The isolation and the structure of new glycopeptide from blood group substances

Vol. 56, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE ISOLATION AND THE STRUCTUREOF NEWGLYCOPEPTIDE FROM BLOOD GROUPSUBSTANCES N.K,Kochetkov,

V.A.Derevitskaga,

L.M.Likhosherstov

and

S.A.Medvedev N.D.Zelinsky Received

Institute of Crganic Chemistry, of Sciences, Moscow, USSR

October

USSR Academy

15.1973

Summary: Three new glgcopeptides with 0-glycosidic and one glycopeptide with N-glycosylaminic carbohydrate-peptide linkages have been isolated after degradation of blood group substances (BGS). Their structure have been determined as 0-(d.-GalNAc)-Ser(I), 0-(GalNAc)-(Pro)-Ser(I1) 0-(GelNAcl-3 GalNAc)-(Thr-Ala)-Ser(III),N-(j3-GlcNAc)-Asn(IV~. The isolation of glyco,peptide I confirmed aC-configuration of 0-glycosidic carbohydrate-peptide bonds. The structure of glycopeptide III with two galactosamine residues is in accordance with the data on the presence of hexosamine core of BGS carbowdrate chains.

Recently

we reported

/1/

on the

isolation

of glycopeptide

0-(N-acetylgalactosaminyl)-threonyl-alanine, products

of blood group

succeeded in isolation

substances of other

die and N-glycosylaminic cidation

of their

Degradation been carried tide

fraction

jected

elution revealed

(A+H)

with

carbohydrate-peptide

C-glycosi-

linkages

and elu-

structure.

out according isolated

to chromatography

from pig stomach lining

to the procedure after

/3/.

gel-filtration

19 peaks (fig,2),

on Aminex AG SOW-X2 resin The fractions

peak were combined and the ratio after

hydrolysis

has

The glycopep(fig.1)

curve was mode up by means of ninhydrin

nes was determined

Now me have

(B&S),

glycopeptides

of BGS isolated II

from degradation

/'l/0

reaction

corresponding

was subThe and to each

of amino acids and hexosami(4N HCl, IOO',

24 h) using

BIOCHEMICAL

Vol. 56, No. 2, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

glycopeptides

% N

0" 2.0.

,amino aci I peptides

/G/--II---i

8 1

Fig.l.Fractionation of BGS degradation P-2 (column 3 x 200 cm, elution

products on Bio-Gel with O.lM acetic acid).

0.5 13

100

Fig.2.

ChromatograpfUI

200 Fraction

15

17

300

400

number

of glycopeptide

fraction

II

(see

fig.1)

on Aminex AG .5OW-X2 column (1.8 x 80 cm), elution with gradient of pyridine-formate buffer, fraction volume 5 ml. Fractions were assayed by absorbance at 570 nm after reaction with ninhydrin.

the amino acid analyzer and 18-19 contained

amino acids,

amount of glucosamine; and galactosamine simplest

(Biotronic

only,

fractions Fraction

amino acid composition 312

LC 4010).

Fractions

galactosamine IO-17

I-9

and small

contained

2,5 and 'I$ (fig.21 and were used for

amino acids had the the isola-

Vol.

56, No.

tion

2, 1974

BIOCHEMICAL

of glycopeptides.

subjected

AND

BIOPHYSICAL

For this

purpose

to preparative

high-voltage

and paper chromatography phoretic

separations

obtaining

/I/.

cation

the fractions

were

paper electrophoresis

In several

cases two electro-

and paper chromatography of glycopeptides

of Xdman degradation

oxidation

(0,015M NaI04,

treated

with

hydrol;yzed

were used Sor

according 20°,

(4N HCl, IOO', The following

gradation

products

2 h);

to /2/

by appli-

and poriodate

oxidation

products

were

or NaBH4 , 20°, 2 h) then

20 h) and analyzed glycopeptides

using

amino acid

were isolated

from de-

of BGS:

(5.3)

Ser

was determined

NaBH,+ (1.5% solution

analyzer.

