Glycosyl transferases in mammalian gastric mucosal linings

Vol. 29, No. 1, 1967

BIOCHEMICAL

GLYCOSYL TRANSFERASES Diane

Zidermall,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IN MAMMALIAN GASTHIC

Sheila

Gompertz,

and Winifred The Lister

Institute

Zeenat

MUCOSAL LININGS Gunja

Smith,

M. Watkins

of Preventive

Medicine,

London,

S.W.l

Received August 22, 1967 A proposed chains

in

the

sequential

mechanism blood-group

addition

of sugar

macromolecule

intermediate

units

macromolecules

blood-group ses of

that

the

compounds

transferases, preparations

from

that

respectively

donate

low molecular katerials

weight

from

6-phosphate

a rich

glycosyl

and glycoproteins, here

the In source

transferaweight

were tested

as

i"or a- and $-galactosyl transferase,in

and rabbit

D-galactose

(uniformly

the

New

(Jourdian

by means

particu-

gastric

mucosa,

and N-acetylglucosamine

of

England

labeled; &clear

and Roseman, (acetic-l-Cl4

and Roseman,

glucosamine-Cl4 samine

into

Low molecular

baboon

carbohydrate

to

and Methods

purchased

Merrick

for

a

1966).

mucosa,

was examined

human,

weight

in

acceptors.

UDP-galactose-Cl4

group

gastric

the

chains

Watkins,

and a @-N-acetylglucosamillyl

late

through

growing

1959;

is presented

carbohydrate

incorporated

specificity.

Evidence

is

low molecular

subsequently

known structure,

acceptors.

to the

hypothesis,

appropriate of

or to

material,

of the

glycoproteins

and Morgan,

this

active

biosynthesis

units

are

(Watkins

to test

the

active

glycoprotein

attempts

for

with

pyrophosphorylase

Corporation.

1962)

labeled

anhydride,

19583) was converted phosphoglucomutase from

120 mC/mM)

bakers'

56

was Glucosamine-

in

65 mC/mM)

the

acetyl

(Distler,

to UDP-N-acetyl-Dand UDP-N-acetylglucoyeast

(Drown and Glaser,

of

Vol. 29, No. 1, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1955). Normal fresh

tissue

m!cosal

ice-cold

from

linings

human stomachs from

baboons

0.15

tissue

-M KC1 containing 10 ml. homogenising

to

trifuged

at 0'

for

20 min.

at

105,3oU

resuspended

in

KCl-mercaptoethanol

at 105,000 the

g.

original

fractioil

Finally volume

was used

examined water

5:2:2

by reference

a-galactosidase liberated

of the

of

foetus from

(Harrap

1 and 2 res-

sugars

were

by paper

elec-

eluted

and

oligosaccharides known structure

linkage

of the

with

(Courtois

formed run

T.foetus

pH 9.5.

on

galactose-Cl4

a @-galactosidase unpublished) and Petek,

and an The

1966).

The

chromatographically.

unpublished)

57

and

acetate:pyridine:

and Watkins,

from

buffer

1 hr.

particulate

of

was identified in borate

one-tenth

J-acetylglucosamine-Cl4

and Watkins,

N-acetylglucosamine-Cl4 electrophoresis

labeled

beans

a $-N-acetylglucosaminidase

a-activity

in

pH 3.6,

ethyl

was identified

containing

for

pilosphates

buffer

aliquots

(Harrap

coffee

galactose-Cl4

oligosaccharides with

formate

The anomeric

by treatment

again

in Tables

radioactive

to compounds

same chromatogram.

was

galactosyl

given

(solvent;

The size

Trichomonas

for

and sugar

-M ammonium

"/y).

This

test are

nucleotides

cen-

source.

neutral

chromatographically

was determined from

the

g.

gauze,

The deposit

was resuspended

used to

incubation

in 0.2

was estimated the

enzyme

in

supernatant

and centrifuged

transferases

from

and the

at 0'.

deposit

mixtures

After

trophoresis

1 hr.

(2.5

through

of KCl-mercaptoethanol.

