A new destruction of blood group substances. Isolation of glycopeptide containing O-glycosidic carbohydrate-peptide linkage

Vol. 52, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A NEWDESTRUCTIONOF BLOODGROUPSUBSTANCES.ISOLATION OF GLYCOPEPTIDECONTAINING0-GLYCOSIDIC CARBOBYDRATEPEPTIDE LINKAGE N.K.Kochetkov,

V.A.Derevitskaya,

L.M.Likhosherstov,

and

S.A.Medvedev N.D.Zelinsky Received

Institute of Organic Chemistry, of Sciences, Moscow, USSR

March

USSR Academy

30,1973

SUMMARY:De radation of blood group substances using chemical and enzymat Pc methods has been carried out. A glyoopeptide with an 0-glycosidic carbohydrate-peptide linkage has been isolated for the first time from biopolymers of such a type. It's structure has been determined as O-(N-acetyl-galactosaminyl)-Thr-Ala. Blood group substances (BGS) are known to be resistant proteolytic

enzymes (1) that makes practically

accomplish

their

structural

studies

regions

degradation

naged to split ans of a highly

impossible

small fragments

of the peptide

of carbohydrate-peptide

that carbohydrate

linkages*

glycosidase

(2).

But attempts

by proteolitic

enzymes failed.

hydrolysis

to hydrolyse Recently,

hods whereas the peptide

the we ma-

from Clostridithe modified

BGS

we succeeded in splitfrom BGS using a new

of chemical

and enzymatic met-

backbone of BGS was practically

unaf-

fected.

The obtained

splitted

by proteolytic enzymes (3). These results enabled us first time to develop a practical procedure of BGS de-

for

the

polypeptide

for

from BGS by me-

preparation

off about 95% of the carbohydrates

approach based on a combination

required

In view of the fact

about XX/o of the carbohydrates active

to

chain and particularly

chains prevent proteolytic

um perfringens ting

into

to

fragment has been easily

Vol. 52, No. 3, 1973

BIOCHEMICAL

gradation

in splitting

resulted

glycoprotein molecular

into

a mixture

and several

of aminoacids, containing,

linkage

with

between N-acetylgalactosamine

This provided

conclusive

and

experimental

evi-

carbohydrate-pep-

in BGS.

6.1 g of BGS (A+H) isolated cording

mixture

glycopeptide

dence for the occurence of the 0-glycosidic tide

and low

in average, one mo-

of an individual

linkage

residues.

peptides,

weight

aminoacid residues0 From this

we succeeded in isolating the 0-glycosidic

RESEARCH COMMUNICATIONS

of the high molecular

weight glycopeptides

nosaccharide

threonine

AND BIOPHYSICAL

to (4) was oxidized

from hog stomach linings

ac-

by aqueous sodium periodate

(0.04M NaJ04, 1.55 1, 5", 24 h), reduced with KBH4 (7.5 g, 5O, 3 days) and hydrolyzed polymer was isolated tion

of a glycosidase

ported in (2). incubated

at pH I.5

(3 h, IOOO). The resulting

on Bio-Gel P-IO and subjected preparation

from Cl.perfringens

The polymer was isolated

with pronase (2O:l)

fraction

determination

Gel P-2, and again treated

with a glycosidase

P-2 chromatography treated

on on Rio-Gel P-2 resulted tion

was isolated

and then the glycopeptide

pH 7.5,

buffer, as followed

hexosamines and aminoacids

Bio-Gel

as re-

on Bio-Gel P-IO and

in 0.2M tris-HCl

for 7 days at 37". The glycopeptide

Cl.perfringens

to the ac-

from

on Bio-

preparation

fraction

isolated

from by

with pronase* Gel-filtrati-

in two glycopeptide

fractions.

Frac-

I (about 400 mg) eluted with the void volume of a column

was not further

investigated,

a lower mobility

contained

only N-acetylgalactosamine samine ratio

ca.lO:l).

fraction

II

along with aminoacids practically (no galactose,

Fraction

graphy on cation-exchange pyridinium form, column

(about 200 mg) with galactosamine$gluco-

II was subjected

to chromato-

resin Aminex AG 5OW-X2 (200-325 mesh, 1.8 x 80 cm equilibrated with 0.05M

749

BIOCHEMICAL

Vol. 52, No. 3, 1973

pyridine-formiate

buffer

with 0.05M the stepwise

elution (300

te buffers,

fraction

gradient

elution

ml + 750 ml).

