On the structure of the carbohydrate chains of blood group substances

BIOCHEMICAL

Vol. 39, No. 4, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ON THE STRUCTURE OF THE CARBOHYDRATECHAINS OF BLOOD GROUPSUBSTWJCES N.K. Kochetkov,

V.A. Derevitskaya,

M.D. Martynova,

L.M. Likhosherstov,

S.N. Senchenkova,

G.S. Kikot'

and

L.S, Bogdashova N.D. Zelinsky Institute of Organic USSR Academy of Sciences, Moscow, Received March

24,

Chemistry US SR

1970

A scheme for the general composition of the carbohydrate chains in blood group substances is suggested. The principal peculiarities of this scheme are the high degree of carbohydrate chain branching and the presence in the region close to the peptide backbone of a fragment built up of only N-acetylhexosamines. The biological

specificity

of blood group substances

(BGS)

is known to be determined by the structure of their carbohydrate chains /l/ which are attached to the central peptide backbone '2' . The nature

of the terminal

des and the structure

nonreducing

of the neighbouring

monosacchari-

regions

has been es-

tablished tial

by means of immunochemical methods as well as by pardegradation of BGS /I/ . No data have been available up to

now concerning

the general

BGS. A structural the carbohydrate

scheme for chains

bone has been recently rio

structure

the nonreducing

and the part put forward

chains

in

parts

of

terminal

close to the peptide by Lloyd,

back-

Kabat and Lice-

13/. On the basis of our present

lated

of carbohydrate

from hog stomach lining

bed procedure

'4')

together

according with

oligosaccharides

isolated

neral

the structure

scheme for

studies

earlier

of BGS (A + H) (isoto a previously

data on the composition /1,3,5,W

descriof

9 we suggest a geof BGS carbohydrate chains.

583

Vol. 39, No. 4, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The main approaches of the carbohydrate degradation,

chains

methylation

zymatic degradation ducts. tion

to the elucidation

The latter rate

of the structure

were as follows: studies,

consisted

products

During

saccharinic

and second periodate

of N-acetylhexosamine

5 hours,

only traces

of galactose

thus demonstrating nonreducing tions

that

termini

result

high degree

into

on the basis

chains. -tetra-, di-,

only from the Smith degrada-

chains

of a high degree

galac/7/*

in BGS should be

and taking into account /3/. Consequently the oxidation

can only be

of branching

of the

chains. and high degree

Several

of BGS are also in accord with of branching

O-methyl-derivatives

3,4,6-tri-, 2,6-di-,

derivatives,

of N-acetylhexosamine

and hexosamines

The data on methylation complexity

contained

of ca. 7Wh of the original

2 determinants

of galactose

carbohydrate

Two successive

of chains.

60 as deduced from '8g10(

the chain branching

products

proceeded

number of carbohydrate

approximately

explained

the oxidation

and ca. 50% of that

The total

weight

within

2.5 and 1.3 moles

and N-acetylhexosamine

in destruction

tose content

hex-2-enose.

to completion

consumed being

per 1000 g of DGS. The lowmolecular

and ga-

of BGS the first

proceeded

the amount of oxidant

units,

(chromogen),

Smith degradation

oxidations

pro-

of the forma-

acids and 2-substituted

the stepwise

and en-

degradation

in the determination

3-acetamido-5-(1,2-dihydroxyethyl)-fursn units,

Smith

a combined chemical

and a study of alkaline

of degradation

lactose

stepwise

2,4,6-tri-,

of its

carbohydrate

of galactose 2,3,6-tri-,

3,6-di-0-bPle-Gal),hexosamines

(2,3,4,6-

2,3,4-tri-, (3,4,6-tri-,

4,63,6-di-,

and fucose (2,3,4-tri-O3-, 6- 0-Me-GN, 3,4,6-tri-0-Ue-GalN) Me-Fuc) were identified among the products of hydrolysis of 584

the

BIOCHEMICAL

Vol. 39, No. 4,197O

the methylated merit

BGS thus revealing /11,12/ . According

linkages

in part,

galactose,

occupy terminal

The presence

C-4 and C-3 or C-2 follows

very

accumulate

with destruction lactose

Galactose

slowly.

that

well

of

of the second 4 hrs

which give

degradation

products

cau-

rise appear

is accompanied

content of N-acetylof galactose /13/ . Hence ga-

to degradation

does not appear

linkage.

attached

to serine

the reducing

It

under conditions

to enter

These data also give

is N-acetylhexosamine.

that

products

at

absent from the carbohydrate

is subjected

the monosaccharide

residues

in

with substituents

Such a treatment

seems to be practically

rate-peptide

ga-

of ca. 3W/o of the total

used, and consequently that

weight

and of ca. 4% of that

chain region

while

are also located

of N-acetylhexosamines

to chromogen production.

and

and 2-acet-

of BGS with 0.05 M Na2C03 at 70" for

destruction

hexosamine

fucose,

also from the identification

among the lowmolecular Smith degradation of BGS /7/* ses rapid

of intermono-

positions,

of galactose

threitol

Treatment

variety

to these data,

and 2-acetamido-2-deoxy-D-glucose

branch-points.

and

a large

2-acetamido-2-deoxy-D-glucose

amide-2-deoxy-D-galact;ose lactose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

should

the carbohyd-

en unambigous proof and/or

threonine

be pointed

end of the carbohydrate

out as

chain consists

of several (no less than 3-4) N-acetylhexosamine residues linked with l-3 bonds, this being the backbone of this chain region.

