Vol.

174,

January

No.

2, 1991

BIOCHEMICAL

AND

BlOPHYSlCAL

RESEARCH

COMMUNICATIONS

Pages

31, 1991

~-SUBUNITS

OF

EQUINE

AN IDENTICAL

CHORIONIC

AMINO

ACID

GONADOTROPIN

SEQUENCE

RAVE

OLIGOSACCRARIDE

Taei

MATSUI, Darrell

Hiromu SUGINO”, N. WARD” , Koiti

sDepartment of Texas,

University Received

December

24,

DIFFERENT

HORMNE

WITH

ASPARAGINE-LINKED

CHAINS

Mari MIURA, George R. BOUSFIELD”, TITANI, and Tsuguo MIZUOCHI’

Institute Program, Wako, Saitama

Research

LUTENIZING

Institute Polymer Science, Health University School Aichi 470-li, Japan

Division of Biomedical Medical Science, Fujita

QFrontier

AND

940-945

for Comprehensive of Medicine, Toyoake,

of Physical and Chemical 351-01, Japan

of Biochemistry and Molecular M.D. Anderson Cancer Center,

Research,

Biology, Houston, Texas

77030

1990

The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a o-subunit with an identical amino acid sequence. The Asn-iinked oligosaccharide chains of eCGB and eLHB were quantitatively liberated as tritium-labeled oligosaccharides by hydrazinolysis foiiowed by N-acetylation and NaBJHd-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCGs contained neutral and sialylated oligosaccharides, while eLHB contained neutral, sialylated, sulfated, and both sialylated and suifated oligosaccharides. In addition, elution profiles on a Bio-Gei P-4 column of the neutralized oligosaccharide mixtures of eCGB and eLHB were different, indicating that the moiecular masses of oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked oligosaccharide chains of eCG@ and eLHfl are different although they have an identical amino acid sequence. 0 1991 Academic mess, Inc.

Gonadotropins non-covalently

are

glycoprotein

associated

(L- and p-subunits

a common a-subunit

which

species.

A unique

o-subunit

role

in determining

the

primary

Recently,

the

complete

chorionic

gonadotropin

to be identical eLH is 1

(3,4).

consistently

To whom correspondence

0006-291X/91 Copyright All rights

hormones

has an identical

amino

of each the acid

(eCG8)

(1,2j. amino

hormone, individual

sequences

and lutenizing

composed

of heterodimers Each hormone

acid

sequence

therefore,

is

hormone hormone

the

two hormones

share

more active

than

eCG in terms

of

be addressed.

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

940

within

a

thought

to play (1).

from

equine

(eLH@) were

Although

should

possesses

specificity

of o-subunits

of

determined

a common receptor,

receptor

binding

Vol.

174,

No.

2, 1991

activity the

BIOCHEMICAL

and stimulation

carbohydrate

of

moiety

activities.

in the from

MATERIALS

AND METHODS

BIOPHYSICAL

steroidogenesis

(5,6j.

of the

present

iiberated

AND

two glycoprotein

study,

Asn at residue

RESEARCH

it hormones

we characterized

13 of each

is

the

COMMUNICATIONS

suggested

that

modulate

these

oiigosaccharide

eCGB and eLHg by hydrazinolysis.

Materials---eCGg and eLHfl were isolated from highly purified eCG and eLH, respectively, as previously described i3,7j. Sraphy iococcus aureus Vi protease was purchased from Miles Scientific iNapervilie, INj and Sephacryl S-200 from Pharmacia Fine Chemicais (Uppsala, Swedenj. 6io-Gel P-4 (-400 mesh) was from Bio-Rad Laboratories (Richmond. CA). NaB:H, (455 GBq/‘nunoij was from New England Nuclear (Boston, MAj. Sialidase purified from drthrobacrer urenfaciens was from Nacaiai Tesque Inc. iKvoto, Jaoan _ _ ,j . Liberation of Asn-iinked oligosaccharides from V; protease fragments of eCGB and eLHg---Each D-subunit was digested with S. aureus Vi protease (E~S=!/W) for 4h at 37 OC after reductive S-carboxymethviation as previously described (3,4j. A fragment (residues 1 to 21’1 containing an-Asniinked oligosaccharide but no Ser:Thr-iinked oligosaccharide was isolated from each digest on a Sephacryl S-200 column and verified by amino acid sequence anaiysis. The fragment (80 ugj thus obtained from each p-subunit was subjected to hydrazinolysis for 9h foiiowed by N-acetylation (iii. The oiigosaccharides iiberated from the polypeptide moiety were reduced Xith NaB,HJ (150 MBqi in 50 ~1 of 50 rmY NaOH for 4h at 30 ‘Y folicwed by further reduction with NaBH, for 2h. ‘The tritium-iabeied oligosaccharides were separated by paper chromatography as described i9j. Analyticai Methods---Paper electrophoresis, sialidase digestion, soivolytic desulfation and Bio-Gei P-ii column chromatography were performed as described (9-ilj. Siaiidase treatment was performed twice for the siaiidase-resistant acidic fractions from eLHB.

