Oxytocin biotransformation in the rat limbic brain: Chemical characterization of two oxytocin fragments and proposed pathway for oxytocin conversion

Vol.

97, No.

December

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATlONS

16, 1980

10051013

Pages

OXYTOCIN BIOTRANSFORMATION CHEMICAL CHARACTERIZATION

IN THE RAT LIMBIC

BRAIN:

OF TN0 OXYTOCIN FRAGMENTS AND

PROPOSED PATHWAY FOR OXYTOCIN CONVERSION J.P.H.

Burbach,

P. Schotman+

and E.R de Kloet

Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, and +Division of Molecular Neurobiology, Institute of Molecular Biology and Laboratory of Physiological Chemistry, University of Utrecht, Padualaan 8, Utrecht, The Netherlands Received

September

17,198O

SUMMARY Two peptide fragments of oxytocin were isolated by high-pressure liquid chromatography from digests of oxytocin obtained after exposure to a SPM preparation of the rat limbic brain. The structures of these peptides, being Gln-Asn-Cys(0)x-Pro-Ieu-GlyNH2 and Gln-Asn-Cys(-S-S-Cys)-Pro-Leu-GlyNH2, were combined with the determination assessed by quantitative amino acid analysis, of N-terminal end groups and cysteic acid residues after performic acid treatment. The fragments comprised the 4-9 and 1,4-9 sequences of oxytocin, respectively. The types of proteolytic enzymes involved in their formation are discussed and a pathway for the conversion of oxytocin by SPM is proposed. INTRODUCTION The neurohypophyseal to affect midbrain

brain

which

molecules,

elicit

tion

of the

activities

related

are thought are devoid central

in the central

which

studies, are

to mediate

effects

as well.

Therefore,

might

have been (1,2).

processes.

endocrine

Limbic

Fragments

effects

be a regulating

and of both

of their

proteolytic factor

shown

parent

fragmentaof their

system.

we have partially

involved

and memory

these

classical

hormones nervous

and vasopressin,

to learning

of the

neurohypophyseal

In previous peptidases

mechanisms

structures

hormones,

hormones , oxytocin

in the proteolytic

characterized conversion

SPM associated of oxytocin

Abbreviations: SPM = synaptosomal plasma membrane(s), HPLC = high-pressure quid chromatography, DNS= dansyl-, l-dimethylaminonaphthalene-S-sulfonyl-. The amino acid residue numbers refer to the primary structure of oxytocin: CysI-Tyr-Ile3-Gln-AsnS-Cys-Pro7-Leu-GlyNH2g. 1 4

1005

(3‘4). li-

0006-291X/80/231005-09$01.00/0 Copyright 0 1980 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol.

97, No.

In these

3, 1980

BIOCHEMICAL

experiments

structure

the accumulation

was observed residue,

it

graphically

distinct

from

We termed

The present from oxytocin previously ed to point preparations

was devoid

paper

described our

this

and their data

routes

of the rat

This

of the

the C-terminal

describes

digests

BIOPHYSICAL

of an oxytocin

in the digests.

glycinamide

and Leu-GlyNH2.

AND

chemical

on oxytocin

in a pathway limbic

residue

and dipeptides,

tentatively isolation

oxytocin

of multiple

characterization. converting

for

fragment contained

tyrosine-2 tri-

fragment the

peptide

RESEARCH

oxytocin

peptidase conversion

COMMUNICATIONS

of unknown the

C-terminal

and chromatoPro-Leu-GlyNH2 x-9

(OXT x-9).

OXT x-9 These

components

and the

activities mediated

allowby SPM

brain.

