Blood-group activity of human Chorionic Gonadotropin

BIOCHEMICAL

Vol. 55, No. 3, 1973

BLOOD-GROUP

ACTIVITY

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF HUMAN CHORIONIC

GONADOTROPIN

L. M&z, 0. P. Bahll Department of Biochemistry J. of Microbiology,

Department

Received

and F. Mohnl Blood-Group

State

School University Buffalo,

September

25,1973

Research

Unit

of Medicine of New York at Buffalo N. Y. 14214

SUMMARY Human Chorionic blood-group tion. in

A-like

inhibition the

Gonadotropin activity

of

.A11 of

the

hormone

contain

any N-acetylgalactosamine,

for

blood-group

the

comprised

(hCG-a

and hCG-6)

15,000

and 23,000,

in

of

D-galactose, acid

(hCG) is

two noncovalently

with

approximate

respectively

subunits

The carbohydrate

(l-4).

it

is does

a component

located

not

necessary

molecular .and each

have moiety

been of

subunits weights

containing

and amino recently

hCG-a

N-acetyl-D-glucosamine

D-mannose,

a glycoprotein

bonded

The monosaccharide both

hemagglutina-

activity

although

Gonadotropin

30% carbohydrate. sequences

the

to have

A activity.

Human Chorionic hormone

found

as shown by the

technique.

a subunit

has been

about acid

established

is made up of and sialic

while that of hCG-6 contains N-acetyl-D-galactosamine This work was supported in part by U. S. Public 1.

Health Population

Service

Grants

Council

AM10273

and HEO5351,

of New York. 717

Copyright 0 1973 by Academic Press, Inc. All rights of reprqduction in any form reserved.

of

and by the

BIOCHEMICAL

Vol. 55, No. 3, 1973

and L-fucose,

in

Furthermore,

addition

the

in the

RESEARCH COMMUNICATIONS

above

part

monosaccharides.

of

features linkages,

the

resembles

mucins

In

on the

proteins

on the

prompted

an examination activities.

this

This

are

the

hormone

and the

serum

structural

the

molecule

glyco-

for

various

presents

and their

prese::t

considerations

communication

such an investigation

serine respect

These of

as two types

N-acetylglucosaminyl

one hand

other.

hCG-$ molecule

inasmuch

and N-acetylgalactosaminyl same molecule.

group

the

structural

carbohydrate-protein

asparagine

of

to

carbohydrate

has some unusual of

AND BIOPHYSICAL

possible

blood-

the

results

physiological

significance. MATERIALS

AND METHODS

Highly

purified

described units

samples

(5) were

of hCG prepared

employed

in

of hCG were prepared

with

8 M urea

followed

A-50

and Sephadex

(LH) was kindly acid

studies.

The sub-

by dissociation

of

by chromatography

G-100

(8,2).

supplied

glycoprotein

these

as previously

luteinizing

Papkoff.

were purified

hormone

on DEAE-Sephadex

Ovine

by Dr.

the

hormone

Fetuin

by published

and ol-

procedures

(6,7).

Determination Serial

(9): buffered (w/v)

of twofold

saline of each

an equal

volume

or lectin

just

ate

3% PBS cell

carried

out

ABH and Lewis serologic

solution, substance

pH 7.35 under

immediately

in

(PBS)

of

examination

maximum

suspensions

group

dilutions

oc one pre-selected causing

blood

in

prior

718

were mixed

agglutination

to

preliminary each

phosphate 1% solutions

dilution

the

activities

assay.

with

of

antiserum

of

approprititration

The sub-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 3,1973

stance-antibody ture

for

15 minutes

minutes were

mixtures

for

incubated temperature

In

examinations

properties,

a lectin

a simple

group of

saline gorse)

Any A-like immune)

0 secretor

activity

1% solutions and group controls.

of porcine

cells

with

group

B salivas. a human

controls

human

Le(a-b+)

and Le(a-b-).

