A new O-glycosidically linked tri-hexosamine core structure in sheep gastric mucin: A preliminary note

Vol.

92, No.

February

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1143-1150

27, 1980

A NEW 0-GLYCOSIDICALLY LINKED TRI-HEXOS!MINE CORE n STRUCTURE IN SHEEP GASTRIC MUCIN: A PRELIMINARY NOTE 5' 1$ Elizabeth F. Eounsell, 14. Fukuda; F.1.E. Fowell: Ten Feizif and S. Hakomori2 1 !Clinical

Research Centre, Watford Road, Harrow, Middlesex, England; and 2Biochemical Oncology, Fred Hutchinson Cancer Research iCenter, and TJniversity of Washington, 1125 Columbia, Seattle, WA 9810!,

Received

December 21,1979

Summary-Oligosaccharides Ii active sheep gastric cm-e structure:

released by reductive alkaline degradation mucins have been shown to contain two types F,3+Galpl,

Rl+GlcNAcBl,, 'CaiNAc 6'

A)

3,GalNAc

El

R2+GlcNAcB1' The novel Structure mucins.

of glycoproteins

!I-glyccsidically

linked

tion

aqueous

in alkaline (1,2).

gland

and hog (8,9)

tide

During Ii

gastric

of either through

active

isolated

carbohydrate solution

(3),

with

0-glycosidic the

course

sheep

gastric

or

secretions

cysts

isolated c&,5)

structure

contain

can be released of reducing

have been

The core

GalBl‘3GalNAc

which

protection

human ovarian

mucins.

from mucous

chains

Such oligosaccharides

hog submaxillary

sisted

RI +GlcTIAc Bl ' '-'

structure, A, was the core of one of the main oligosaccharide fractions. B is identical to that reported previously in various human and animal

The majority

reduction

of blood group of branched

by B-elimina-

groups

and characterized

and human

of these

by borehydride

(6),

horse

frcrn (7)

oligosaccharides

ccn-

attached

to pep-

fr~>m blood

group

GalBl'j[!;lcNAcBljh]GalNAc

linkage. of studies mucins

of oligosaccharides an oligosaccharide

released with

a new type

cf core

struc-

*

This investigation was supp-yrted in part by the U.S. National Cancer Institute Grant CA19224 and CA23907. IEFH was supported by an !*mC (UK) training fellowship. Abbreviations: Galol: galactitol, GlcNAcol: N-acetylglucosaminitol, GalNAcol: Nacetylgalactnsaminitol, GlcU: glucuronic acid, SA: sialic acid, CLC: gas liquid chromatography. 0006-291X/80/041143-08$01.00/0

1143

Copyright ‘r 1980 A II rights of reproduction

by Academic Press. Inc. in an-v form rerervcd.

Vol.

92. No.

4, 1980

ture

as seen below

core

described

BIOCHEMICAL

was identified

above.

AND

BIOPHYSICAL

in addition

RESEARCH

to others

having

COMMUNICATIONS

the

common branching

GlcNAc@l\ GlcNAcf31' MATERIALS

AND METHODS

OZigosaccharides Studied: The preparation of blood group Ii active sheep gastric glycoproteins, reductive alkaline degradation in the presence of tritiated sodium borohydride and sequential oligosaccharide fractionation on a Biogel P4 column, paper chromatography and electrophoresis will be described in detail elsewhere. Two major oligosaccharide fractions LE2 and LD2 are described in this report. StandaZard Otigosaccharides: Fuc~l~2Gal~l~~GlcNAc~l~3~alol, GlcNAcf31+3Galol (both from H2-glycolipid), Gal~l+~GlcNAc~1+3Galol (from paragloboside) were obtained by endo-&galactosidase digestion with subsequent borohydride reduction in the presence of NaB[3H]4 (10). These samples were donated by Mrs. Michiko N. Fukuda of this laboratory. Gal~l-t~ClcNAc~l-t2Man~l~6[~Gal~l~~GlcNAc~l~2Man~l~3]GlcNAc~l~~ [FUC~~&]G~CNACO~ was obtained from imtnunoglobulin by hydrazinolysis (29) followed by reduction with NaB[3H14. Manal+3[Manal+6]GlcNAc~1+~[Fuccll+6]GlcNAcol was obtained by E-galactosidase and B-N-acetylglucosaminidase digestion of the above oligosaccharide. C?womatographic Methods: Biogel P4 chromatography was carried out on a 2OO-)iOO mesh Biogel P4 column (0.9 x 150 cm) at room temperature (2C-30'C)using distilled water as solvent with flow rate 4 ml/hr and 2 ml fractions. The column was calibrated in "hexose units" according to the data of Yamashita et al (11) using nligosaccharides of known structure containing hexose (1 unit) N-acetylhexosamine (2 units) and fucose (0.7 units). Oligosaccharides were
LE2 chrcmatographed corresponding

