^cation

exchange chromatography.

Nonreduced BLV labeled with 14

C-lysine

^{or with 3}

H-lysine

^was

electrophoresed

^{in SDS}

gels

under nonredu-

cing

conditions. VGP I and VGP II were eluted from the

gels

^and

reelectrophoresed

^under

reducing

conditions. 14

C-lysine

^labeled gp 60 and ³

H-lysine

^labeled gp 30 were mixed and

subjected

^to

trypsin digestion.

^The

trypsin digestion products

^of the gp 30 and _gp 60

were

applied

^{to a column} of Chromo Beads

Type

P and eluted with a

pH gradient

^{of 2.5 to}

4.5.

Fig.

^{2 shows the}

analysis

^of

14 C-lysine

labeled

tryptic peptides

of gp 60 and ³

H-lysine

labeled

tryptic peptides

^of gp 30. A

compari-

son of the

tryptic peptides

obtained from gp 60 and gp 30 shows that about 1 /3 of

peptides

of gp 30

co-migrate

^with

peptides

^of the gp 60.

This

experiment

suggests that gp 60 and gp 30

are

partially

^{related to each other. Since}

tryptic glycopeptides

^{did not bind to} the Chromobead P column we decided to

analyze

^the

tryptic

glycopeptides

of the gp 60 _{and gp} 30

by

^DEAE

cellulose

chromatography.

^{For this} purpose 3 H-fucose and !4 C-fucose labeled BLV was

reduced with

dithiothreitol, alkylated

^with

iodoacetamide and

subjected

^{to SDS-PAGE.}

The gp 60 and gp 30 were eluted from the

gel

and sialic acid residues were removed from both

glycoproteins by

^{mild acid}

hydrolysis.

Desialated gp 60 labeled with 14C-fucose and desialated _gp 30 labeled with ³H-fucose were

mixed, digested

^with

trypsin

^{and co-}

chromatographed

^on DEAE cellulose.

In

Fig.

^{3 the}

analysis

^of

tryptic glycopepti-

des of _gp 60 and gp 30

by

^ion

exchange

^chro-

matography

is shown : five

glycopeptides

^of

gp 60 and two separate

glycopeptides

^of gp 30

were resolved

by

DEAE cellulose chromato-

graphy.

^{The 3}H-labeled material of fractions

3,4

^{and 5 was not bound to} the DEAE cellu- lose.

Furthermore,

^two

glycopeptides

^of gp 30

co-migrate

^with

glycopeptides

^of gp 60. The detection of common

glycopeptides

in gp 60 and gp 30 confirms the

suggestion

^{of a}

partial

relatedness between gp 60 _{and gp} 30.

Discussion

The purpose of these studies was to exa-

mine the ^{structure of} the BLV

glycoprotein

and to resolve the

complexity

^{of this}

protein.

The results

presented

here allow us to con-

clude,

^{that two}

glycosylated polypeptide

chains

(gp

^{60 and} gp 30) ^{are linked}

by

^disulfide bonds in structures that we have

designated

as VGP I and VGP 11.

Although

the values of the calculated mol.

wts. of VGP I and VGP II must be

regarded

^as

an

approximation,

the results obtained

by

SDS PAGE under

reducing

^and

nonreducing

conditions suggest ^{a two} gp 60 two gp 30 model for VGP I and a one gp 60 one gp 30 model for VGP II. The model is confirmed

by

the

findings,

that the molar ratio _{of gp} 60 and gp 30 is

equal

ⁱⁿ VGP I and in VGP II. The

question

whether VGP I and VGP II represent the structures which form the surface

projec-

tions of BLV remains to be answered. A com-

parison

^{of the 3}

H-lysine-labeled peptides

^of

gp 60 and gp 30 shows that _gp 60 and _gp 30 share about 1/3 of the

lysine-containing pepti-

des. The resolution of a relative low number of

peptides

^of gp 60 could be

explained by

^the

fact,

^that 35 % of the total

radioactivity

of gp 60

trypsin digestion product

^{did not bind to}

the Chromobead P column. In contrast,

only

7 % of the

radioactivity

^of gp 30

trypsin diges-

tion

product appeared

^{in the flow}

through.

Since the flow

through mostly

^{consists of}

glycosylated peptides

^{(results not}

^shown),

^the

glycosylated tryptic peptides

^were

separately analyzed.

^An

analysis

^{of the}

tryptic

^fucose-

glycopeptides

demonstrated that the gp 60

trypsin digestion product

^is resolved into 5 components and the gp 30

trypsin digestion product into 2 components. Furthermore,

gp 60 and _gp 30 share two

glycopeptides.