MATERIALS AND METHODS Synthetic oxytocin, C$s-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2, and its fragments 1-8, l-7, 1-6, 7-9 and 8-9 were kindly provided by Dr. H.M. Greven and Dr. J.W. van Nispen (Organon International B.V., Oss, The Netherlands). [9-GlyNH2-l-l4 C] -oxytocin was a generous gift of the late Dr. R. Walter (University of Illinois, Chicago, USA). A SPM fraction from rat limbic brain tissue (hippocampus, hypothalamus plus septum) was pre ared by the method of Zwiers et al. (5). Oxytocin (2x10 -E M; 4~10~ dpm of [14C-GlyNH2]-oxytocin was incubated with SPM preparations (1.7 mg protein/ml) at 37OC for 3 h in 1.5 ml 25 mM sodium phosphate buffer, pH 6.9 . The reaction was terminated by heating the suspension in a boiling water bath for 10 min and membranes were removed by centrifugation. The supernatant was subjected to HPLC fractionation. Reversed-phase HPLC of the oxytocin digests was carried out on a uBondapak Cl8 column (0.39 x 30 cm), which was eluted with a linear gradient over 25 min of 5% to 40% acidified methanol (1.5 ml acetic acid per liter methanol) in 10 mM ammonium acetate, pH 4.15, at a flow rate of 2 ml/min. The eluate was monitored continuously by UV absorbance at 210 nm and fractions of 1.0 ml were collected. Aliquots of the fractions were subjected to scintillation counting. At the guidance of the radioactivity distribution fractions were pooled and lyophilized after removal of methanol in vacua at 60°C. Freeze dried samples (circa 2 nmoles) were hydrolyzed with 6 M hydrogen chloride containing 1 I.~M thioglycolic acid in evacuated glass tubes in a constant boiling toluene bath (11OOC) for 22 h as described by Zwiers et al. (6). The amino acid composition was determined by automated analysis (TSM, Technicon) using a stainless steel column (0.21 x 18 cm) packed with Durrum DC-5A resin and fluorescence detection with o-phtaldialdehyde (7). Quantification using a computing integrator (Pye Unicam, DPlOl) was achieved by comparison to norleucine as internal standard and to standard mixtures of amino acids. The oxidative cleavage of disulfide bridges with performic acid was carried out as described previously (8,9). Briefly, performic acid was prepared by mixing 1 volume of 30% hydrogen peroxide and 19 VOlUmeS of 99% formic acid and was allowed to stand at room temperature for 2 h in a closed vessel. Performic acid (50 ~1) was added to the peptide material (3-4 nmoles) which was dissolved in 25 ~1 99% formic acid containing 5 ~1 anhydrous methanol.

1006

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BIOCHEMICAL

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The reaction was carried out at -5OC to -1OOC for 2.5 h and was terminated by adding 400 ~1 distilled water. The samples were subsequently lyophilized. Dansylation of N-terminal amino acids was carried out essentially. according to Gray and Smith (10). After hydrolysis and lyophilization the residues routinely were taken up in 25 ~1 distilled water and extracted with 10 ~1 ethylacetate. For the identification of DNS-cysteic acid 10 ~1 pyridine-water (1 : 1, v/v) or ethanol-water-acetic acid (100 : 5 : 3, v/v/v) was added to the dried residues. DNS-amino acids were identified on doublefaced micropolyamide F-1700 sheets (5 x 5 cm; Schleicher & Schbll), which were developed in a three solvent system (11). DNS-cysteic acid was resolved from DNS-OH with a fourth solvent, 1 M ammonium hydroxyde-ethanol (1 : 1, v/v), run in the second dimension (11).

Incubation limbic

of

brain

yielded

was seen from of the

(fig.

1B).

glycinamide

while

intact

which

was previously

phoresis,

material

for

as indicated

by the

14 C-oxytocin

incubations. the yields

Dansyl glutamic pective peptides

fractions

volume

after

were

residue.

faintly

of radiolabeled from

1B).

by W in

fig.

(fractions

3-6), OXT x-8,

paper

electro-

was resolved

These

fractions

to prepare

sufficient

of oxytocin,

omitting

B and C

were

products

from parallel

the specific

B and fraction

of 14C-

activity

C from a single

obtained

incubation

respectively.

determination

of fractions

demonstrated that residue

fluorescent

incubations

, as

53-57).

It

In order

out and fractions

As calculated

and indicated

E (fractions

products.

HPLC distribution

This

of the column

HPLC fractionation.

analyses,

of fraction

detectable as indicated

C (fig.

rat

HPLC fractionation

peak by high-voltage

B and fraction

carried

also

pooled

in fraction

digestion

was the glutamine-

an additional,

the void

as a single

and 1.8 nmoles,

end group

acid

were

of the

14C-glycinamide after

were

Fractions

structural

oxytocin,

1.6 nmoles

in

fraction

radiolabeled

oxytocin,

1A).

heterogenous

the principal

a SPM preparation

the C-terminal

components

was collected

two components,

peptide

were

Several

detected

appeared

represented

retaining

eluted

oxytocin

with

of the radioactivity

at 210 nm (fig.