as a sharp

to 9 "hexose

of LE2 was estimated

peak on the

units".

Together

Biogel with

as galactose/N-rcetylglucosamine/N-acetyl-

3:2:1.

1144

P4 column GLC analysis

(Fig

1) the

Vol.

92, No.

4, 1980

BIOCHEMICAL

“Hexose

AND

Exe-G-~~a~actusidase

1.5

times

ated

The

diEestiCon

major

frament

relative

alditol

t.o

acetates

standard

ir?ucnsamine)

with

iE21

from

LE$

the

a core

1.5/l.

regiYn

of

frc-ibmennents

on paper

chromatography

.

gave

Analysis

only tn

the

LE,lI-

five

and at

0f

the

LE2? a distanct-:

partially

methy

3,4,6-tri-O-methyl-2-jeoxy-2-

nan-rerlucicr

terminal

N-acetyl-

~-(~,I-methyl)acet3mi?n-~alsctitol' substitute11

Direct

spectrum

units"

labeled

Cal+CicNAc~al~~i

(derived

of

gave

being

migrated

N-acetylGalactosaminitl~1

ratio LE?l

two

1,4,5-tri-O-methyl-~-deoxy-,

to

approxim&te

the "hexose studied.

(corresponding

and

(c~:~rresp~:~nding

indicate structures

gave

LEzl

(N-methyl)acetamidc-qlucose

frwpent

number

Biogel P4 chromatography of original oligosaccharide fraction LE -Ol -; products LE21 and LE22 obtained after exe-g-galactosidase treats&t of products LE2a -O-Oand LD2a -D-Oobtained after LE2 -O-b-; sequential exo-g-galactosidase and exe-B-glucosaminidase treatment of LE2 and LD2 respectively. The column was calibrated in "hexose units" indicated as numbers (arrowed) using the following reduced tritiated oligosaccharides: Gall~~GlcNAc1+2Man1~3~Gall~~GlcNAc1~2Man1~6~Man1~~ GlcNAcl+~[Fucl+6~GlcNAc (13.3 hexose units) and Manl+6[Manl+31Manl+4 GlcNAcl+~[Fucl~]GlcNAc(7.7 hexose units) obtained from immunoglobulins; Gall~~GlcNAcl~3~Ga11~~GlcNAc1~L6GGal (7 hexose units) kindly provided by Professor S. David (Universite de Paris-Sud, Orsay, France) and Fuel-+2 Gall+4GlcNAcl+3Gal (4.7 hexose units) obtained from H2 glycolipid, kindly provided by Mrs. Michiko N. Fukuda.

1.

The figures in parenthesis labeled oligosaccharide

1).

COMMUNICATIONS

70

&I Fraction

(Fit:

RESEARCH

units”

50 Fig.

BIOPHYSICAL

probe shown

mass

in

Fig.

trisaccharide GlcI,IAc

spectrometry PA.

with

at

All the

C6 and of

these following

C3)

in

an

permethylateri

data

sre

consistent

structure:

61 ~ ?GalNRcol

h

GlcNAcfil

'The anil

mass Tuppy

spectrum (19).

of

this

(1erivstive

'

confirmed

1145

that

recently

reported

by

Urann

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

BlOPHYSlCAL

RESEARCH

COMMUNICATIONS

:521 i qH,OMe

260;

219: 464 I Gal+O-GlcNAcj 1930 ' 0,: ,O,GalNAc

B 100 >5oox5 432

l&

Fig.

2.

Direct probe mass spectra of permethylated fragment LEpl (A) formed by exo-8-galactosidase treatment of LE2; and permethylated intact oligosaccharide LE2 (B). Samples were analyzed in a Finnigan 3300 mass spectrometer; mass range 100-1000 amu, electron energy 70 volts, ion extractor 3.5 volts, lens 16 volts, emission 0.5 millienergy 3 volts, ampere, electron multiplier 1800 volts.