^The

relative

amplitudes

^{of these two common} pep- tides are very similar in the _gp 60 and in the _gp 30

sample.

^The

chromatographic analysis

^of

lysine

^and

fucose-peptides provides

^evidence

for the existence of common sequences in gp 60 _{and gp} 30. A more detailed

analysis

^{will be}

necessary ^to

identify

^these

homologous regions.

The most

striking finding is,

^{that VGP} I of BLV is

composed by

^two molecules of _gp 60 and two molecules of _gp 30 held

together by

disulfide bonds. This structure is _very similar to that of an

immunoglobulin

(Cohen and

Milstein,

1967) ^or the

major histocompatibility

surface

protein

(Shreffler and

Chella,

1975).

Based on the recent

finding

^{that B-}

lymphocytes

^{are the} target cells for BLV (Paul

et

al., 1977;

Weiland and

Straub,

1975) ^we

can assume that ^{the viral}

glycoprotein

^{of BLV}

is

exposed

^on the cellular membrane of B-

lymphocytes.

The close structural

similarity

between VGP I and

immunoglobulins

may suggest that the viral

glycoprotein

^{can act on}

the surface of

B-lymphocytes

^{as an} receptor

analogous

^to

immunoglobulin

receptors ^which

play

a role in the

triggering

^of

B-lymphocytes

for

replication

^and differenciation (Vitetta and

Uhr, 1976).

Summary

The structure of the

glycoprotein

^of bovine leukemia virus (BLV) was

investigated. Analysis

^of

BLV under

nonreducing

conditions revealed two viral

glycoproteins

^with apparent molecular

weights

^of

162,000

(VGP I) and

85,000

(VGP II1. After reduction and

alkylation,

^two

glycosylated polypeptide

^{chains of} apparent ^molecular

weights

^of

60,000 (gp

^{60) and}

30,000 (gp

^{30) could be}

demonstrated in VGP I and VGP

II ;

^VGP

I appears

^to contain two molecules of _gp 60 and two molecules of _gp

30,

and VGP II consists of

only

^one molecule of gp 60 and one molecule _{of gp} 30 linked

by

^{disulfide bonds.}

Comparison

^{of the}

tryptic peptides

^of gp 60 and _gp 30

provided

evidence for the existence of _par- tial _sequence

relationship

between _gp 60 _{and gp} 30.

The structural

similarity

^{of the BLV}

glycoprotein

^{molecule to}

immunoglobulins

is discussed in

regard

^{to the}

possible

^{function of this}

protein

^{in the}

pathogenesis

^{of bovine leukemia.}

References

COHEN S., MILSTEIN C., 1967. Structure and Biological Properties ^of Immunoglobulins. ^Adv.

lmmunol., 7, 1-79.

LEAMNSON R.N., ^HALPERN M.S., ^{1976. Subunit structure of the} glycoprotein complex of a-

vian tumor virus. J. Virol., 18, ^956-968.

MADORE H.P., J.M. ENGLAND, ^1977. Rabies virus protein synthesis ^{in infected} BHK-21 cells.

J. Virol., 22, 102-112.

PAUL P.S., POMEROY K.A., ^CASTRO A.E., ^JOHNSON D.W., ^MUSCOPLAT C.C., SORENSEN D.K., 1977. Detection of bovine leukemia virus in B-lymphocytes by ^the syncytia

induction assay. J. Natl. Cancer Inst., 5, 1269-1272.

RHODE W., PAULI G., PAULSEN J., HARMS E., BAUER H., 1978. Bovine and ovine leukemia viruses. I. Characterization of viral antigens. J. Virol., 26, 159-164.

SHREFFLER D.C., ^CHELLA S.D., 1975. The H-2 major histocompatibility complex ^{and the} immune response region : ^Genetic variation, function, and organization. Adv. Immunot, 20, 125-195.

VITETTA E.S., UHR J.W., 1976. Antigen-specific receptors ^on murine bone marrow ― derived B

lymphocytes.

^{In : Cell membrane receptors for viruses,} antigens ^and antibodies, polypepti

de

hormorls

and small molecules. R.F. Beers and E.G. Bassett, eds. Miles international

Symposium Series Number 9, 303-312.

WEILAND F., ^STRAUB O.C., ^1975. Frequency of surface immunoglobulin bearing ^blood lympho- cytes in cattle affected with bovine leukosis. Res. Vet. Sci., ^{19, 100-102.}