Isolation of colitose-containing oligosaccharides from the cell wall lipopolysaccharide of Escherichiacoli

Vol.

21,

No.

6, 1965

BIOCHEMICAL

ISOLATION

OF

THE

WALL

FROM

CELL

AND

D.

Edstrom’

Received

November

The the

Hopkins

cell

case

of --E. coli

0111-84

biosynthetic

studies

sequentially

in the 1964).

a procedure

which

polymer.

The

contains

suggested order;

stantial

yield,

method

Gal (6,750

isolated

cprrjp from

I. 2.

was

the

the

Olll+4

(1959),

produces

of the

polymer

Gal

antigenic

yet

when

the

component

been

established

Heath,

4/6

radioactive

not

formation

approximately

require from

the the

However,

by

cd

in sub-

0111-84.

established,

Although the

results

I.

was

radioactive

(Edstrom

to isolate,

LPS of:.

as follows

:

of an equivalent

of LPS isolated and

colitose

precluded

possible

detectable

(c)

colitose

been

the

transferred

oligosaccharides

the

LPS,

polysaccharide

it has now

of Gal

product

and

hydrolysis.

Figure

with

were

acid

have

3-6

incomplete

glucosamine,

from

in

(3,6-dideoxy-L-

by partial

in

(Elbein

polymer; only

colitose

bound

which

3-deoxyoctulosonate,

which

glycosidically

outlined

degraded

as the only

and

on the

oligosaccharides

destroys

of --E. coli

was

N-acetyl

carbons

mo I e ) o f w h ic h was

Gal

glucose,

(LPS)

hexosamine,

of colitose-containing

are

from

(b)

untreated

constituents

et -- al.

of the

totally

threitol

sugar

of the

analyses

product;

the

golactose

oligosaccharides

of threitol

mutant

I-C14-Gal, threitol

details

procedure

contained

phosphorus,

oligosaccharides

structural

merase-less

Maryland

a lipopofysaccharide

mutant

isolation

colitose-containing

in the

acids,

determinations

of Goldstein

structural

threitol

the

acid-lability

the

degradation

that

the

utilizing

origin

Baltimore,

contain

glucose,

structural

permits

of colitose-containing

The

’

Heath

of Medicine,

fatty

galactose,

Precise

extreme

of preliminary

C.

COLI.

Chemistry

a UDP-gal-4-epimerase-less

isolation

complete

Edward

bacteria

heptose,

Using

Heath,

School

of Gram-negative

0-phosphorylethanolamine,

and

and

ESCHERICHIA

23, 1965

walls

xylohexose).

OF

of Physiological

University

COMMUNICATIONS

OLIGOSACCHARIDES

LIPOPOLYSACCHARIDE

Department Johns

RESEARCH

COLITOSE-CONTAINING

Ronald

The

BIOPHYSICAL

from

1965)

that

component,

of that preceding of the

of Gal experiment LPS and,

(a)

the

amount

of

a UDP-gal-depiwas

grown the

(U,OOO

on U-C

specific

I4

activity

cphmole) was performed

after

degradation,

unlabeled.

Supported by grants (AM-88318 and AM-06278) f rom the National Institutes of Health and a grant from the National Cystic Fibrosis Research Foundation. U.S.P.H.S. Postdoctoral Fellow. Present address, Department of Biochemistry Medical School, University of Minnesota, Minneapolis, Minnesota.

638

-

with

Vol.

21, No.

BIOCHEMICAL

6, 1965

AND

BlOPHYSlCAL

RESEARCH

COMMUNICATIONS

NaBH4 Hydrolysis

(V)

GlcNAc-Glc-Threitol

a-Glc-ase Glc

Figure

I:

Summary

Material in

and

of oligosaccharide

2 N HCI

for

2 hours.

N H2S04

for

previously

described

tography for

are

Descending

systems

(4:4:2);

5

ethyl

alkaline

were

silver

nitrate

sugars was

and

used:

Authentic Health.