Galol

(Fig

labeled

digestion

on the Biogel obtained

700

750

&IO

obtained

1.4 times

disaccharide

from Jack

characteristic

after

(Fig

LE2 with a single

1).

950

exo-B-galactosidase

relative

to

standard

GlcNAcSalNAcol,

GlcNAc+

formed

mass spectrometry

fragments

as shown

labeled

The single

product partially

identified

LE2a,

which

methylated as that (30)

of permethylated in Fig

in

followed

2B.

1146

intact

by

eluted alditol

of 1,3,4,5,6confirming

N-acetylgalactosaminitol. probe

by

contaminant

exo-6-palactosidase

penta-O-methyl-2-deoxy-2-(N-methyl)acet3mido-hexosaminitol

Direct

900

bean meal.

from LE2a had a mass spectrum

as unsubstituted

850

an exo-B-N-acetylglucosaminidase

gave

P4 column

650

component

of fraction

exo-B-N-acetylglucossminidase

600

on paper

preparation

Consecutive

550

to be a core

of LE21 with

&galactosidase

acetate

500

1) chromatographed

digestion

late

450

the minor

and was considered

partial the

464

250

treatment

400

!726

&

LE22,

350

7 669

lil

Fragment

300

GallO-GalLO-GlcNAc 219: 4231

>7oox50

LE2 gave

several

LE2a

Vol.

92, No.

BIOCHEMICAL

4, 1980

Analysis LE2 showed

of the partially the

galactitol;

presence

alditol

RESEARCH

acetates

COMMUNICATIONS

derived

from

methylation

and 2,4,6-tri-O-methyl

and d) the

data

are

fraction

was linked

of fragment

at Cl&; c) galactose

present

galactose

residue

analysis

fraction

of 1,4,5-tri-O-methyl-2-deoxy-2-(N-methyl)acetamido-

N-acetylgalactosaminitol

the

methylated

BIOPHYSICAL

3,6-di-O-methyl-2-deoxy-2-(N-methyl)acet3mido-glucose;

tetra-O-methyl-

linked

AND

was the

remaining

consistent

LE21;

the

which

indicated

that

only

a) the

at C3 and C6, in agreement

b) the N-acetylglucosamine

galactose

with

and 2,3,il,6-

non-reducing residues

following

residues

terminal

were

structure

were

monosaccharide

mainly for

with

linked

at C3.

the main

All

component

of

LE2: GalBl+4GlcNAcB11 316 GalNAcol Galal'3GalB1~4GlcNAcal/

Fraction have the

LD2 was estimated

composition

Consecutive

(Fig

Biogel

of this gave the

1) in an intermediate

fraction

single

with

product

position

relative

Fragment

GlcNAcSalol

and was shown by GLC to contain

and N-acetylgalactosaminitol.

that

of LE2 in having

galactitol ~3

of the partially

LD2 showed

that

Further

evidence

region

an N-acetylglucosamine of LD

2

the

followed

chromatographed

0.9 times

approximately core

residue

linked

methylated

alditol

presence

indicating

and ~6.

that

the

on paper

Thus the

a galactose

3:2:1.

region

P4

and LE,a I-

relative

equal

by exo-

on Biogel

to LE22 (GlcNAc+GalNAcol)

LD2a chromatographed

tose

fraction

and GLC to also

exo-B-galactosidase LD,a which

(GalNAcol).

Analysis

P4 chromatography

galactose/N-acetylglucosamine/N-acetylgalactosaminitol

digestion

B-glucosaminidase

from

to

amount

standard

of galac-

of LD;, differed

from

to the N-acetylgalactosaminitol. acetates

derived

from

intact

of 1,4,5-tri-0-methyl-2-deoxy-2-(N-methyl)acetamil N-acetylgalactosaminitol

which residue

will

be described

was the

substituent

was shown to be GalBl, GalNAcol GlcNAcBl'

1147

residue in detail

was substituted elsewhere

at ~6 and thus

indicated the

core

at

Vol.

92, No.