Characterization

degradation

of BGS proved

tion

of carbohydrate

viz.

substituted

responding nes with

of chromogens formed to be of value

chain structure.

and nonsubstituted,

to the presence and without

of l-

substituents 585

upon alkaline

in further

elucida-

Two types of chromogens, were formed,

3 linked

these cor-

N-acetylhexosamiat C-6 /13/o

Vol. 39, No. 4,197O

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Alkaline

degradation

end borohydride

reduction

amounts of nonsubstituted explained is linked treatment

formed

and which is subject

of the terminal

in the side chain of 1 -3 to degradation

linked

that N-

upon

with alkali.

Examination

of the kinetics

chromogen formation

accumulation

of the former

(fig.

of substituted

linkage

and unsubsti-

I)

showed that the curve of had a lag-phase /14/ . It follows,

u.nequivocally,thatN-acetylhexosamine tein

oxidation

residues

the chain fragment

acetylhexosamines

oxidation

in formation of decreased chromogen /14/. This fact can be

N-acetylhexosamine with

periodate

results

as being due to periodate

nonreducing

tuted

of BGS after

at the carbohydrate-pro-

bear no branching.

lg(Dm-D)

1.1

Fig.

1. The kinetics of the substituted and unsubstituted chromogen formation from BGS treated with 0.05 M aqueous Na2C05 at 70". - unsubstituted chromogen - substituted chromogen. 586

Vol.39,No.4,

1970

BIOCHEMICAL

A combination followed

of degradation

by enzymatic

glycosidase

hydrolysis

preparation

molecular

weight

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

'15')

fragment

(6OA) and hexosamines

methods (Smith degradation by a Clostridium perfringens -----a--------e m-w resulted in isolation of a high-

consisting

mainly

(34%), the 2-acetamido-2-deoxy-D-galac-

tose to 2-acetamido-2-deoxy-D-glucose

7.5 : 1 '16'.

Alkaline

degradation

this

revealed

that

fragment

rate-protein

linkage

ratio

being

and periodate

equal to oxidation

in most of 0-glycosidic

a single

do-2-deoxy-D-galactose,was

HexNAc

of amino acids

monosaccharide,

of

carbohyd-

viz.

2-acetami-

present.

I

I R NH : I / Hefl,Ac I-3 HexNAc I-33 HexNAc I-3 GalNAc-OCRCH 6 6 \ f c=o i I .

f3

I Fuc I-4 GNAc

Gal1 6 \

?

peptide chain

I

GNAc 3-I Fuc 4 t I Gal 261 Fuc

3

Fuc I-2

?

t I Gal

3

3

t I

The scheme for presented

here may, of course,

ned in the light details

the carbohydrate

be completed,

of newer experimental

of attachment

cal activity

chain structure

of the part

to the fragment

built 587

of BGS

altered

data,particularly

responsible

for

or refion the

the biologi-

up of R-acetylhexossmines.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 39, No. 4,197O

We should

like

scheme, viz. -

to emphasize two principal the high degree

and the presence of the fragment

of carbohydrate

in the region built

peculiarities

of this

chain branching

close to the peptide

backbone

up of only N-acetylhexosamines.

References 1. Watkins, W.M., in ltGlycoproteinsl' (Ed. A. Gottschalk) p. 462, Elsevier Publishing Company, Amsterdam, London, New York, 1966.

2. Koch&k&, N.K., Derevitskaya, V.A., and Kara-Murza, S.G., Carbohydrate Res., 2, 403 (1967). 3. Lloyd, K.O., Rabat, E.A. and Licerio, E., Biochemistry, 2, 2976 (1968). 4. Likhosherstov, L.M., Derevitskaya, V.A. and Fedorova, V.I., Biokhimiya, 4, 45 (1969). 5. Rege, V.P., Pain% er, T.J. Watkins, W.M. and Morgan, W.T.J., Nature, 200, 532 (1963j. 6. Lloyd, K., mat, E.A. Lagug, E. and Gruezo, F., Biochemistry, fz, 1489 (1966). 7. Likhosherstov, L.M., Bogdanova, L.S., Derevitskaya, V.A. and Kochetkov N.K., Iav. Akad. Nauk SSSR, Ser. Khim. (in press). 8. Zarnite, M.L. and Kabat, &:.A., J. Am. Chem. SOC., 82, 3953 (1960). 9. Marcus, D.M., Kabat, E.A. and Schiffman, G., Biochemistry, 2, 443 (19fJQ). 10. Tuppy, H. and Standcnlsher, W.L., Biochemistry, 2, 1742 N K Kikot' 11 . Ko:i%% G.S., Bogdanova, L.S., Likhosherstov, LfM.*&i Dereviiska a, V.A., Izv. Akad. Nauk SSSR, Ser. Khim., 2, 2085 (1968 3 . 12. Likhosherstov, L.M., Kikot', G.S., Derevitskaya, V.A., Arbatsky, N.P., Fedorova, V.I. and Kochetkov, N.K., Dokl. Akad. Nauk SSSR (in press, 1970). 13. Kochetkov, N.K., Derevitskaya, V.A., Likhosherstov, L.M., Martynova, M.D. and Senchenkova, S.M., Carbohydrate Res., (in press, 1970). 14. Likhosherstov, L.M., Senchenkova, S.N., Derevitskaya, V.I., and Kochetkov, N.K., Dokl. Akad. Nauk SSSR (in press). 15. Likhosherstov, L.M., Martynova, M.D., and Derevitskaya, V.A. Biokhimiya, 2, 1135 (1968). 16. Kochetkov, N.K., Derevitskaya, V.A., Likhosherstov, L.M. and Martynova, M.D., Dokl. Akad. Nauk SSSR, 186, 1195 (1969).

588