1B. Free

into

products

the distribution

digests

absorbance

['4C-GlyNH2]-oxytocin

the

the purity

of peptides

N-terminal

amino

of oxytocin. spot

B and C showed

was visible,

1007

acid

In fraction which

a single

in the resof both C, however,

did

DNS-

not

co-

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

10

s '; E 0"

6.

0

6

16

24

32

&O

46

56

61

72 froctm

nmba

Fisure 1. HPLC fractionation of a digest of oxytocin which was obtained by incubation with a SPM preparation for 3 h at pIi 6.9. Chromatography was performed by gradient elution with acidified methanol and ammonium acetate buffer. The HPX eluate was monitored continuously by IJV absorbance (A) and aliquots of the collected fractions were counted for radioactivity (B). For experimental details see Methods. Fractions were pooled as indicated by the horizontal bars (fractions A to E). The vertical arrows show the elution position of synthetic oxytocin and related fragments.

migrate of

with

fraction

B and

fraction

B as

oxytocin, two cystine

one

well

of

the

reference

fraction as

DNS-amino

C shown that

in

in

table

fraction

and

differed proline

in residues

mobility

in were

The

I indicated

C comprised

Gln-Asn-(Cys)-(Pro)-Leu-GlyNIi2.

peptides

acids.

not

the

This

finding

HPLC

system.

detected

1008

the

with

amino

acid

that

the

4 to was However,

the

compositions peptide

9 sequence surprising cysteine,

o-phtaldialdehyde

in of as

the

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

Table AMINO amino

ACID

COMMUNICATIONS

I

CCHPOSITION

position

acid

RESEARCH

OF HPLC FRACTIONS

in

fraction

B

fraction

C

oxytocin

Crs+ Tyr

1,6 2

0.0

0.1

1le

3

0.2

0.3

GlX

4

1.0

1.0

ASX

5

0.7

0.8

pro+

7

Leu

8

0.6

0.7

GUY

9

1.3

1.2

+Cysteine

and proline are not detected method. The indicated numbers of residues were obtained in the analyses by the highest The amino acids are those contained in

reagent

in

between

the To

C

subjected

to

this

experimental

fraction

acid

and

design,

in

the

reference

it

was

absent

in

showed

additionally

and

peptide

samples Based

on

cysteic

amino

acid

Thin

as

described

in

acid

content,

spot,

peptides. fraction

the

compositions, the

following

fraction

B:

Gln-Asn-Cys(0)x-Pro-Deu-GlyND2,

fraction

C:

Gln-Asn-Cys(S-S-Cys)-Pro-Ieu-GlyND2.

these

these

experiments l-6,

layer

was

expected,

method

ortho-DNS-tyrosine

performic fractions

arginine-vaso-

section. acid

that as

treated

was In

residues,

with

identified the

peptides,

chromatography

co-migrated

spot

As

difference

in

DNS-glutamic

which

This 8.

the

oxytocin.

with

the

amounts

residues.

oxytocin

acid.

pmole

reaction In

oxytocin,

to

of

bridge

cleavage

addition

the

the

cysteine

hydrolysis.

a fluorescent

from

the

cysteic

acid

fluorescence

that

a disulfide

synthetic

DNS-cysteic

C contained

tained

and

cystine

identify

in

of oxidative

o-phtaldialdehyde

anticipated

located

the

with

vasotocin,

used

and

to

compared

be

the

calculated by dividing common factor. the primary structure

was

possibility

dansylation

were

pressin,

acid

could the

to

It

analyses.

components

were prior

B and

acid

investigate

samples acid

amino

by

ob-

DNS-cysteic reference

derivative.

radioactivity,

N-terminal

structures

are

(x

= 0,1,2,3),

proposed:

end

groups

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION Oxytocin

is

preparations lytic

obtained

process

accumulate.

amino

acid

as a single

were

resolved

primary

However,

the peptides acid

residues

suggested

is

and

in their

prior

to isolation.

is probably

very

residue

occurs

to determine

14

C-radioactivity,

peptides,

was attached

The remaining

sulfhydryl

to oxidation. oxidation,

residue

of

was absent

peptide

group

was

of cysteine-6,

As in the experiments it

form,

to the peptide

residue

of this

of

dansylation

the cysteine-1

bridge

in an oxidized

glycinamide

acid,

cysteine-1

the disulfide

with

The presence

performic

residue

the

respectively.

residues.

with

This

susceptible

of both

C-terminal

and the

detected

assessed.

acid

may represent

have been made to prevent

cysteine-6

were

was in agreement

to cysteine-6. that

fragments

composition,

treatment

it

proteo-

electrophoresis

structures

cysteine

this

this

have been

The 4 to 9 sequence

glutamic

that

which

allowed

SPM

peptide

paper

techniques

acid

Therefore,

linked

fragments,

high-voltage

with

During

and several

by HPLC and their

C after

cleaved

is

suggested

indicated

that

as CYST

no

the in the

structure.

