Immunochemical studies on dextrans: Distribution of α1→2 and α1→6 specific determinants of dextran NRRL B1397

BIOCHEMICAL

Vol. 73, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IMMUNOCHEMICAL STUDIES ON DEXTRANS: DISTRIBUTION SPECIFIC Keiko

OF al-t:!

AND al+6

DETERMINANTS OF DEXTRAN NRRL El397

Sakakibara,

Mitsuo

Torii,

and Akira

Blesila

P. Albert0

Misaki*

Department of Immunology, Research Institute for Microbial Diseases, Osaka University, Yamada-kami, Suita, Osaka 565, and Faculty of Osaka City University, Sumiyoshi-ku, Osaka 558, Science of Living*, JAPAN

Received

September

21,1976

SUMMARY: A rabbit anti-dextran serum was separated into two fractions, specific for cl+2 and al-+6 glucose linkages, respectively. Dextran NRRL B1397 containing al+2 and al+6 specific determinants was fractionated by step-wise precipitation with the al+2 specific antibody fraction to see whether the cl-t2 and ul+6 specific determinants were both in the same molecule. Most of the dextran fractions were shown to have two specificities by quantitative inhibition experiments with kojibiose and isomaltose as inhibitors. The precipitin lines in aqar of dextran fractions with antibody fractions specific to cl+2 and al+6 fused. These results strongly suggest that these two antiqenic determinants are present in the same molecule. Chemical indicated

studies

that

C-2 are mainly than

directed

units

to al+2

fractionated

this

different

contents

with

fractions concanavalin fractions whether dextran

glucose

concanavalin in C-2 branches

had both

molecules, This antiboty

with

A and a homologous reacted

slightly

Inhibition

and al+6

study

determinants

we fractionated to examine

and whether reports

fraction,

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

both

this the

fractionation

Recently, we A (3). Several

concanavalin rich

of C-3 branches in C-2 branches

anti-dextran or not

serum, at all

but reacted

whereas

with

showed

that

the

However, we did not determine were both present in the same dextran

by step-wise

addition

of determinants

are present

on the preparation of dextran

741

of more

(2).

contents

distribution

determinants

experiments

composed

at

by injecting

experiments

specificity.

specific

antibody, paper

Fractions

have the branches

had two specificities

respectively

had similar

antiserum.

al+2

molecule. In the present

dextran

all

points;

prepared

rabbits

linkages,

of C-2 branches.

A or the

NRRL 81397

at C-3 are

sera into

by interaction which

the al-+2 and al+6

molecule.

Copyright All rights

dextran

obtained

dextran

and those

Anti-dextran

both

specific

units

NRRL B1397

poor

of al+2

specific

(1).

and al+6

were

branched

has C-2 and C-3 branching

D-glucose

mesenteroides

fractions well

dextran

single

two glucose

Leuconostoc

on the highly

this

in the

in the same

of the al+2

B1397 by interaction

Vol. 73, No. 3,1976

with

the

BIOCHEMICAL

specific

antibody

AND BIOPHYSICAL

and chemical

RESEARCH COMMUNICATIONS

and immunochemical

analyses

of the

fractions. MATERIALS

AND METHODS

Dextran 81397 prepared from Leuconostoc mesenteroides NRRL Bl397 was a generous gift from Meito Sangyo Co., Ltd., Nagoya, Japan. The bacterial strain was kindly provided by Dr. A. R. Jeanes, Northern Regional Research Laboratories, U. S. Department of Agriculture, Peoria, Illinois, U. S. A. Dextran B512 was also supplied by Dr. A. R. Jeanes. Dextran 2000 was a commercial product from Pharmacia (Uppsala, Sweden). Kojibiose (O-a-D-glucopyranosyl-(1+2)-D-glucose), isomaltose (O-cc-D-glucopyranosyl-(1+6)-D-glucose) and isomaltooligosaccharides up to heptaose were the preparations described previously (4, 5). Methyl u-D-glucopyranoside was a commercial product from Pierce (Rockford, Illinois, U. S. A.). Rabbits were injected with &. mesenteroides NRRL B1397 suspension as described previously (2). Seven days after the last injection, blood was taken and antisera were prepared by the usual method. The two sera obtained Anti B1397 (156~) (2) was also used. (199D and 204D) were combined (Pool 1). Quantitative precipitation and inhibition assays were performed in the Antibody nitrogen in the specific precipitates was determined usual way (6). by the ninhydrin method (7). Generally, quantities of serum which gave 3 to The 5 ug of precipitable antibody N were used for inhibition assays. precipitin reaction in agar was carried out by Ouchterlony's double diffusion technique with Bacto Special Agar Noble (8). Periodate oxidation and methylation studies were performed as described Sugar was measured by the phenol-sulfuric acid method (9). previously (3).