RESULTS

AND DISCUSSION

Lewis tion

blood-group technique.

salivas

of

activities The hormone

salivas

inac-

salivas

as

was determined serum

and group

B

was examined cells

using

as

Le(a+b-),

was examined

719

(non-

and nonsecretor

phenotypes

showed

(9).

Al

activity

by the

al.

and human group

and Le(a+b-)

Gonadotropin

samples

with

secretor

the

single

AZ erythrocytes

anti-B

blood-group serum

was used with

a natural

property

human

of human Lewis

Human Chorionic

and group

as

europaeus

by Mohn --et

A substances

(nonimmune)

anti-Lea

Ulex

of

using

any B-like

controls

prepared

(10)

and nonsecretor

Similarly

a natural

with

serum

A2 secretor

agglutination.

and nonsecretor

was detected

anti-A

of

consisted

as reported

at

inhibitory

previously

The controls

human

seeds

were

centrifugation

specificity

the

tubes at room

macroscopic

anti-H of

and clarified

30 minutes

H antigen-like

as described

0 cells.

tivated

with

extract

human group

with

for

cells

the

following for

and 30

detector

contents,

15 or

read

tempera-

ABH inhibitions

their

and,

were

at room

appropriate

an additional

3 min,

the

(common

mixing

accordingly

600 g for

for

and then

After for

incubated

in tests

Lewis,

added.

were

for

hemagglutination a significant

A,B,H

and inhibi-

cross-reac-

4 f

2 P

(11

+t

++++ +++ +++ tt +t

33rl

&

++++ +++t ++++ +t+ +t

++++ +++t ++++ ++-I-+ +++t

Cl

+t++ +++ ++ +t

++++ ++++ ++++ -I-+++ +++t

sasuezpqns

LPT-Sfl

++++

Q

aurarod

600-S3

++++

as

&

++

++++ +++t ++++ +++ ++t

ZQ dno;cs

++++ t++t +4-t+ t+t+ t-t++ +++t +ttt

+t++ +t+t +ttt +++t ++t+

SPAJ'IBS

as

A

+++t +tt ++t tt

IQ dnoxg

as

++++ ++++ t+++ ++++ ++++ ++++ ++tt

++++ +tt+ t+++ ttt+ +t+t

8ZT:T v9:'I ZEET g-c:1 8:T tr:-E z:T

auras P-LOT :'I ITS:1 9SZZT

= as

= as

pa3n~rtwn

zoqaxoas

zoziamasuou

Vol. 55, No. 3,1973

tivity

with

almost

all

BIOCHEMICAL

blood-group of

the

and,

on a weight

than

in

the

the

f3 subunit

conditions human

blood-group hCG used

these

all

which

does

mine,

a necessary

of

the

not

Blood-group

not

studies

component is

(12)

to this

contain

with

the

present

in

hCG-a.

Since

inhibit

the

ally

appears the sary

that

possible

the

this

the

unit

A substance. with but

nonthere

to a recent

(CEA) of

was found

the

human

to possess

A-like

any N-acetylgalactosamine of

the

mannose

these

of

antigen

are

monosaccharides

structure impart

cross-reactivity

the with

also

individu-

reaction, of the

were

and L-fucose

L-fucose,

hemagglutination

must

since

a subunit

according

exception

overall

carbohydrate for

rule

N-acetylglucosamine,

do not

in

components

which,

of

N-acetylgalactosa-

antigen

acid,

and

Further-

(ll).,

sialic

serum,

purified.

N-acetylgalactosamine

not

in

hCG preparations

associated

The monosaccharide

l

in

The antigen

and did

activity

fetal

generally

system.

a subunit

assay

of blood-group

on carcinoembryonic

digestive

the

be due to any contaminant

any detectable

A activity

u subuni‘t

show any activity.

was located

contain

the

similar

was highly

activity

terminal

not

Furthermore,

The activity

bovine

present

seems to be exceptions report

from did

could

more,

reducing

fetuin

A substance in

in in

I).