II.

Pro-Ser

(5.1.2.1)

I

lioC-GalNAc III.

COMMUNICATIONS

homogeneous glycopeptides.

The structure

I.

RESEARCH

0-d-GalNAc

Thr-Ala-Ser

IV. fsn (2.3)

(14.4.2)

I C-d-GalNAcj-I

GalNAc

N-J3-GlcNAc 5.3 3-f)

C-(A-N-acetylgalactosaminyl)-serine

(I).

with

was isolated

by electro-

oxidation

of I results

GalN:Ser

phoresis

= 1.0:0.98

at pH 1.9 (zone 3). Periodate

in destruction of this

molar ratio

of N-acetylgalactosamine

glycopeptide

which completely of serine

was confirmed

excluded

Glycopeptide

only.

The structure

by Edman degradation

the structure

with

two residues

and N-acstylgalactosamine.&-Configuration

O-glycosidic

carbohydratepeptide

the high positive H20;calculated

optical

linkage

rotation

from the determination

of

was deduced from

value@]? of serine

+

117'

using

(c 0.015;

amino

*) iilhen numbe ring glycopeptides the first figure is the number and of a fraction after ion-exchange chromate raphy (fig.2) next figures are the numbers of the zone 7from the less mobile) after successive paper electrophoresis and chromatogrwhy

l

313

BIOCHEMICAL

Vol. 56, No. 2, 1974

acid analyzer).

This result

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is in accordance

obtained

by Donald et al.

guration

of carbohydrate-peptide

with

the data

who have established

/3/

linkages

d-confi-

in EGS from the ot-

her source. 0-(d-N-acetglgalactosaminyl)-(prolyl)-serine copeptide

5.1.2.l

with

ed by electrophoresis

at pH

2) and subsequently figuration

GalN:Ser:Pro

(zone

bond for

has been accepted

Gly-

was isolat-

1.0:0.85:1.0

and at pH 3.6 (zone

I>,

by paper chromatography

of 0-glycosidic

des described

1.9

=

(II).

this

(zone and othor

by analogy

with

A-Con-

I).

glycopepti-

glycopeptide

I.

O-/~-O-(hr-acetylgalactosamingl)-d-N-acetglgalactoseming1/threonyl-alanyl :Thr:Ser:Ala at pH te

-serine =

(III

) . Glycopeptide was isolated

2.0:1.08:1,0:1.2

when

III

was subjected

(N HCl, 4 h, IOO'),

NaBH4 treatment

ed by GLC analysis

using Fye series

graph (3% OV-17 on GasChrom &, 0.4 x 150 cm, 215'), tose in III.

by electrophoresis

oxidation

threonine

to serine. galactosamine on alkaline

the absence of galacresults

in the comple-

and only 5O% of galactosamine. residues

bonds in a disaccharide,

The presence residues

follow-

mesh, column size

of III

two N-acetylgalactosamine or 1 --t4

/4/

model 64 gas cbromato-

104

100-120

aceta-

to hydrolysis

and acetylation

thus demonstrating

Periodate

te destruction I-3

with GalN:

(zone 4) and at pH 3.6 (zone 2). No galactitol

1.9

could be detected

quently

14.4.2

of l-3

degradation

of methylamide

with

which is attached

linkage

was established

are linked

Conse-

between N-acetyl-

by comparison

of data

of glycopeptide

III

and of model compound 0-/4-O-(J3-acetglglucosaminyl)-~-N-acetylglucosaminyl/-N-benzyloxycarbonyl-Lserine /5/.

while

In the former in the latter

case 70% of galactosamine case

(1

-4

linkage)

314

methylamide was destroyed, glucosamine

was

not

Vol. 56, No. 2, 1974

affected.