N-acetylglucosaminyl

separated

for

the

as the

The reaction

pectively.

g.

filtered

1500 r.p.m.,

and

were homogenised

J" mercaptoethanol

fluid),

centrifuged

by gastrectomy

and rabbits

0.05

at

removed

were treated which and the

by chromatography

was free

from

liberated and by

Vol. 29, No. 1, 1967

Table

BIOCHEMICAL

D-galactosyl

1

from

Sugar

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transferases

rabbit

stomach

in

mucosal

fractions

lining

Size of Saccharide formed

Acceptor

particulate

IncorporatioLl of Gal- Cl4

Hydrolysed by Galactosidase

w

GNAc a-Me-GXAc P-Me-GNAc @-GNAc-(l-4)-GNAc ManBAc G @-Gal-(1+4)-G:?'Ac a-Fuc-(1+2)-p-Gal-(1+4)-G a-Fuc-(1+2)-@-Gal-(1+4)-GNAc' a-Fuc-(1+2)-Gal @-Gal-(1+4)-G @Gal-(1+3)-GNAc Lacto-K-fucopentaose I2

DiDiDiTriDiDi-

63 40 76 73 14 5

TriTetraTetraTriTriTriHexa-

33 33 25 25 12 6 5

a

P

+ + + + + + + + + + + + +

-

Reaction mixture: UDP-Gal-C14, 0.002 wale (25C,OGCl c.p.m.); Tris-HCl pH 7.2, 1.25 voles; MnCl2, f).J lJj,lole; ATP, 1 pole; gastric mucosal particle suspension, 25 sugar acceptor 1 wale; Mixtures incubated 16 hr. at 37'. Tile Total volume 75 ~1. 11.1. following sugars did not accept Gal-Cl4 in this test system (less than 0.1% incorporation); Gal, Gal;?Ac, a-Me-Gal, $-Me-Gal, a-hieN-acetylnes.raminic acid, a-GalGalXAc, $-Me-GaliJAc, FUC, a-Gal-l-P, (1+3)-Gal, @-Gal-(l&)-G.JAc, lactodifucotetraose*, lacto-N-fucolacto-N-difucohexaose I*, lacto-N-difucohexaose II*. pentaose II*, Abbreviations: GNAcT N-acetyl-D-glucosamiae; Gal, D-galactose; G, D-glucose; Fuc, L-fucose; ManNAc, N-acetylmannosamine; N-acetylgalactosamine; Me, methyl; P, phosphate. GalNAc, Results

and Discussion Incubatioa

of particle

UDP-galactose-Cl4 yielded

neutral

were judged the

aild

acceptor

a series

radioactive

from

their

suspensions of

low molecular

compouilds

chromatographic

by one added

sugar

from

residue

of which mobility (Table

rabbit

tissue

weight

with

acceptors

the main

fractions

to be larger 1).

These

than labeled

1. Serologically active trisaccharide isolated from human H substance (Rege, Painter, Watkins and Morgan, 1964). 2. Oligosaccharide isolated from human milk (cf. Kuhn, 1957).

58

Vol. 29, No. 1, 1967

products ric

BIOCHEMICAL

could

linkage

of

kaximum observed

be divided the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i.ito

two groups

galactosyl-Cl4

residue.

incorporation

of

acceptors

containing

with

8-glucosaminyl

residues.

galactose-Cl4

The labeled

co-chromatographed

acetylglucosamine

and was distinguishable

isomers

galactosyl ive

of this

transferase

and maximum bation

incorporation

for,6

hr.

for

1).

The a-galactosyl

ions,

but

was about

picture

the

the

human and baboon

that

added

stomachs

with

@-

as effectfrom

6 to 8

after

incu-

with

enzymes

given

groups

from

donors,

fl-galactosyl

a-linkage

enzymes.

preparations transferases or p-N-acetyl-

Enzymes ia

(Table

human or

rabbit

to d-acetyl-D-glucosamine

of galactose-Cl4

+acetylglucosamine

L-fucose

un-

by Mg++ and Yn++

by the

of the

contained

either

activators.

D-glucosaminyl-(1+4)-N-acetyl-D-glucosamine.

catalysing

to 8-D-galactosyl-(1+4)-

and a-L-fucosyl-(l-2)-@-D-galactosyl-(1*4)-

glucose

were demonstrated

stomach

and in particle

Incorporation

terminal

the

times

residues

effective

to that

galactose-Cl4

occurred

N-

in a-linkage

was activated

more

ABC blood

from

rides

of

occurred

C2 position

obtained

Irrespective

addition

with

3- and 6-

pH optimum

@-D-galactosyl

were the

was similar

the

the

ten

D-galactosyl-Cl4

in

baboon stomachs

of

X-acetyl-

formed

Activation

transferase

Mn++ ions general

from

had a broad

non-reducing

or substituted

Tile

was

B-D-galactosyl-(ld)-N-

of D-galactosyl-Cl4

sugars

terminal

substituted

anome-

at 37O.