The two isolated of the oxytocin acids

oxytocin

activity

of the peptide

involved

in its

is

about

known

peptides

molecule.

from

aminopeptidase bridge

after

differed

B, indicating

amino

two main

amino

in fraction

precautions

released

from different

bridge.

which

off

are

study

in fraction

by a disulfide

proposed

(3,4).

limbic

by dansylation

hydrolysis

however,

brain

of the N-terminal

as detected

oxytocin,

the rat

by their

residues

and acid

are associated

of the two peptides.

was indicated

cysteic

which

component

data

structure

the presence

by peptidases,

and isolated

The combined

which

from

In the present

previously (4),

converted

the tyrosine

In previous

studies

oxytocin

1,4-9

do not

aminopeptidases

(3,4).

prior

with

1010

successive

residues release

and the involvement

indicates

require

and isoleucine the

has been observed has been suggested

formation brain

lack

of an

The conserved that

of these

disulfide

the aminopeptidases

reduction

such a specificity.

of this

bond.

Pliska

Little et al.

Vol.

97, No.

8lOCHEMlCAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

OXYTDCIN

CVS-TVR-ILE-GLN-ASN-CYS-PRO-LEU-

GLvNH~

CP / PCE

5.2

Gin-Aan-Cyr-Pm-Lcu-GlyMM*

E”P I

XI = -s-s-cy* x2 - -5”. -S(Ol,

LN

+

GW,

Figure 2. Pathway for oxytocin conversion by SPM preparations of rat brain. The proteolytic events occurring in the N-terminal and C-terminal parts of the oxytocin molecule are presented as separate routes. Several types Of activities, which nay be involved in the conversion process, are indicated: Amp= aminopeptidase (12) CaP = carboxamidopeptidase (18,19) PCE = post-proline cleaving enzyme (20) CP = carboxypeptidase ExP = exopeptidase TPDO = thiol:protein-disulfide oxidoreductase (16.17)

have demonstrated ring

portion

the

of oxytocin

studies

suggest

can act

on oxytocin

ed slowly

cleavage

that

(13,14,15).

the tyrosine

Under relatively

suggesting our

by minced

arylamidases,

cerebral enzymes

that

while

with

residues

and acted

the

reduced

conditions the

cortex

isoleucine

rapidly hormone the

in the

preparations

purified

intact

(12).

on open nonaserved

activity,

and octa-

of oxytocin rapidly

releas-

in the presence

as substrate

appears

Other

from brain,

from oxytocin

degradation

residue

1011

bond

aminopeptidase-like

One such enzyme,

agents

experimental

slowly,

cysteinyl-tyrosyl

and isoleucine

of sulfhydryl-reducing peptides,

of the

(15). proceeds after

the

Vol.

97, No.

release

3, 1980

BIOCHEMICAL

of tyrosine

nonapeptide

oxytocin

initial

step,

rapidly

proceeding This

mechanism

indicates

aminopeptidase

action

(3,4). Leu-GlyNH2

supporting

the view

activity

(20)

digests,

which

activity

(18,19).

The various

pathway

and several

types

Several

molecules synthetic

are

distinctly

Leu-GlyNH2

(1,2).

for

Therefore,

may have a modulatory

(16,17)

is

low amounts

a

present.

of the C-terminal

of glycinamide

were

by a post-proline

was a minor

found,

cleaving

enzyme

by unidentified component

in the

of a carboxamjdopeptidase

routes

are

enzymes,

summarized

which

the neurohypophyseal

peptides

different

that

in a proposed

are known

to act

on

may serve

as

2). that

fragments

and suggests

to

portion

of the involvement

(fig.