E t $ N

14 13 JJ 1.

Pl P2

8 5 al 0,5? z

Ia 0

I.__:ii._...._, 10

20

30

40 TUBE

50 60 NUMBER

70

80

90 -100

Figure 1. Fractionation of rabbit anti-dextran pool 1 into two distinct The arrow indicates change from sodium aside-saline to 1 M glucose fractions. Fractions of 20 ml were solution in sodium azide-saline for elution. collected at a flow rate of 30 ml per hour.

RESULTS Fractionation ml) was dialyzed

of Anti-dextran against

sodium

B1397 aside-saline

(Pool

1).

Anti-dextran

Pool

1 (20

(NaN3 0.02 %, NaCl 0.9 %) and The column was eluted first applied to a column of Sephadex G75 (2 x 30 cm). with sodium azide-saline (1.1 l), and then with 1 M glucose solution in 742

BIOCHEMICAL

Vol. 73, No. 3, 1976

AND BIOPHYSICAL

0.01

0.001

0.1 " INHIBITOR

pmole

RESEARCH COMMUNICATIONS

1 ADDED

Figure 2. Inhibition by oligosaccharides of precipitation of fraction Pl and P2 by dextrans B1397 and B512. a, Pl and B1397; b, P2 and B1397; c, P2 and B512; 8, kojibiose; glucose; A , isomaltose; A, isomaltotriose; ?! lsomaltopentaose; , isomaltotetraose; 0, isomaltohexaose; isomaltoheptaose; k 8 , cc-methyl D-glucopyranoside. sodium

azide-saline

and P2) were was removed

1).

eluted.

As shown

The very small by chromatography on the

to be contaminated separately

(0.9

only

with single

Pl. peaks

in Fig.

1, two peaks

of material

(Pl

amount of P2 contaminating fraction PI same column. Fraction P2 was shown not

On rechromatography of fractions of Pl and P2, respectively, were 743

Pl and P2 obtained

Vol. 73, No. 3,1976

suggesting

BJOCHEMICAL

that

these

containing these sodium azide-saline

Pl and P2.

11) only

of the

two

greatly

was

However,

precipitated

assays

Pl

showed

The precipitations

more strongly

inhibited showing

Fraction

Pl.

stepwise

by successive

14, 14,

14,

separated saline

Dextran

14 and 7 ml)

and treated

after

the were

fractions

shaken

with

ether

The dextran were:

it

B1397 with

showed

al+2 P2 were

both

than

by

at 4'C.

treated

mixtures

were

and dialyzed contents

of the

A 9.1 mg, B 26.6

of fraction excess

The final with

Pl

was

supematant acid.

and the

superna-

The dialyzed

water.

Sup in order

fractions

(7,

1 M glucose-

trichloroacetic

centrifuged,

against

Antibody

Each precipitate

by adding

acid.

was also

Specific

was precipitated amounts

in water

named A, B, C, D, E, F and the

precipitation.

specificities

and B512 with

with

increasing

10 % trichloroacetic

acid-treated

were

determination

with

6 to 8 days each

precipitations

that

B1397

ml of saline)

dissolved

with

serial

The trichloroacetic tants

for

precipitation of dextran

by Precipitation

(100 mg/50

by centrifugation,

ci1+6 specificity

the serological

indicating

both

specificity.

B1397

incubation

with

and isomaltooligosaccharides

P2 had cl-+6 B1397

has only

well

Quantitative

of dextrans

of Dextran

Quantitative

reacted

2, precipitation

by isomaltose

that

Fractionation

~2.

to determine

As seen in Fig. by kojibiose,

inhibited

specificity. kojibiose,

out

B1397

The fractions against

dialyzed

Pl and P2.

B512 which

fractign

carried

materials.

to 20 ml,

dextran

dextran

with

were

fractions.

adjusted

that

RESEARCH COMMUNICATIONS

different

of Fractions

experiments

inhibition

each

SpeCifiCitie.5

Precipitation fraction

contained

materials were and stored.