RESULTS AND DISCUSSION Paper

eiectrophoresis

eCG@ and eLHg---When subjected

to paper

the

three

acidic

fractions,

ratio

of N, CAl,

: 6.4

from

sialidase,

the (Fig.

acid

di-,

fractions indicating

Fractions

from

Asn-iinked

saccharides shown

in Tabie

fraction

(LNj : 43.3

zero

their

complex-type

sialic

with

converted nature

into

These

: 26.5

neutral

same areas

as

respectiveiy, results

indicated

a mixture

residues

The

was due to sialic

in the

of eCGB are acid

ia;.

Upon incubation

oiigosaccharides,

chains

to three

(Fig.

: 4i.ii

acidic

and human FSH (i3j.

CN, and

to be 25.1

CAZ, and CA3 migrated

oligosaccharide

containing

of oligo-

in the

ratio

I.

Radioactive i6.5

CA:,

human CG ii2j

completely

that

and trisiaiylated

obtained the

were

fraction,

obtained

radioactivities.

ib),

that

a neutrai

CAZ, and CA3, were

from

of eCGg were

CA2, and CA3 was caicuiated

acidic

residues.

mono-,

CAi,

iiberated

oligosaccharides at pH 5.4,

incorporated

components

oiigosaccharides

radioiabeled

eiectrophoresis

moiar

their

cf Asn-linked

oligosaccharides and two acidic

: 40.2

by paper

of eLH@ were fractions

iLA1

electrophoresis 941

separated

and LA2) (Fig.

lcj.

into

in a moiar

a neutral ratio

Upon incubaticn

of

Vol.

174, No. 2, 1991

BIOCHEMICAL

-

b

CN

---

AND BIOPHYSICAL

CA1

CA2

RESEARCH COMMUNICATIONS

CA3

’

A

n

LNl

d

e

LSI

A i\_ m

-

LN2

f

LNS

As2

3

J 1

0

10

DISTANCE

30

20

FROM

ORIGIN

(cm)

Fig. 1. Paper eiectrophoresis of the radioactive Asn-iinked oligosaccharides liberated from eCG@ and eLH,g by hydrazinolysis. The radioactive oligosaccharide mixture was subjected to paper electrophoresis at pH 5.4. Arrows indicate the positions where authentic lactitol (lj, sialyllactitol (2), and bromphenol blue (3j migrated. a, radioactive oligosaccharide mixture from eCGB; b, acidic oligosaccharide mixture in a (CAl, CA2, and CA3) after incubation with sialidase; c, radioactive oligosaccharides from eLHfl; d, fraction LA1 in c after sialidase digestion: e, fraction LSl in d after solvolytic desulfation; f, fraction LA2 in c after sialidase digestion: g and h, fractions LNS and LS2. respectively, in f after solvolytic desulfation. with sialidase, but the remaining This

ldj. verted

a portion portion

sialidase-resistant

to a neutral

suggesting

that

TableI.

(34%j of the LA1 fraction became neutral was not susceptible to enzyme digestion acidic

component

the

Comparison

acidic

after

nature

of Asn-linked

fraction solvolytic

of

(LSij

was completeiy

desulfation

LSl was due to sulfate,

oligosaccharides

(LN1) (Fig.

(Fig. which

conlej, has been

derived from eCG@ and eLHP

Oligosaozhatides neutral

monosialylated

eCGj3

26.1%

41 .O%

eLHj3

16.5%

14.7%

disialylated

trisialylated

monosialylated & monosulfated

26.5%

6.4%

0

4.4%

0

942

12.1%

s:Pf::d 0 26.6%

su&?d 0 23.7%

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

7.22018 16 14 tttttttttt In m

BIOPHYSICAL

12 10 t t t t

RETENTION

8 t 1y

TIME

RESEARCH

COMMUNICATIONS

’ i

t

I

(hr)

Fig. 2. Bio-Gel P-4 column chromatography of the neutral fractions of eCG6 and eLH0 obtained by either desialylation or desulfation. Neutral or neutralized radioactive oligosaccharide fractions from eCG0 (a) and eLHfl (b-ej were subjected to Bio-Gei P-4 column chromatography. Big arrows indicate the elution positions of glucose oligomers (numbers indicate the glucose unitsj added as internal standards. Small arrows indicate the I, Gal., GlcNAca Man3 GlcNAc elution positions of standard oligosaccharides: (Fuc)GlcNAcoi (subscript OT indicates NaBsHa-reduced oligosaccharidesj; GalZGlcNAcyMan3GlcNAc(Fuc) II, Ga13GlcNAc;Man7GlcNAc(FucjGlcNAcnl ; III, Gi cNAco i ; IV, ManlGlcNAc(Fuc)GlcNAcn, . a, neutral oligosaccharide mixture obtained from total eCG0 oligosaccharides by sialidase digestion; b, neutral oligosaccharide mixture (corresponds to a mixture of fractions LN, LNl, and iN2j obtained from total eLHB oligosaccharides by sialidase treatment; c, d, and e, neutral oligosaccharide fractions from LSl, LS2, and LNS, respectively, by solvolytic desulfation.

