Immunoblotting detection of lectins in gluten and white rice flour

Vol. 142, No. 3, 1987 February 13, 1987

BIOCHEMICAL

IMMUNOBLOTTING

DETECTION

OF LECTINS

Elisabeth

Jan Kolberg",

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 71J-723

IN GLUTEN AND WHITE RICE FLOUR

Wedege**,

and Ludvig

Sollid***

*Department of Immunology and **Department of Methodology, National Institute of Public Health, Geitmyrsveien 75, 0462 Oslo ***Laboratory for Immunohistochemistry and Immunopathology, The National Hospital, 0027 Oslo 1, Norway Received

December

11,

4

1986

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WGA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour. o 1987 Academic Press, Inc.

The mechanism(s) intestine

of patients

Douglas which

(1)

put

binds

theory

forward

amounts

of

VGA) (7) t and recently higher

antibody

or healthy

levels

controls

that

WGA has also

intestine

similar

same effects

through

The aim of ence

indicating

Abbreviations: dodecyl sulphate;

with

with

other

with

WGA is

biologically

the

present

study

was to get

an immunoblotting

agglutinin

the

disorders

pathogenesis changes

CD (9,lO).

of in

the

The lectin

active

after

(11).

further

evidence

technique.

CD, coeliac disease; SBL, soybean WGA, wheat germ agglutinin.

717

germ

morphological

tract

of these

CD had significantly

of WGA in

patients

pro-

small

intestinal

to induce

in man since

activity

of wheat

shown

human gastrointestinal using

patients

by gluten

contains

seen in

the

of WGA in gluten

to that

lectin

CD. This

activity gluten

and

a toxic with

show such

the small

Weiser

patients

that

an effect

been

to those

contains

not

similar patients

disease.

passage

did

demonstrated (8),

gluten in

reported

to WGA than

the

the

studies

affects

unknown.

of lectin-like

properties

rat

may have

that

gluten

(CD) is

glycoproteins

previously

with

protein

disease

by reports other

We have

a lectin

cereal

the hypothesis membrane

However,

(5,6).

the

coeliac

supported

(2.3,4).

proteins

with

to altered

was later

teins

by which

lectin;

for

the pres-

WGA has also

been

SDS, sodium

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights oJ reproduction in any form reserved.

Vol. 142, No. 3, 1987

reported

to have

like

a lectin

been

confirmed

lectins

BIOCHEMICAL

physicochemical

isolated

from

properties rice

by the extensive

(15). The rice

fractions

(17) which

determine

whether

interest

in

patients

given

symptoms

(18).

lectin are

rice

relation rice

not flour

to the flour

in

AND BIOPHYSICAL

acid

was isolated used

as human

also

contained

theories their

and sugar

(12,13,14). amino

on the gluten-free

MATERIALS

RESEARCH COMMUNICATIONS

This

foodstuff.

between (12.16)

pathogenesis remain

This

these and bran

We therefore

lectin. diet

has recently

homology

the embryo

this

specificity

similarity

sequence from

binding

wanted

would

to

be of

of CD because free

of disease

AND METHODS

Materials. WGA, N-acetyl-D-glucosamine-agarose and rabbit antibodies to soybean lectin (SBL) were from Sigma Chemical Company (USA). N-acetyl-Dgalactosamine-agarose and rabbit antibodies to WGA were from E.Y. Lab. Inc. (USA). SBL was obtained from Pharmacia (Sweden). Swine immunoglobulins to rabbit immunoglobulins were delivered by Dakopatts (Denmark). Gluten was from ICN Nutritional Biochemicals (USA). Rice bran and white rice flour were obtained from commercial mill companies in Norway. Isolation of lectins. Rice bran, 0.25 kg, was defatted in 800 ml aceton for 30 min. at room temperature, and re-extracted once after filtration on a Buchner tract. The bran was then suspended in 0.1 M Na-acetate buffer, pH 4.0, in a final volume of 1.5 1, stirred for some min. and re-extracted twice after centrifugation. Ammoniumsulphate was added to the combined extracts to 60% saturation. The precipitated proteins were dialysed against the extraction buffer and then against the buffer used for affinity chromatography. The isolation was also performed without lipid extraction of the rice bran because we observed that most of the lipids in the sulphate fraction was removed by paper filtrations and a clear solution was obtained by subsequent freezing and thawing. Lectins in rice flour was isolated by suspending 0.5 kg in 0.1 M glycine/HCl buffer, pH 1.5, in a final volume of 1.5 1. Preliminary experiments indicated a higher activity yield by extraction at this pH instead of pH 4.0. The isolation procedure was then the same as described for the rice bran. Lectins in gluten was isolated as described for rice bran. Affinity chromatography. The proteins were applied to a N-acetylglucosamine-agarose column in 0.01 M Tris/HCl containing 0.2 M NaCl, pH 8.0. The following eluants were used for the rice bran lectin: 1) Tris/HCl buffer as described above, 2) 0.2 M HAc, 3) Tris/HCl buffer as above, 4) 0.2 M HAc, 5) 0.4 M N-acetylglucosamine in Tris/HCl buffer as above. Eluants 3 and 4 were omitted for desorption of rice flour and gluten lectins. The rice flour proteins in the unbound fraction from the N-acetylglucosamine-agarose were applied to a second column with N-acetylgalactosamine-agarose and the following eluants used: 1) Tris/HCl buffer as above, 2) 0.1 M galactose in Tris/HCl buffer as above, 3) 0.1 M acetate pH 4.0 and 4) eluant 2 once more. The collected fractions were dialysed against 0.1 M acetate, pH 4.0, followed by 0.01 M HAc, and freezedried. SDS-polyacrylamide gel electrophoresis and immunoblotting. The electrophoresis was performed in a Laemmli system (19) with 1.5 mm thick slab gels containing 4% acrylamide in the stacking gel and 15% in the separating gel. 2-mercaptoethanol. ImmunoSamples were boiled for 5 min. with or without blotting was performed as described (20). No antibody binding was seen in control dot blot experiments performed either without antibodies against the Furthermore, no lectins or without peroxidase-conjugated immunoglobulins. staining was detected when normal rabbit IgG was used instead of lectin implying that the antibody binding observed on the blots were antibodies, 718