SerOlOgiCal

(10,

fractions

AND BIOPHYSICAL

of their

estimated

by glucose

mg, C 7.2 mg, D 7.2 mg, E 2.6 mg, F 0.5

mg and Sup 3.2 mg. Periodate 1'2

Oxidation

and Methylation

and l-+3 branching

oxidation

points

and methylation

The results

are

chromatography separated,

shown

branch

analyses in Tables

2,4-dimethyl

from that points

whereas

the

(4.48

to 5.95

8.34

contents

using

were

glucose

points

of total in

branches. fraction

and 7.40

%) except

the Sup

were

(3). gas not

by subtracting The linkages. the content

of 1+3

As seen A (26.29

in Table 2, the content of l-t2 in fractions B, %), intermediate

%, respectively)

of 1+3 branch

previously used for

derivatives

was calculated

of

by periodate

the methods described Under the conditions

1 and 2.

of l-+3 branch

determined

1+3 linkages from that of total l-t3 points was calculated by subtracting

was high

C and D (9.70,

fractions

and 3,4-dimethyl

so the content

the content of linear content of l-+2 branch branches

in the

The contents

of the Fractions.

points (9.97

%).

744

and low were

similar

in the in all

Sup (1.78 the

%),

fractions

BIOCHEMICAL

Vol. 73, No. 3,1976

Table

1.

O-Methyl

Molar

Ratios

glucose

of Methyl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sugar Components Fractions

2,3,4,6-Tetra-

2,4,6-Tri-

in Methylated

2,3,4-Tri3,4-

Linkage

indicated

Glcp*

+3Glcpl+

Dextran

Mixture of and 2,4-di-

+6Glcpl+,

-+GGlcpl+

'6Glcpl'

+2

Dextran:

Table

2.

Fraction

Original

1.00

0.05

A

1.00

0.10

1.15

0.96

B

1.00

0.05

4.11

0.93

C

1.00

0.03

4.97

1.15

D

1.00

0.03

5.99

0.93

SUP

1.00

0.0-l

6.44

0.97

Percentages

Branch

Original

pointa

1+3 Linkageb

1+3 Branch

%

Dextran

pointC

Fractions

1+2 Branch

8 5.72

12.66

A

30.77

7.56

4.48

26.29

B

14.66

5.69

4.96

9.70

C

14.29

6.38

5.95

8.34

D

12.47

5.44

5.07

7.40

9.97

1.78

E

5.56

F

7.64

11.75

10.79

Calculated from the quantity of non-reducing terminal by methylation analysis. Glucose residues resistant to periodate oxidation. Calculated as (b - percentage of linear l-*3 linkages). Calculated as (a - c).

Precipitation

Quantitative dand P2 reactivities of the shown in Fig.

dextran,

and slightly

3.

of Dextran

precipitation fractions Pl reacted less with

Fractions

experiments with

serum

fractions

were

with

groups

estimated

Serum Fractions

performed

to examine

Pl and P2.

The results

more with fraction A than with fraction B than with the original

745

pointd

%

6.64

Quantitative

are

in the

0.88

18.38

SUP

b c d

3.39

of l-+2 and l-t3 Linkages

%

a

‘3

the original dextran.

Pl the

Vol. 73, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

‘&if& 20

0

40

60

,ug

DEXTRAN

ADDED

)rg

DEXTRAN

ADDED--

60 _.

-Figure 3. Quantitative precipitin curves of serum fractions Pl and P2 by dextran fractions. a, Pl (20 ~1); b, P2 (14 ~1); 0, original; a, fraction A; X, fraction B; A, fraction C; A, fraction D; 0, fraction E; a, fraction F; a, fraction Sup. The precipitating order Fig.

abilities

and the 3a.

were

similar

were

different. Quantitative

specificities

final

With

of fractions

fraction

Sup did

P2, the precipitation

with

those The

with

Pl,

Sup precipitated

Inhibition

C, D, E and not

whereas with

of Precipitation.

of the precipitations

give

curves

decreased

any precipitate, of fractions

those

of fraction

fractions

in this as seen in

A, B, C, D and E F and the

P2 although it did To characterize

of the dextran

746

F

not with the with

Sup Pl.

Pl and P2,

BIOCHEMICAL

Vol. 73, No. 3,1976

Table

Inhibition Fractions

3.