shown

for

portion

eLH I i4). was converted

sialidase the

another

same position

was about areas

as LSi

li

: 30 : 53.

where

mono-

electrophoresed,

it

disiaiyiated

was suggested

oligosaccharides (Figs.

soivolysis at

the

(at same

lg

as LSl

positions

susceptibility

2hj,

of LSi,

they

of LN2, which

derived

mrgrated

from

mono-

neutral

shownj.

after peak

Based

desulfation

solvoiytic observed

on the and their

monosulfated

oiigo,saccharides and LS2 consists of disulfated oiigosacSince LNS was derived from LA2 by sialidase treatment, it that

LNS is

derived

from

monosialylated 943

that

to mild was

on paper

charides.

was suggested

and

Both

mobilities

suggested

to were

LS2 was subjected not

resistant

oiigosaccharides derived

at

LNS, and LS2

respectiveiy.

LNS and LS2 to solvolytic it

(iS2i

intermediate

and LNS idata

appeared

LN1 and LNi were

were

When fraction a radioactive

eiectrophoresis,

fraction

complex-type that

a smali

:iNSi

respectiveiy,

LNS and LS2 became

and lhi.

25 OC for

exhaustive

ratio

in LA1 and LA2,

fractions

after

fraction

fractions

and disialylated

siaiidase-resistant desulfation

neutral

oniy

acidic

The molar

LA1 and LA2,

although

LiNij

acidic to the

if,.

Since from

LA2,

component

radioactive

(Fig.

treatment

fraction

in addition

digestion

by sialidase

case of

to a neutral

treatment,

to the enzyme

the

In the

LSl and LNS are

and monosuifated

was oligo-

Vol.

174,

No.

2, 1991

saccharides

in

LAZ.

monosialylated,

The presence

sulfate

and sialic Although

eLHB (84%j three

sialic

contents

presence

acidic

order

were

that

Gel

column

in the

mono-

ratio

shown

containing reported

(IS!.

in eCG@ (74%j

and

The acidic

nature

different. to the

in both

presence

of eLH0 was mainly

of one to

due to the

Ij.

to analyze

oiigosaccharides

neutral,

and both

oligosaccharides

of neutralized

neutral P-4

while

(Table

profiles

coiumn---In

chains

groups

COMMUNICATIONS

eLHB contains

oligosaccharides

of eCGB was due solely

of sulfate

Elution

that

and human LH has been

of acidic

residues

RESEARCH

disulfated,

Asn-linked

in ovine

similar,

acid

was suggested

oligosaccharide

of the

oligosaccharides

BIOPHYSICAL

monosulfated,

acid

the were

AND

it

and monosulfated

I.

of the

Finally,

disialylated,

sialylated Table

BIOCHEMICAL

oligosaccharides

the

size

obtained

of oiigosaccharides

as described

of eCG@ and eLiiB,

above

to biooligosac-

gave region

muitipie of complex

peaks

be bi-,tri-, without with

with

eiuting

obtained

(Fig.

2b-2ej

elution

by either

siaiidase

were

different

totai

of

fractions

from

LSl,

and ie,

respectiveiy)

complex

elution

In the in their

present

and size

sequence.

glycosylation

site,

structures

is

hormones

markedly

of adenylate receptor-binding

indicated. cyclase

and the

positions

that

both

there

to

treatment

of

oiigosaccharide (Fig.

units.

is

on the

(corresponding

2c,

Aithough

2d, their

much microheterogeneity some peaks

were

the presence

in

eluted

at

of a

groups. the

diversity

of structures

on eCGB and eLHB and the

these

two giycoproteins

glycoproteins

is well

that

944

oniy

their

deglycosylation

although

of

difference an identicai

one N-

oiigosaccharide

effectiveness, without

and that

with

contain in

known

biological

(17,18)

Eiution

desulfation

microheterogeneity the

2aj.

neutral

suggesting

between

desulfation

by sialidase

fractions,

chains

oligosaccha-

mixture

9 to 16 giucose

and steroidogenesis,

activity

to

or

eiution

solvolytic

solvolytic

charge

It

reduces

or

obtained

we demonstrated

Since a high

neutralized

of eLHfl.