(750

was performed using 0.2M pH 3.6

Then

volume 5 ml. The elution

corresponding

reaction

curve was made

and revealed

19

peaks. The

to each peak were combined and the ra-

of aminoacids and hexosamines was determined

after

hydroly-

sis

(4N HCl, 24 h, IOCP) using the aminoacid analyzer

tronic

LC 4010). Fraction

ing by galactosamine Val 0.2,

paper chromatography

parative

threonine

was subjected

to preparative

45:30:9:36).

The zone with galactosaratio

acid-acetic

The homogeneity of this

fers with pH 1.9,

acid buffer

and electrophoresis 3.6 and 6.5).

according

ter hydrolysis glycopeptide

and consequently

The

alanine

1.02,

molar

was proved by pa-

(80 V/cm, 1 h in bufof the glycopep-

of two successive

to (5) with estimation

obtained show threonine

pH 1.9.

(relative

I.0

The structure

(4N HCl, 20 h, 100")

by pre-

(Whatman 3MM,

glycopeptide

was determined by application

The results

was purified

paper electrophoresis

0.97 and N-acetylgalactosamine

gradations

system butanol-py-

thus obtained gave upon hydrolysis

per chromatography tide

Pro 0.5, Ala 0.4,

= 1.0:1.2:0.3:1.4

60 V/cm, 1 h) in formic glycopeptide

Ser 0.1,

(Whatman 3bQI, solvent

high-voltage

(Bio-

between 132.5-1450 ml consist-

1.0, Thr 1.2,

acid-water

mine:Thr:Ser:Ala

ratio).

eluted

Leu 0.2 (molar ratios)

ridine-acetic

ml),

(300

ml) and 0.3M pH 6.3 (300 ml) pyridine-formia-

up by means of ninhydrin tio

RESEARCH COMMUNICATIONS

pH 2.6) using linear

0.2M, pH 2.6 buffer

0.2M pH 4.3

fractions

AND BIOPHYSICAL

Edman de-

of aminoacids af-

using aminoacid analyzera to be on the N-terminus

the glycopeptide

has the struc-

ture 0-(N-acetylgalactosaminyl)-T&Ala. Thus, the new procedure led to isolation glycopeptide

from BGS. Further

of first

short

study of other glycopeptide

750

of

BIOCHEMICAL

Vol. 52, No. 3, 1973

fractions

obtained by this

investigating

the detailes

chain in the regions eve that this lytic

AND BIOPHYSICAL

method seems to be promising of the structure

of carbohydrate-peptide

approach to BGS degradation

enzymes (papain and trypsin)

glycopeptides

suitable

RESEARCH COMMUNICATIONS

of BGS peptide linkage.

lative

We beli-

using other proteo-

would give rise

to larger

for a more thorough study of the pepti-

de chain of BGS, It should be noted that the structure peptide

for

chain of BGS has been subjected

of the

so far only to specu-

discussion, REFERENCES

1. Watkins,

W.&I*, in "Glycoproteins" (Ed.by Gottschalk,A*) Elsevier Publishing Company, Amsterdam, London, New York, 1966, pe493. 2. Kochetkov,N.K., Derevitskaya,V.A., Likhosherstov,L.M., and Martynova,Md.D., Dokl.Akad.Nauk SSSR, 186, 1195 (1969). Likhosherstov,L.&, Martynova,M.D., and 3. Derevitskaya,V.A., Kochetkov,N.K., Dokl.Akad.Nauk SSSRx]3, 700 (1972). 4. Likhosherstov,L.M. Derevitskaya,V.A., and Fedorova,V.I., Biokhimiya J4, 45 11969). 5. Gray,W.R., in 'Methods in Lnzymology~* (Ed.by Hirs, C.H.W.) ~01.11, Academic Press, New York, 1967, p.469.

751