4 soluble

form

D-threitol

method

with

(2:1:2). (Trevelyan

was a gift

hydrolysis

of the

were

determined was

(1959).

isolated

from No.

(8:2);

water;

i,

after sample

by paper

chroma-

values

presented

same

1 paper.

in 0.2 as

The the

hydrolysis

quantitatively

isolated

of Korn

chromatogram. The

following

butane-I-oI:pyridinewater

_D, butane-I-oI:propane-2-olwater

Sugars

on chromatograms

et c- al.,

1950)

or periodate-benzidine

---LPS

was

described

by treatment

determined

Whatman

A_, propane-2-olrwater with

were

Threitol’

to glucose

performed

saturated

as previously

sugars

by the

of Lipopolysaccharide 0111-B

after

1965).

compared was

colitose

determined

Heath,

determined

as ratios

except

Individual

100”.

acetate:pyridinerwater

to a more

3.

A> and

butane-l-01

Preparation E. coli --

at

chromatography

solvent

&,

bound

(Elbein

given

degradation

Colitose

20 minutes

(Solvent

threitol

---All

Methods

+ Threitol

isolated

(Elbein

and

with

alkali

from

Dr.

prior

N.

639

K.

from Heath,

(1:7:2);

were

(Gordon

with --et al.,

washed

cell

wall

preparations

1965).

The

product

was

to degradation.

Richtmyer

detected

of the

One

National

gram

1956). of

converted

of LPS was

Institutes

of

Vol. 21, No. 6, 1965

dissolved solution

BIOCHEMICAL

in 200 ml of 0.2 N NaOH to pH 5 with glacial

790 mg of powder

which

RESEARCH COMMUNICATIONS

and heated at 60’ for 30 min.

acetic

contained

AND BIOPHYSICAL

acid,

precipitation

After-adjusting

the

with 6 volumes of ethanol

85% of the colitose

originally

yielded

present in the untreated

LPS. Degradation

of LPS--- To 410 mg of alkali-treated

LPS (Fraction3

in 50 ml of water Afer

justed to pH 6.3) were added 60 ml of 0.1 M sodium periodate. at 2S”, 3.35 The solution

ml (60 mmoles) of ethylene was cooled

the mixture

was maintained

room temperature remaining distilled

to 4’,

16 hours in the dark

were added to destroy the excess periodate.

4.3 g (120 mmoles) of sodium borohydride

at 4” for 2 hours.

for an additional

sodium borohydride. water;

glycol

2 hours. The solution

the water was changed

twice

(ad-

The solution Acetone

were added,

was then permitted

and

to stand at

(20 ml) was added to destroy the

was dialyzed

for 3 days against

The dialyzed

daily.

material

100 volumes of was concentrated

to 3 ml -in vacua and adjusted to pH 2.0 with 2 N H2S04. After heating at 100” for 20 minutes, the solution was centrifuged to remove the slight precipitate, adjusted to pH 9 with

NH40H

containing

and applied fractions

to a column

were eluted

from the column between colitose

--Fraction

II) and concentrated

between to 3 ml.

threitol.

The identity

paper with solvent!

of the threitol e_t Ql.,

(96 pmoles) containing

500 ml and 640 ml; these fractions

the presence

bound

were pooled

of an acid hydrolysate

of colitose,

was confirmed

emerged

glucosamine,

by chromatography

of

glucosepnd

on borate

treated

threitol,

I .50; and the polyol

R values were: glycerol, 3.86; erythritol, 9lc from the oligosaccharide, 1.48. Quantitative analysis of

--Fraction

11 (Table

that all of the constituents

equimolar

(Cabib

Two colitose-

A peak of material

Paper chromatography

II in solvent ,A and B_indicated

G-25.

Free colitose

with 0.015 N NH40H.

660 ml and 740 ml of eluate.

(123 pmoles) was eluted

(Fraction

(4.3 x 53 cm) of Sephadex4

I) indicated

quantities;

the values obtained

than the other sugars, suggesting saccharides.