amounts

in the C-terminal

l-8

of proteolytic

is

of the disulfide

of the dipeptide

convertive

residue

cleavages

amounts

oxytocin

has been postulated

precursor

oxytocin

in favour

are indicated

It

cases

is

Woreover,

and of

of rat

of Leu-GlyNH 2 formation

(15).

of a slow

SPM preparations

constant

cleavage

the open

in isolated

studies

by rapid

detect

in equimolar

enzyme

over

and accumulating

followed

exopeptidases

oxytocin,

occurs

that

COMMUNICATIONS

bond,

reduction 4-9

prevails

In these

not

the glutamine

require

reduction

we have demonstrated

of oxytocin

till

of oxytocin

that

we did

cysteinyl-tyrosyl

action

does not

RESEARCH

may be indicative

oxidoreductase-like

Recently,

dipeptide

experiments

of the

the detection

1,4-9

BIOPHYSICAL

arguments

the cleavage

thiol:protein-disulfide

brain

These

aminopeptidase

However,

oxytocin

1n these

1,2-9.

being

encountered. bridge.

(3,4).

AND

with

retain from

specific

the enzymes, role

functions

behavioural oxytocin

in the

hormones

in the brain

activities, itself,

which

in

(21,22). some

such as the dipeptide

involved

in the

conversion

central

effects

of this

of neuro-

peptide. REFERENCES 1. van Ree, J.M., Bohus, B., Pharmacol. 27, 1793-1800. 2. de Wied, D., and Versteeg, 3. Burbach, J.P.H., de Kloet, supp1. 225, 250.

Versteeg,

D.H.G.,

and de Wied,

D.H.G. (1979) Fed. E.R., and de Wied,

1012

D. (1978)

Biochem.

Proc. 38, 2348-2354. D. (1979) Acta Endocrinol.

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

4. Burbach, J.P.H., de Kloet, E.R., and de Wied, D. (1980) Brain Res., in press. H., Veldhuis, H.D., Schotman, P., and Gispen, W.H. (1976) Neurochem. 5. Zwiers, Res. 1, 669-677. 6. Zwiers, H., Schotman, P., and Gispen, W.H. (1980) 3. Neurochem. 34, 16891699. 7. Zwiers, H., Verhoef, J., Schotman, P., and Gispen, W.H. (1980) FEBS Lett. 112, 168-172. Vol. XI, Enzyme Structure a. Hirs, C.H.W. (1967) In: Methods in Enzymology, (Hirs, C.H.W., ed.), pp. 197-199, Academic Press, New York and London. E., and Fontana, A. (1975). In: Molecular Biology, Biochemistry 9. Scoffone, and Biophysics, Vol. 8, Protein Sequence Determination (Needleman, S.B., ea.), pp. 162-201, Springer Verlag, Berlin. (1979) Anal. Biochem. 33, 36-42. 10. Gray, W.R., and Smith, J.F. 11. Narita, K., Matsuo, H., and Nakajima, T. (1975) In: Molecular Biology, BioVol. 8, Protein Sequence Determination (Needleman, chemistry and Biophysics, Springer Verlag, Berlin. S.B., ea.), pp. 30-103, 12. Pliska, V., Thorn, N.A., and Vilhardt, M. (1971) Acta Endocrinol. 67, 12-22. H-D. (1975) Acta Endocrinol. 78, 634-648. 13. Kuhl, H., and Taubert, 14. Kuhl, H., Sandow, J., Krauss, B., and Taubert, H-D. (1979) Neuroendocrinology 28, 339-348. 15. Marks, N., Abrash, L., and Walter, R. (1973) Proc. Sot. Exp. Biol. Med. 142, 455-460. L.A., Ferrier, B.M., and Celhoffer, L. (1972) Can. J. Biochem. 50, 16. Branda, 507-509. W.B. (1974) Nature 251, 237-239. 17. Small, C.W., and Watkins, Schwartz, I.L., and Walter, R. (1969) Proc. Natl. Acad. Sci. 18. Glass, J.D., USA 63, 1426-1430. 19. Simmons, W-M., and Walter, R. (1980) Biochemistry 19, 39-48. 20. Koida, M., and Walter, R. (1976) J. Biol. Chem. 251, 7593-7599. 21. Walter, R. (1974) In: Psychoneuroendocrinology (Hatotani, N., ea.), pp. 285-294, Karger, Basel. 22. de Wied, D. (1977) Life Sci. 20, 195-204.

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