BIOCHEMICAL

Vol. 142, No. 3. 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

not caused by lectin-glycoprotein interactions. Antibodies to WGA did not react with SBL and antibodies to SBL did not react with WGA. Haemagqlutination. The tests were performed with human erythrocytes treated with papain as described previously (7). Determination of protein concentrations. The method of Lowry et al. (21) WCS used.

RESULTS AND DISCUSSION In a previous contained gluten

lectin,

with

the

get

further

carried

report

a lectin

with

isolated

out.

was demonstrated

The blots

addition

the presence (Fig.

1) showed

67

KK\

40

K-

36

K-

wheat

to that showed

to some other

of WGA in similar

gluten

protein

gluten

of WGA (7). bands

in

bands. patterns

SDS-gels

of

was anti-WGA

ANTI-SBL

*’

30K-.we. 26

K’

A

B

C

D

E

F

G

NON-REDUCED

Fig. 1. Immunoblot of lectins separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose filters and incubated with antibodies to WGA (l:lOO), lanes B-E, or antibodies to SBL (l:l,OOO). lanes F and G. The samples in lanes B-G were not treated with 2-mercaptoethanol. Antibody binding was visualized with peroxidase-conjugated swine antibodies against rabbit immunoglobulins (1:500). Lane B shows purified WGA (0.5 pg) from Sigma, lane C the gluten lectin (0.5 pg). lane D the rice bran lectin (0.5 ug), lane E the rice flour lectin (1 ug) isolated from N-acetylglucosamine-agarose. lane F purified SBL (0.5 pg) from Pharmacia and lane G the rice flour lectin (1 pg) isolated from N-acetylgalactosamine-agarose. Lane A shows blotted standard proteins (reduced with 2-mercaptoethanol) and stained with amido black. The protein concentration of the sample in lane C was determined by comparing its haemagglutinating activity to that of WGA. the sample in lane E to that of rice bran lectin and the sample in lane G to that of SBL.

719

The

In order

immunoblotting

binding

ANTI-WGA

45

the

similar

chromatography,

as WGA, in for

that

specificity

by affinity

same mobility evidence

it

a sugar

to

Vol. 142, No. 3, 1987

BIOCHEMICAL

94

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

K

67K

=(I

43

K

30K

-

20

K

14K

am

1

2

3

4

REDUCED

5

NON-REDUCED

Fig. 2. SDS-gel electrophoresis. Samples in lane l-3 were treated with 2mercaptoethsnol, whereas those in lane h-5 were untreated. Lane 1 shows standard proteins, lane 2 and 4 purified WGA (6 pg) from Sigma, lane 3 and 5 rice bran lectin (18 ng) purified on N-acetylglucosamine-agarose. to purified

WGA and the

together

with

the

indicate

that

gluten

affected

by reduction

bound

less In

ported

in

a contamination

other

hand,

detected are

from

the found

latter

whether

after

in

the

protein low

members not

of the while

used

rice

flour

from

electrophoresis

(Fig.

is

re-

the

the

flour

of a WGA-

and might

repres-

process. which

On the

has not

(Gramineae).

of rice

flour

been

We were also

bran

2) after

720

therefore

About

tolerated

contains

a lectin

interested

contained this and was isolated with

Wheat pro-

are

as human foodstuff,

protein,

100 g rice

Our finding

endosperm

same family those

on N-acetylglucosamine-agarose.

was isolated

whereas

the milling

the

it

concentration.

to WGA (12.17).

as a reference

in

but milling

surprising

during

WGA

treatment.

industrial

as gluten.

was

against

portion,

fraction,

therefore

proteins

of its

After

(7)

lectins

lectins

antibodies

embryo

germ

such is

since

the

(22).

wheat

proteins

these

of the

mercaptoethanol

in

endosperm

results

between

bonds

found

fraction

is

white

was used

50%. SDS-gel

are

which

related

chromatography lectin

WGA is

to CD patients,

bran,

structurally vestigate

proteins

because

and rice

(18). Rice

lectin

to the

WGA may be a natural

toxic

similarities

disulphide

grain

immunoblotting

WGA. The antigenicity

of embryonal

earlier

Wheat teins

reported

endosperm the

These

of the

are

contains lectin

lectin.

contains

wheat

proteins

fraction ent

dry

to be absent

embryonal like

previous

distinctly

the

gluten

to in-

lectin. The rice by affinity 2.5

an activity mercaptoethanol

bran

mg of purified yield

of about

treatment

of the

Vol.