However,

in solvents A through material

actually

p-N-Acetyl

of a mixture

glucosaminidase

colitose)

was treated

supplied

by Dr. H. Heymann,

4.

Pharmacia

below,

GlcNAc, Treatment

with P-N-acetyl

Fine Chemicals

except

colitose

in this fraction

were consistently

higher

of oligo-

by paper chromatography

of --Fraction II showed that this

study of -Fraction of a tetrasaccharide (Col, GlcNAc, Glc,

1.98;

were present in

was composed of a mixture

was not resolved

E. As indicated

(Co12,

for colitose

that the fraction

this mixture

consisted

and a pentasacchrzide

1953).

further

Glc,

threitol)

threitol).

of Fraction

II ---A

glucosaminidase

(Findlay

Ciba Pharmaceutiaal

Co.,

Inc.,

N.J.

New Market,

640

portion

Summit,

of Fraction II (41 pmoles -and Levvy, 1960~kindly N.J.).

in 20 hours at

11

Vol.

21,

370,

No.

70%

6, 1965

of the

no further column The

BIOCHEMICAL

N-acetyl

liberation (I.5

glucosamine

of

x II0 cm)

N-acetyl

III,

bound

calitose.

N-acetyl

Another

of these

containing

The

moles

threitol.

Since

the

acetyl

glucosaminidase

acetyl

glucosamine

eluted

the

of colitose, N-acetyl

been

removed

suggesting

5.

Bio-Rad

by

and

developed

contained

Fraction

proposed

that

22 pmoles

of free

one

I) and

the

was colitose

of

elution

a pentasaccharide

glucosamine,

pentasaccharide

2).

peaks:

23 pmoles

awas

of N-acetyl

(Figure

contained

u(Table

in

to a

water

distinct

IV

resulted

was applied with

in two

fland

each

enzyme

mixture

Fraction

column

in the

it was

i-%1

glucose

resistant

was

and

to 8-N-

attached

to the

N-

14!lN-ACETYL-

Einstein

College acid

that the colitose

GLUdSAMlNE fl,

A

equal to the

Laboratories,

mole

of more

column

that

t

resistant

mild

the

the

i’ I I 120 140 MILLILITERS

of products of &N-acetyl P-2, Fraction Ill,

IV contained

Albert

was

indicated

one

E;xhiin 11, 80 100

Osborn,

column

COMMUNICATIONS

oligosaccharide.

0.4

wos

incubation

from

from

glucosamine

r

saccharide

The

colitose

gel,

0.5

p-Fraction

the

RESEARCH

Addition

of Fractions

and

treatment,

Figure 2: Separation on a column of B&Gel through 158 ml.

released.

eluted

compositions from

in this

and

of bound

traction

compounds

two

was

I6 pmoles

glucosamine.

pattern

P-25

material

contained

was

BIOPHYSICAL

glucosamine.

of W-Gel

colitose-containing

--Fraction

AND

amounts action

c 200

glucosaminidase lo8

ml through

glucose

treatment 130 ml;

and

a-glucosidase

(kindly

New

However,

of Medicine,

hydrolysis,

all

was linked

to glucose

Richmond,

160

of colitose,

of yeast

’ I I80

of the

California.

641

York).

glucose

was

in --Fraction

threitol.

liberated

IV.

of Fraction

Fraction --

1Vm

This

supplied after by the

by

triDr.

colitose enzyme,

II -

M.J. had

Vol.

21,

Mild

No.