The acceptor contained

$-linkage

disaccliaride

disaccharide.

The enzyme

to the

non-reducing

with

by kin ++ ions

as Mg++ ions.

in

terminal

acetylglucosamine

position

according

in preparations suspensions

from

a human

from several

of N-acetyl-D-glucosamine-Cl4 with

non-reducing

low molecular

weight

P-D-galactosyl

into acceptors

residues.

59

baboon

group

B

stomachs. oligosaccha-

containing With

the

rabbit

Vol. 29, No. 1, 1967

Table

BIOCHEMICAL

2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

N-acetylglucosaminyl fractions

Sugar

transferase

from

rabbit

stomach

in mucosal

Size of Saccharide formed

Acceptor

B-&lie-Gal @-Gal-(1+3)-GalJAc @-Gal-(1+4)-GNAc B-Gal- (la3)-GiJAc P-Gal-(1*6)-GNAc P-Gal-(1+3)-G P-Gal-(1+4)-G

particulate lining

Incorporation of GiTAc-Cl4 w

DiTriTriTriTriTriTri-

12 7 6 4 3 3 3

Reaction mixture: UDP-GHAc-C14, 0.006 wale (175,000 c.p.m.); ATP, 2 wales: EDTA 2 wales; Tris-HCl buffer pH 7.2, 2.5 poles; sugar acceptor 1 wale; gastric mucosal particle MnC12 1 vole; suspension 25 ~1. Total volume 35 ~1. Mixtures ijlcubated 16 hr. at 37'. All the radioactive oligosaccharides formed were hydrolysed by $-N-acetylglucosaminidase. The following sugars did not accept GXAc-Cl4 in this test system (less than 0.1% incorporation): Gal, GXAc, a-Ye-Gal, @-G:?Ac(1+4)-GNAc, a-Gal-(1+3)-Gal, a-Gal-(1*6)-GNAc, a-Fuc-(1+2)-Gal, a-Fuc-(1*2)-@-Gal-(1+4)-G, lacto-M-fucopentaose II, sucrose, a-nitrophenyl mannoside. D-mallnose, Abbreviations as in Table 1.

enzyme with

@-methyl

enzymes

galactoside

froni

human and baboon

X-acetylglucosamine

was a better

Substitution

side. L-fucose

of the

destroyed

the

by EIIn++ ions,

demonstrable rabbit

occurred

capacity

acceptor

stomachs acceptor

terminal

and slightly

in the

stomach

samine-Cl4

best

than

into

preparation the

of

the

at pH 7 in

The significance

maximum

to accept transferase

ions,

but

but

glycowith

N-acetylis

activity

activais

or Ca++ ions.

incorporation formed

2),

methyl

residue

sugar

of big++ ions

disaccharide

the

galactosyl

by Zn"

presence

(Table

p-D-galactosyl-(1+4)-

The N-acetylglucosaminyl

glucosamine-C14. ted

was the

With

of N-acetylgluco-

with,

p-methyl

galactoside

4 hr. of these

transferases

60

not

in

relation

to

the

the

Vol. 29, No. 1, 1967

biosynthesis but

their

age of the required carbohydrate Watkins, This Council.

BIOCHEMICAL

of blood-group specificity sugar

substances

with in

to build

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

regard

acceptor

up some of the

chains

of the

to

must the

molecule units

blood-group

await

nature is

further and anomeric

consistent

known to occur active

work,

with in

glycoproteins

linkthat

the (cf.

1966). work

was supported

by a grant

from

the Medical

Research

References Browa, D.B. and Glaser, L., proc.Nat.Acad.Sci., 4l, 253 (1955). (eds. Courtois, J.E. and Petek, I?., in Methods in Enzymology Xeufeld ana Ginsburg) 8, 565 (1966). Distler, J.J., Merrick, J.M. and Roseman, S., J.biol.Chem., 230, 497 (1958). Jourdian, G.W. and Roseman, S., Biochem.Preparations, 2, 44 (1962). 60, 23 (1957). Kuhn, R., Angew.Chem., Rege, V.P., Painter, T.J., Watkins, W.M. and Morgan, W.T.J., ,:ature, Lond., 203, 360 (1964). Watkins, W.M. and Morgan, W.T.J., VOX Sang., 4, 97 (1959). Watkins, W.M., Science, 192, 172 (1966).

61