Dextran

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by Two Disaccharides of Precipitation of Dextran with Two Specific Serum Fractions Pl and P2

fraction

With

al+2

specific

Kojibiose

With

Pl

Kojibiose

Isomaltose

specific

P2

Isomaltose

la

153a

la

0.34a

A

1

318

1

0.32

B

1

78

1

0.37

C

1

82

1

0.31

D

1

63

1

0.21

E

1

97 1

0.22

Original

SUP a

al+6

Relative inhibition.

amount

of disaccharide

inhibitor

required

for

50 %

Figure 4. Precipitin reaction in agar. Pl and P2, serum fractions Pl and 156D, anti-dextran B1397; B, dextran fraction B; S, saline; p2 I respectively; 2000, dextran 2000.

quantitative points

precipitation

indicated

have

two specificities

were

used as inhibitors.

more inhibitory fractions isomaltose fractions that

the

precipitation

inhibition

by arrows (Z),

than

inhibitor

determinants

3, with

the

systems,

specific than specific

for

fractions

A, B, C and D, which

with

antibodies,

al+2

for

kojibiose

747

kojibiose PI,

were

out

and isomaltose

kojibiose

indicating linkage.

at the shown to

was much

indicating that the al-t2 linkage.

the al+6

had antigenic

carried

was previously

two disaccharides

in all

determinants

were

The dextran

As seen in Table

was a stronger contained

3.

so the

isomaltose

had antigenic

four

experiments

in Fig.

that

all

the

With the

P2,

The fact

specifically

separated

determinants

specific

by for

Vol. 73, No. 3,1976

the al+6

BIOCHEMICAL

linkage

two distinct

suggests

antigenic

the double

technique

Precipitation dextran

B512,

is

4a).

between

dextran

results

were

obtained

The latter

dextran

4.

specificity

between with

the two antigenic

fused

B and antiserum

antiserum

other

with

determinants

by

with

B

fraction

was derived

from

11).

The

line

between

(156D)

fused

(Fig.

in the

Precipitation

the

A, C and D.

are both

had

(10, with

a spur

fractions

fractions

this.

The results

to have al+6

2000 and the obtained

to confirm

B and Pl completely

The line

in these

in the same molecule.

in Agar.

known

between

RESEARCH COMMUNICATIONS

molecules

was performed

shown in Fig.

which

line

P2 (Fig.

that

Reaction

2000 are

precipitin

the dextran

determinants

diffusion

and dextran

that

AND BIOPHYSICAL

with

4b).

B and

the line

Similar

These

results

indicate

same molecule.

DISCUSSION The following

structure

has been

proposed

for

'6Glcpl+6Glcpl+6Glcpl'-----+6Glcpl+6Glcpl+ 2 4

chains

joined

are mostly,

linkages

may be longer

glucose

residues.

Previously interaction in

than 1'2

side

properties.

on the

nature first

highest all

content

with

this

dextran

fractions

that

previous Fractions

specificities

al+2

molecules

of dextran

antigenic

determinants.

experiments,

as shown

D-

for

al+2

study

few

to differ

serological

sample

was composed

showed

the hetero-

A which

precipitated

fraction, (1.78

by

Pl,

contained did

the

not

%) l-+2 branches.

However,

to 5.95 %) of 1+3 branches, on l-t3 branches coincide well

(3).

A to D, which for

by al+3

of three

found

and the Sup which

had similar contents (4.48 results %). The latter

findings

were in their

Fraction

antibody

the Sup (9.97

of the

joined

fractions

the dextran

The present

a very

those responsible

fractions

more clearly.

only

quarters

consist

several

and also

%) of l-t2 branches

Pl contained

whereas

apparently

These

specific

three

specificity. into

points

indicated

of cl+2

long,

are

al-+6

A (3).

occupy

and probably

chains for

dextran

(26.29

with

the

except

one unit

population.

of this addition

precipitate

side

findings

of a microheterogeneous geneous

all,

of C-2 branching

These

which

linkages,

chains

concanavalin

contents

(1):

3 4

two residues,

we separated

with

their

not

Thus

and l-t3

specificity,

by al+2 if

branches,

NRRL B1397

Gl~pl+6Gl~pl+6Gl~p

Glcp The side

dextran

precipitated with al+2 specific antibody, as shown by inhibition experiments.

and a1+6, in

fractions A to D each seem to contain This was confirmed by double diffusion

in Fig.