BIOCHEMICAL

Glycopeptide

methylamide

since free

j3-elimination pyridine

/6/.

mixture

diazomethane (36 h,, 0') III

III

had to be first

carboxyl 20 min,

and treated

with

methylamine

isolation

converted

group of serine

It was acetylated (l:l,

/5/;

wi.thout

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

acetic

into

inhibits anhydride

and then esterified

100')

8% methylamine

by

in abs. methanol

was removed and methylamide

was treated

with

-

of

0.05M aqueous Na2C03

(4 h, 70’). 0+3-N -acetglglpcosaminyl)-I-serine a model compound for selection of amidation

was used as and degradation

conditions. _N'-( -N*-ace jtylglacosaminyl)-asparagine de 2.3 with

GlcN:Asp = 1.0:0.93

sis at pH 1.9 (zone 3)* Its chromatography A-6 resin, buffer,

Glycopeptiwas isolated

elution

by electrophore-

time upon ion-exchange

(47 min, column 0.9 x 5.5 cm packed with

elution

with

0.2M sodium citrate-hydrochloric

pH 2.2 at 60°, SOml/h) and electrophoretic

at pH 3*6 and 1.9 were identical

hydrolase

/7/

determination

with

subsequent

of aspartic

some preliminary

structure

of peptide

regions direct peptide

of serine

evidence

here and in the previous

conclusions

of glycopeptide

chains

linkages. and threonine acids.

O(-configuration 315

the

of BGS near the The isolation

1)

/I/

of 0-glycosidic

both hydroxyamino

I confirmed

paper

can be made concerning

and carbohydrate (I-III)

and quantitative

acid.

of the presence

bonds with

of

bond were confirmed

identification

of carbohydrate-peptide

glycosides

sample

with ~-aspartyl-glycosylamine-amido-

From the data presented /'l/

mobilities

The structure

of N-glycosaminic

by enz:ymatic hydrolysis

acid

to those of synthetic

of N-(P-Nf-acetylglucosaminyl)-L-asparagine. IV and configuration

Aminex

is the first carbohydrate-

2) The isolation of these bonds.

of

Vol.

56, No.

2, 1974

BIOCHEMICAL

3)

The structure

of glycopeptida

data on the presence substances adjacent lated

AND

built

BIOPHYSICAL

III

hydrophobic

acids - alanine

recent

suggestion

rate-peptide isolated

The isolation /9/

that

bonds alongside

residues

and proline

with

from pig stomach lining

which is

acid linked

with

car-

IV supports

our

are N-glgcosylaminic O-glycosidic according

iso-

are bound either

of glycopeptide there

the

4) In the glycopeptides

with NH*- or COOH-group of hydroxyamino 5)

with

core in blood group

up of N-acetylhexosamine chain /8/.

COMMUNICATIONS

is in accordance

of the carbohydrate

to the peptide

bohydrates.

RESEARCH

to

carbohyd-

bonds in BGS /IO/.

REFERENCES

1, Kochetkov,N.K.,

and Medvedev,S.A.,

(1973).

Derevitskaya,V.A., Likhosher&tov,L.M., Biochem.Biopbys.Res,Commun, 52, 74&

Gray,W.R., in "Methods in Enzymology" (&l. by Hirs,C.H.W.), vol,ll, Acadwmic Press, New York, 1967, p.469. Creeth,J,W., Morgan,W,T,J., and Watkins,W.M,, 3. Donald,A.S,R. 125 (1969). I, Sloneker,J.H,,acd Jeans,A., Anal,Chem.z, 4. 2.

5. Likhosherstov,L.M., Izv,Akad.Nauk

Novikova,ii.S.,and Derevitskaga,V.A., SSSR, Ser.Khim. 2, 652 (1971).

6, 7.

Kaverzneva,E.D,,

and Tiutrine,G.V,,

Biokhi-

8.

Martinova,M,D.,and Dokl,Akad.Nauk SSSR 203, 700 (1972). Kochetkov,N.K., Derevitskaya,V,A., and Fedorova,V.I., IO. Likhosherstov,L.M. Biokhimiya 34, 45 (1969).

9.

316