Five

additional

shown

to

BIOCHEMICAL

4, 1980

oligosaccharides

have

this

Gall+3CalNAcol

core

cbbtained

in

the

core

BIOPHYSICAL

from

Two

structure.

linkage

AND

,&her

RESEARCH

the

sheep

COMMUNICATIONS

gastric

i~lignsaccharides

mucins had

were

nn

alsl

unbranched

region.

DISCUSSION In have of

been the

been is

contrast

to

the

extensively

core

region

elf

systematically limited. or

sidic

linkage

residues

involves

I.

serine-

clr

threonine-linked

have

linked

Table

classified

types been straight

through

and of

carbohydrate

chain

a Gal+Gal+Xylose

the

of

Table

I).

oligosaccharides sequence

chain.;

their

The

has

structural

first

containing to

regions structure

serine

rlr

not

variation

C-v~Lycnsidically

chain

(see

core

chemical

carbohydrate

knowledge

our

reported

whose

(20 ,21,22)

and

main

threonine

oligosaccharides

studied

investigated Three

serine

asparagine-linked

linked type

of

uranic

tl-> 0-~iycoacilj

threonine.

Structure of four groups of 0-glycosidically linked carbohydrate (The Group 4 with tri-hexosamine core is chains in glycoproteins. the new addition through this study. Residues which are not always Core regions are surrounded by present are shown in parenthesis. dotted lines). References

Group 1 R-Flcupl-~~lpl-~~G~li~~~y~~-Ser/Thr in-----------------.

(23,241

Group 2 r----------1 ( SAaZ)+alpl-3GalNAc,-StSerlThr ---f’”

(251

Group 3 R--Gal@43

GlcNAcpll

~----.--~ 3 Galp1~3GlcNAcpl~~~G~~~~ I 1 ‘3 GalNAckerlThr GlcNAcpl Y6 I ’ f6 ( Gal p I-%.%%&3l) L----------------:

R-Galpl-4 R-G’cNAcpl\ R-.GlcNAcpl

(4.281

__--__----, /’

( R-Galgl&G~~Nk~) I_----

pa’p1k3 ; /

Ga(N&Qr,Thr / J

‘6

I I GalNA+Ser/Thr ’

(9)

Group 4 R-Galpl+4GlcNAcpl I / I! R-+Galpl+4GlcNAcpl L____

f3 ---------1

Present study

1148

Mary

Vol.

92, No.

prcitengYjjcans sh"rt

BIOCHEMICAL

4, 1980

contain

this

rlig;asaccharide

chains

-nay tie substituted :'!II? thirtl :i,R,fi,

with

releasrd

rP,K'C~rlS were

off" often

not

,:rt~~ps by borohy~iride ~-~li~r~,s~charides structures type

identified. reduction

of varying

that

belong

'-)f structure

with

iras-tric

?lnder

the

new trihexosamine

oligosaccharide

I

blood

conditions

region

have been

which

per-

the

cr-ire

ccre

.::I‘ reducirq

to he studied

and

isolated

with

the

the

fourth

Vie nclw report

(2-9).

("Group

fractions

gr,-.u?

such oligosaccharides

protection

core

and complexity

which (25-:l;i).

end and therefore

recently

';'I in Table

zarryinc

studies

the

structure

L",

obtained

Table from Ii

I)

which

active

wa; sheep

nrlcin. ::arbchylirate

t:ilre.:nine that

length

the

T'-,l.~nd in rbne of the main

More

include!;

Fi and A determinants

reducing

has enabled

to "Croup

group

degradation

from the

nf residues

group

as the basic

In earlier

by alkaline

COMMUNICATIONS

The second

~sligosaccharilies

(4,9,28).

from peptide

~r,jt?;e
or blood

complex

highly

RESEARCH

(23,2.+).

G,316l+jCalI:.4c

acid

I activities

BIOPHYSICAL

structure

having

sialic

fyroup includes

Lewisand

were

core

AND

are well

:)-jrl:Jcosiriically

sacchnriles t*: .leterminc oi' 7ntcins

attachecl

knc'wn tc' occur

, simple

silrilar

Tt will

chains

end complex

in various

their

the

on secreted carbohydrate

to systematically

trihexiisamine

roles

O-glycosidic

membrane-associated

be of interest with

thro!lgh

tc_? serine

or

Flycoyt~oteins.