4. 748

had So, the

two distinct precipitation

BIOCHEMICAL

Vol. 73, No. 3,1976

The precipitation for

curves

fractionation

P2 were

of the dextran

of the dextran

unexpected

(Fig.

sample

fraction

3):

and contained

the highest

and fractions

B, C, D and E, which

this for

order,

also

these

or not were

at all

not

content

showed

results

the

in unknown.

with

because

The precipitations (Fig.

combining

This site

antibodies than

triose not

of P2 is

binding

eluted

precipitated

with

Heterogeneous

populations

precipitation,

chromatography fractions

were

heterogeneous genie

not

properties.

geneous

studied.

nature

would

polysaccharides

heterogeneous

are produced

antibodies have been

if

any,

which

fraction

Pl and P2. demonstrated

of these

and present

B1397 was studied

be interesting

116 specific

structures,

and concanavalin

antigenicities

In the previous

of dextran It

the

antibody of 1+6

oligosaccharides

that

in both

dextrans

interaction

However,

1+6 specific Elution

possible

most

and -heptaose

to larger

although

recovered

of native acid

is

studies

inhibited

to such large

solution,

mostly

boric

(13-15).

So, it

P2,

available.

both

of the

Pl

slightly

Further

P2.

-hexaose

complementary

complementary

1 M glucose

B1397 were

P2 were

with

with

with Pl in The reason

P2. reacted

of samples

-pentaose,

those

most with

reactivities

with

was used

easily

reacted

to isomaltotriose.

were (12).

sites

with

alcohol

amounts

and B512 with

sites

However,

also

curves

the maximum size

has been reported with

precipitin

which

most

of reactivities with F and the Sup which

complementary

combining

Pl,

precipitated

decreasing

-tetraose, that

with

as expected.

branches

showed

limited

of B1397 suggests

whose

antibodies were

of the

by isomaltotriose,

2bc).

were

same order Fraction

RESEARCH COMMUNICATIONS

fractions

A which

of al+2

Pl gave unique

possible

and equally

AND BIOPHYSICAL

work

in relation

by

A-Sepharose dextran the microto the

anti-

to know why and how such hetero-

by microbes,

and the

role

of these

biopolymers.

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9.

Miyaji, H., Misaki, A. and Torii, M. (1973) Carbohyd. Res., 2, 277-287. Torii, M., Sakakibara, K. and Kabat, E. A. (1973) J. Immunol., 110, 951958. Torii, M., Sakakibara, K., Alberto, B. P. and Misaki, A. (1976) Biochem. Biophys. Res. Conunun., in press. Sakakibara, K., Torii, M., Misaki, A. and Miyaji, H. (19721 Carbohyd. Res., 2, 443-451. Torii, M. and Sakakibara, K. (1974) J. Chromatogr., 96, 255-257. Kabat, E. A. (1961) Kabat and Mayer's Experimental Immunochemistry, 2nd ed, pp. 22-95, Charles C. Thomas, Springfield, Illinois. Schiffman, G., Kabat, E. A. and Thompson, W. (1964) Biochemistry, 2, 113120. Ouchterlony, ii. (1949) Acta Pathol. Microbial. Stand., 26, 507-515. Dubois, M., Gilles, K., Hamilton, J. K., Rebers, P. A. and Smith, F. (1951) Nature, 168, 167.

749

Vol. 73, No. 3, 1976

10. 11. 12. 13. 14. 15. 16.

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Kabat, E. A. and Berg, D. (1953) J. Immunol., 2, 514-532. Kabat, E. A. and Bezer, A. E. (1958) Arch. Biochem. Biophys., 78, 306318. Schlossman, S. F. and Kabat, E. A. (1962) J. Exp. Med., 116, 535-552. Arond, L. H. and Frank, H. P. (1954) J. Physical Chem. z, 953-957. Wilham, C. A., Alexander, B. H. and Jeanes, A. (1955) Arch. Biochem. Biophys., 2, 61-75. Kobayashi, M., Shishido, K., Kikuchi, T. and Matsuda, K. (1973) Agr. Biol. Chem., 37, 357-365. Kennedy, J. F. and Rosevear, A. (1973) J. Chem. Sot. Perkin Trans. I, 19, 2041-2046.

750