142,

BIOCHEMICAL

No. 3, 1987

proteins

revealed

lectin

showed

This

one band

one band

supports

the

ally

cleaved

probably

chain

into

one of these

rice

WGA starting

at its

mercaptoethanol

chains

bounds

(M

(23).

in absence

It

is

like

different

no.

folding

by a procedure

blotting

analysis

WGA showed 1). ein

the

bands

(Fig.

amount

pattern

used

of

2). in

rice

lectins

were

specific

contained 0.025

latter

bran

a rice

bran-like

to that

of the

antibody

levels

healthy

controls

support

the

proposal

(1) * ion of rice

flour

both

the passage

lectins

contains

rice

molecules

with

bran

to the

(Fig.

blue

revealed

showed

bands,

its

against

much higher

lectin

in

finding

that

Immuno-

lectin,

Coomassie

flour

rice

the

prot-

a broad

the

same

that

M

both

white

rice

flour

haemagglutinating

we found

that

it

l/100

of the

corresponded amount

to

isolated

by

with

CD had significantly intestinal

disorders

or

and the

detection

of WGA in

gluten

may be involved

lectin

of WGA in

cause

the

are

pathogenesis

unaffected

able

that to elicit

lectin

intestinal are

in rice might

be

permeability

antigenic. immune

of CD

by ingest-

CD. One possiblity

increased

molecules are

in

diet

of a WGA-related

role

higher

other

demonstration

that

the rice

and the

on gluten-free

of larger

white lectin.

antibodies

suggested

patients

observation

the

a

bran.

pathogenetic

WGA and the

permits

gluten

(18),

WGA which

indicate

bran

distinct

This,

was about

that

as CD patients

questions

which

that

1).

patients

This

(8).

chain

with with

By comparing

rice

shown

to WGA than

However,

flour

from

of

than

M

rice

from

rice

for

The rice

lectin,

which

the

was related

same sugar bran

g flour

previously

the

without

lectin.

chromatography We have

proteins

(Fig.

rice

of

disulphide

had higher

was isolated

rice

method.

the

bands

to a part

and might

of the SDS-gel

lectin

M

sequenEe

intrachain

chains

for

activity

for

mg lectin/lOO

affinity

that

the

The low

identical

concerns

used

discrepancy

binding

as the

rice

37 and 40.000)

This

antibody

region

activity

(M

only

lectin

staiiing

(15). terminal

chains.

to that

bands

protein

synthesi:ed

M of WGA in absence of r 2) was unexpected because the two

treatment

the non-reduced

two main

whereas

three

of

M .

The higher

specially

specific similar

size

to be almost

why the

polypeptide

bran

of lower

originally

The amino

mercaptoethanol

of these

rice

band

as WGA, and post-translation-

contain

This

agent.

A N-acetylglucosamine flour

18,000

is

equal

shown

95 (15).

uncfear

WGA after

lectin

chains.

24,00O)(Fig.

of M

also

rice

COMMUNICATIONS

the

and a broad

of about

has been

RESEARCH

WGA, whereas

same length

cleaved

about

of reducing

migrated

the

residue

WGA subu%ts

the

chains

these

for

mobility

that

with

BIOPHYSICAL

18,000

similar

smaller

represented

identical

of M

with

assumption

as one polypeptide

AND

However,

responses

only

leading

to CD. The unbound amine-agarose

were

rice

flour

found

proteins to contain

after

chromatography

an agglutinin.

721

Sugar

on

N-acetylglucos-

inhibition

analyses

r

Vol. 142, No. 3, 1987

BIOCHEMICAL

showed that this agglutinin

AND BIOPHYSICAL

was specific

RESEARCH COMMUNICATIONS

for N-acetylgalactosamine.

The

fraction

was therefore applied to an affinity column with the latter sugar The eluted lectin was compared to soybean lectin (SBL) which as ligand. also has a specificity for this sugar. Immunoblots (Fig. 1) demonstrated binding of anti-SBL to proteins

from rice flour with the same electrophoretic mobilities as purified SBL. These findings probably indicate that the tested batch of commercial rice flour was contaminated with soybean oil meal. We isolated about 0.2 ug SBL/lOO g rice flour. A yield of 160 mg SBL/lOO g soybean meal proteins has been reported (24). These values would correspond to a contamination

of about 0.1 mg soybean proteins/100

g rice

flour.

ACKNOWLEDGMENTS. We thank Torunn Mellum and Irena Juniszewski for skilful technical assistance.

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