There

are

chains

rlccur

linked

may also

glycnproteins

investigate

and hexose-fii-hexosamine

in the formation

and Iof membrane-associated

linkage

of highly

branched

indicatial

such as glycophorir:

the

incidence core

structures

carbohydrate

cf (:lig-:and ckains

slycoproteins. REFERENCES

1. Iyer, R.N., and Carlson, 2. Arllerson, E:., Rovis, L.,

D.M. (1971) Arch. biochem. Eiophys. &, 101.-105. and Rabat, E.A. (1972) Arch. Biochem. Biophys. 1/p,,

304-314.

2. Csrlson, D.M. (1968) J. Biol. Chem. m, 616-6~6. -i. Rovis, L., Anderson, R., iiabat, E.A., Gruezo, F., and Liac, J. (1973) Eiochemistry g, 5340-5354. 5. r;laisl~nl,@u@e-McAuliffe, F., and 'i;abat, E.A. (1976) Arch. 6iochem. Eiophys. 90-113. a, 6. "lates, M.D.G., Kosbottnm, A.C., and Schrager, J. (Is'/;.) Carbohyd. Res. 3, 115-137.

1149

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

7. Newman, W., and Kabat, E.A. (1976) Arch. Biochem. Biophys. 172, 535-550. 8. Slomiany, B.L., and Meyer, K. (1972) J. Biol. Chem. m, 5062-5070. 9. Derevitskaya, V.A., Arbatsky, N.P., and Kochetkov, N.K. (1978) Europ. J. Biochem. 6, 423-437. 10. Fukuda, M.N., Watanabe, K., and Hakomori, S. (1978) J. Biol. Chem. a, 6814-

6819. 11. Yamashita, K., Tachibana, Y., and Kobata, A. (1977) J. Biol. 5411. 12. Bhatti, T., Chambers, R.E., and Clamp, J.R. (1970) Biochim.

222, 339-347.

Chem. z, Biophys.

5408Acta

13. Hakomori, S. (1964) J. Biochem. (Tokyo) x, 205-208. 14. Karlsson, K.A., Pascher, I., Pimlott, W., and Samuelsson, B.E. (1974) Biomedical Mass Spectrometry 1, 49-56. 15. Watanabe, Ii., Hakomori, S., Childs, R.A., and Feizi, T. (1979) J. Biol. Chem. ;154, 3221-3228. 16. BjErndal, H., Hellerqvist, C.G., Lindberg, B., and Svensson, S. (1970) Angew. Chem. Int. Ed. Engl. 9, 610-619. K., Saito, H., and Hakomori, S. (1973) Arch. Biochem. Biophys. 155, 17. Stellner, 464-472. 18. Li, Y-T., and Li, S-C. (1972) Methods Enzymol. 18, 702-713. 19. Wrann, M.M., and Tuppy, H. (1978) Europ. J. Biochem. 92, 105-110. 20. Kornfeld, R., and Kornfeld, S. (1976) Annu. Rev. Biochem. 45, 217-237. (1979) in Glycoconjugate Research 21. Montreuil, J., and Vliegenthart, J.F.G. Vol. I, 35-78, Gregory, J.D., and Jeanloz, R.W. Eds. Academic Press, New York 22. Kobata, A., Yamashita, K., and Tai, T. (1979) ibid. 193-197. 23. Anderson, B., Seno, N., Sampson, P., Riley, J.G., Hoffman, P., and Meyer, K. (1964) J. Biol. Chem. m, ~~2716-~~2717. (1968) Biochemistry of Glycoproteins and Related Substances; Cystic 24. Rod&, L. Fibrosis Pt. II, 185-202. Rossi, E., and Stoll, E., Eds., Karger, Basel. (1969) J. Biol. Chem. 2&, 5943-5946. 25. Thomas, D.B., and Winzler, R.J. 26. Carlson, D.M. (1966) J. Biol. Chem. a, 2984-2986. Chem. m, 608627. Takasaki, S., Yamashita, K., and Kobata, A. (1978) J. Biol. 6091. 1, 2976-2990. 28. Lloyd, K.O., Kabat, E.A., and Licerio, E. (1969) Biochemistry (1976) J. Biochem. 80, 1223-1232. 29. Fukuda, M., Kondo, T., and Osawa, T. 30. Finne, J., and Rauvala, H. (1977) Carbohyd. Res. 2, 57-64.

1150