Under

glycoprotein

activity

resided

(Table

was negligible.

al-acid

I).

was much greater

hormone

LH,

RESEARCH COMMUNICATIONS

(Table

activity

basis,

ovine

This

A substance

A-like

intact

AND BIOPHYSICAL

whole

it or part

conformation

blood-group

necesA

substance. Although the

structural

more

work

components

is

needed required

721

to determine for

the

precisely A-like

activity,

of

BIOCHEMICAL

Vol. 55, No. 3, 1973

the

carbohydrate

the

serological

in

the

are since

activity.

located the

hCG with its

of hCG-cr does

hCG-anti-hCG

not

in

sequential

binding

the

Also,

least

part

of

the

the

been of

potentially postulated

sion

of

the

lymphocytes hormone

not

that not

of

the

very

molecule,

the

from

much alter by radio-

carbohydrate,

necessary

its

that

determinants

as determined

is

for

testicular

at

the

and ovarian

significance but

during

endometrium

by the

immunoresponse

has been

in view of

of

The question

which

those

of

is fetal the

by binding

to

exhibit

the

in

fetus has

acceptance

mother.

with that

the

antigens

be involved

be noted

present

fascinating

implantation

the

found

the

suppression

since

hCG might

may

of

biologists

from

It

is

the

(13).

mother

that

(13).

it

the

transplantation

which

to note

monosaccharides

does

with

different

been

the

of hCG in

function

fetus

it,

clear,

role

puzzling

the

of

yet

possible

on maternal

of

physiological

is not

lymphocyte

part

with

receptors2.

The precise

of

carbohydrate

appears

hormone

membrane

findings

antigenic

antibody2 it

interesting

the

glycosidases

-with

a major

is

removal

immunoassay.

plasma

the

RESEARCH COMMUNICATIONS

seem to be associated

It

reaction

specific

binding

AND BIOPHYSICAL

are It

the

has suppres-

maternal

hCG is

the

first

blood-group

activity.

2.

Manuscript

in

preparation.

ACKNOWLEDGMENT The authors

wish

to thank

Dr.

suggestions.

722

V. Ginsburg

for

helpful

BIOCHEMICAL

Vol. 55, No. 3,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

Bahl,

0.

P.,

J.

Biol.

Chem.,

244,

575

(1969).

2.

Bahl,

3.

Bellisario, Chem.,

4.

Carlsen, R. B., them., -'248

Bahl, 0. P., and Swaminathan, (1973), in press.

5.

Bahl,

Biol.

6.

Spiro,

7.

S&mid,

8.

Swaminathan, Commun.,

9.

Mohn,

0. P., in Hormonal Proteins and Peptides, (1973), C. H. Li (Editor), Academic Press,

0.

R., 248,

P.,

J.

R. G., K.,

Carlsen, (1973),

J. J.

R. B., and Bahl, in press.

Chem.,

Biol.

Chem.,

Am. Chem.

244, 235,

Sot.,

N. and Bahl, 0. 40, 422 (1970).

567

P.,

0. P.,

J.

N.,

Biol.

J.

Biol.

(1969).

2860

-75,

Vol. I p. 171.

(1960).

60 (1963). Biochem.

Biophys.

Res.

J. F., Cunningham, R. K., Pirkola, A., Furuhjelm, and Nevanlinna, H. R., VOX Sang., 24, 385 (1973).

10.

Milgrom,

F.,

Mohn,

11.

Feizi,

T., Marsh,

12.

Gold,

J. Biol.,

13.

Adcock, E. W., Teasdale, F., August, C. S., Cox, Meschia, G., Battaglia, F. C., and Naughton, Science, 181, 845 (1973).

Kabat, L., J. M.,

J.

F.,

and Loza,

E. A., Vicari, Immunol., 106,

Freedman, S. O., 239, 80 (1972).

723

U.,

VOX Sang.,

G., Anderson, 1578 (1971).

and Gold,

P.,

B., Nature

U.,

in press. and New S., M. A.,