6, 1965

Acid

BIOCHEMICAL

Hydrolysis

of the

Oligosaccharide

pmoles

of --Fraction

II by dilute

paper

chromatography

with

equimolar not

amounts

cleaved

Isolation

acid

of N-acetyl

Mixture---Removal (0.2

A, yielded

glucose

COMMUNICATIONS

of the

N HCI,

I7 pmoles

glucosamine,

---Treatment

permitted

the

chromatography

the disaccharide

of and

glucosamine

to equimolar

in

suggest

that

v,

of 3.0

15 min.,

colitose

IOOO)

Trisaccharide threitol.

colitose

from

followed

25 by

_V which

contained

The trisaccharide

was at C-6

and

was

Description

I

Alkali-treated

II

Tetrasaccharide

LPS***

1.17

and

1.24

(Fraction --

the

in the

in -Wand

of N-acetyl

Cal

8-N-acetyl-

complete

VI)

hydrolysis

of

threitol.

resulted

destruction v,

glucosamine,

but

not and

of N-acetyl in

Ill. -

that

These

the glucose

I

Molar Fraction

with

of glucosyl-threitol

destroyed

TABLE

pmoles)

catalyzed

oxidation

glucose

is linked

pmoles

of glucose

---Periodate while

V (3.4

a-Glucosidase

A.

quantities

Studies -III and

of Fraction --

isolation

in solvent

Oxidation

results

RESEARCH

by a-glucosidase.

glucosaminidase

Periodate

BIOPHYSICAL

hydrolysis

solvent

of Glucosyl-threitol

by paper

AND

Ratio

GlcN

Glc

0.95

1.00

1.00

Threitol (Galactose)*

Recovery**

(0.W

100%

1.00

1.02

34

Pentasaccharide

t ** ***

II1

Pentasaccharide

2.14

0.98

1.00

0.88

I4

IV

Trisaccharide

0.99

-

1.00

0.94

20

V

Trisaccharide

1.08

I .oo

I.04

-

VI

Disaccharide

1.00

0.90

-

The value given in parentheses Recovery from alkali-treated Ratios for this material reflect portions of the LPS molecule.

residue these

of the positions

glucosamine date

oxidation

pentasaccharide and

colitosyl-N-acetyl

is further in the

substituted

refers to galactose in the intact polymer. LPS was based on co litose. the presence of glucose and glucosamine

(111) is substituted -

at Cd2

glucosamine in intact

LPS,

polymer.

642

and

at the since

this

C-4,

other. constituent

in other

by colitose Presumably is resistant

at one

of

N-acetyl to perio-

Vol.

21,

No.

BIOCHEMICAL

6, 1965

AND

Discussion---Th

e primary

colitose-containing

the

of E. coli --

0111 LPS by

degradation

obtained

as a mixture

further

chemical

structural 1 acetyl

unit

P

and

of a pentasaccharide enzymatic

a and

the

RESEARCH

oligosaccharides

(Fraction --

procedure and

analysis

of the polysaccharide

?-glucosyl-I glucosamine

the

BIOPHYSICAL

of these

is composed 4-golactose;

glucose

of Goldstein

oligosaccharides

colitose

II) resulting

et al. --

a tetrasaccharide.

of the

COMMUNICATIONS

trisaccharide, is glycosidically

(1959)

As shown indicated

were in Figure

that

N-acetyl bound

I,

a basic glucosaminylto both

residues.

References--Cabib, E., Leloir, L.F. and Cardini, C.E., J. Biol. Chem. 203, 1055 (1953). Edstrom, R.D. and Heath, E.C., Biochem. Biophys. Res. ComKns. I& 576 (1964). Elbein, A.D. and Heath, E.C., J. Biol. Chem. 240, l9f9 (1965). Findlay, J. and Levvy, G.A., Biochem. J. n, m(l960). Goldstein, I.J., Hamilton, J.K. and Smith, F., J. Am. Chem. Sot. fi, 6252 (1959). Gordon, H.T., Thornburg, W. and Werum. L.N., Analyt. Chem. 28, 849 (1956). Korn, E.D. in Methods of Biochemical Analysis, edited by David GKk, Interscience Publishers, N.Y. 7, 179 (1959). Trevefyan, W.E., Procter, D.P. and Harrison, J.S., Nature I66 444 (1950). -’

643

from

the