different study,

of the

structure

oligosaccharide

charge acid

between

among these

the Asn-iinked

2bi

and LNS by indicated

with

their

of eCG0 (Fig.

and iN2j

oligosaccharide

common structure

(iG,i6i.

suggested with

comparing

oiigosaccharide

LNl,

were

same position

that

(Fig.

profiles

the Asn-iinked

amino

LN, LS2,

after

treatment

neutral

eLHB oiigosaccharides

fractions

were

oligosaccharides

profiles

from

of the

units

units

(lO,i6j.

case of eLHs,

P-4 coiumn

a mixture

repeats

oiigosaccharides

rides

M-acetyiiactosamine

17, and 19 giucose compiex-type

N-acetyilactosamine authentic

Bio-Gel

to five

14,

and tetraantennary

In the

the

two

at about

2a,

subjected neutrai

oligosaccharides

in Fig.

were the

Major

As shown

F-4

charide mixture obtained from eCG@ by siaiidase digestion peaks at positions between i3 to 23 glucose units in the type

chromatography.

on a Bio-Gel

such

interfering the

of the as stimuiation with

two hormones

the share

Vol.

174,

No.

2, 1991

a common receptor, receptor

binding

BIOCHEMICAL

AND

eLH is consistently activity

BIOPHYSICAL

more active

and stimulation

RESEARCH

COMMUNICATIONS

than eCG in terms

of steroidogenesis

(5,6j.

of the The

present study suggests that the structural differences in the oligosaccharide chains of these two glycoprotein hormones with an identical amino acid sequence have a significant effect on their biological activities. date, oniy the major component of oiigosaccharides in eCG has been structuraily

investigated

(19,iOj.

structures

of the oiigosaccharide

to ciarify

the biologicai

TO

It wiil be necessary to eiucidate the moiety of eCG and eLH in detaii in order

function.

ACKNOWLEDGMENTS ‘This work was supported in part by Grants-in-Aid for the Scientific Research from the Ministry of Education, Science and Cuiture of Japan and from Fujita Health University. REFERENCES 2. i. Ryan, Pierce k ;.G., and Parsons, T.F. (198lj Ann. Rev. Biochem. 50, 465-495. . ., Chariesworth, M.C., McCormick, D.J., Miiius, R.P., and Keutmann, il.?‘. (1988j FASEB. J. 2, 266i-2669. 3. Suqino, H., Bousfield, G.R.. Moore, Jr., W.T., and Ward, D.N. (i987i J. Bioi. Chem. 262, 8603-8609. 4. Bousfieid, G.R.. iiu. Chem. 262, ~alio, 86iO-8620A*;*ly*;g~;~:y: ::;*yY?;:: ;:;:, y:yo”i;;;* I 5. iicht, P., . ^__. and Papkoff, H. :IY!YJ J. Endocrinoi. 83, 311-322. 6. Stewart, F., and Aiien, W.R. (19793 J. Reprod. Fertii. 27 (Suppl.j, 43i-440. 7. Bousfieid, G.R., and Ward, D.N. (i984j J. Biol. Chem. 259, i91i-i921. 8. Takasaki, S., Mizuochi, ,i., and Kobata, A. (i982i Methods Enzymoi. 83, 263-268. 9. Mizuochi, T., Fujii, J., Kisiel. W., and Kobata, A. ii981j J. 6iochem. 90, iO23-iO3i. i0. Yamashita, K., Mizuochi, T., and Kobata, A. (i982j Methods Enzymoi. 83, 105-i26. il. Yamashita, K., Ueda, I., and Kobata, A. (1983j J. Bioi. Chem. 258, i4i44-i4147. i.2. Mizuochi, T., and Kobata, A. (198Ol Biochem. Biophys. Res. Commun. 97, 772-778. i3. Renwick, A.G.C., Mizuochi, T., Kochibe, N., and Kobata, A. i1987j J. Biochem. 101, 1209-i221. 14. Ward, D.N., Wen, T., and Bousfield, G.R. (i987) J. Chromatogr. 398, 255-264. 15. Baenziqer, J-U., and Green, E.D. (1988) Biochem. Biophys. Acta. 947, 287-306. i6. Kobata, A., Yamashita, K., and Takasaki, S. (1987j Methods Enzymol. i38, 84-94. i7. Keutmann, H.T., Mciiroy, P.J., Bergert, E.R., and Ryan, R.J. (i983j Biochemistry 22, 3067-3072 i8. Sairam, M.R., Bhargavi, G.N. (1985j Science 225, 65-67. 19. Bahl, O.P., and Wagh, P.V. (19863 Adv. Exp. Med. and Biol. 205, l-51. 20. Damm, J.B.L., Hard, K.. Kamerling, J.P., van Dedem, G.W.K., and Vliegenthart, J.F.G. (i99Oj Eur. J. Biochem. 189, 175-183.

945