Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma

Vol.

146,

No.

July

31,

1987

2, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

Pages

AMINO ACID SEQUENCE OF ATRIAL NATRIURETIC IN HUMAN CORONARY SINUS PLASMA Tim Yandle'*,

Ian Alan

'Department

of

'Department

of Cardiology, Christchurch,

The Princess New Zealand

of

sinus phase 99-126 being ratio

11,

Espiner',

Margaret

Hospital

Margaret

Biochemistry, University Dunedin, New Zealand

of Pathology, Christchurch,

832-839

PEPTIDES

Gary Nicholls', Eric and Stephen Brennan4 The Princess New Zealand

4Department

June

Crazier', Carne3,

Endocrinology, Christchurch,

3Department

Received

COMMUNICATIONS

Hospital

of Otago

Christchurch New Zealand

Hospital

1987

Two atria1 natriuretic peptides were purified from pooled human coronary plasma by Sep-Pak extraction, immunoaffinity chromatography and reverse HPLC. The amino acid sequences of the two peptides were homologous with human atria1 natriuretic peptide (hANP) and 106-126 hANP, the latter most probably linked to 99-105 ANP by the disulphide bond. The molar of the peptides in plasma, as assessed by radioimmunoassay was 10:3.

0 1987 Academic

Press,

Whereas in rat

pro-rANP

atria

103-126 erent

(1,2),

peptides

99-126

- pro-hANP,

suggest

IR-ANP

in humans

plasma

of

sequenced the

are

To establish plasma,

the

main

main

and report

the

to be the in rat

ratio

from

to

(7).

9:l

atria1

circulating 99-126

Recently the

storage

are

(3,4). tissue

of

(2).

BhANP may also

99-126

hANP has been

secreted

with

renal

and circulating

of

ANP

rANP and

human,

99-126

3 diff-

hANP)

and

Chromatographic

and secreted

hANP.

form

99-126

In the

dimer

(5,6,7)

of patients of

main

plasma

antiparallel

ultrafiltrates structure

been

in

BhANP (the

similar

definitive

appears

components

extracted

the

plasma

has not

purified

been

that

some subjects from

rANP)

major

are present

hANP - have

humans

(1-126 the

rANP which

studies

ever

Inc.

(5)

forms

be present extracted

in the and

failure forms

of

(8).

How-

of ANP in

determined. the

structure

of

immunoreactive their

* To whom correspondence

amino should

ANP secreted forms

acid

by the

of ANP present

human heart,

we have

in human coronary

sequence.

be addressed.

ABBREVIATIONS: ANP: Atria1 Natriuretic Peptide, IR-ANP: Immunoreactive-ANP, Labelled ANP: [ 125I] Tyr- monoiodo 99-126 ~ANP, PMSF: Phenylmethysulfonyl fluoride, PTH: Phenylthiohydantoin, RIA: Radioimmunoassay, TFA: Trifluoroacetic acid. $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights qf reproduction in any jbrm reserved. 0006-291X/87

832

sinus

Vol.

146,

No.

MATERIALS

2, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

General Pooled coronary sinus plasma from 11 patients was extracted using SepPak cartridges and further purified by inununoaffinity chromatography and reverse phase HPLC prior to determining the amino acid sequence using a gas phase sequencer. ANP peptides were obtained from Peninsula Labs (Belmont, CA) or Bachem (Torrance, CA). BhANP was kindly supplied by Dr H. Matsuo, and 101126 rANP by Merck Sharp & Dohme (Rahway, N.J.). [oxidised Met"'] 99-126 hANP was obtained from Peptide Institute (Osaka, Japan). Radioimmunoassays

and affinity

column

preparation

HPLC fractions or samples were assayed without extraction by two different RIA's. The first used an antiserum supplied by Peninsula Laboratories directed to the carboxy-terminal region of 99-126 hANP. This assay was performed as previously described (5) except that ['251]Tyr-monoiodo 99-126 hANP was used and all reagents were incubated together in a single overnight incubation at 4'. The second RIA used an antiserum (R27) raised in a rabbit against 101-126 rANP conjugated to bovine thyroglobulin. This antiserum recognised the middle region of 99-126 hANP, with the following cross-reactivities at 50% displacement:99-126 hANP lOO%, 105-126 hANP lOO%, 111-126 ANP < 0.03% [oxidised Metl"] 99-126 hANP 63%, @hANP 21%, 103-123 rANP 116%. Angiotensin I, angiotensin II, arginine vasopressin, and oxytocin all cross-reacted less than 0.003%. Assay conditions were the same as those for the first RIA except that the bound counts were precipitated by addition of polyethyleneglycol. For the affinity column, antiserum (R22) was raised in a rabbit as described for antiserum R27. R22 antiserum had the following cross-reactivities measured at 50% displacement:99-126 hANP lOO%, 105-126 hANP 83%, 111-126 ANP 20%, 103-123 rANP 66%. The gamma globulins in this antiserum were precipitated with anunonium sulphate and further purified by binding the anti-ANP antibodies to a lOl126 rANP-Sepharose gel. They were then eluted with a pH gradient according to Hodgkinson & Lowry (9). These anti-ANP antibodies were coupled to CNBractivated Sepharose 4B (Pharmacia) and excess active groups blocked with ethanolamine. The gel was packed in a Poly Prep column (Bio-Rad Richmond, CA) with a 0.8 ml bed volume and an approximate capacity of 500 pmol ANP. Prior to use, the column was subjected to at least two equilibration elution cycles and finally equilibrated with buffer A (0.05 M Tris-HCl pH 7.4, 0.5 M NaCl, 0.1% Triton X 100) _ Patients

and blood

collection

After obtaining informed written consent, coronary sinus blood was collected as described previously (10) from two groups of patients undergoing routine cardiac catheterization. The first group consisted of 6 patients with congestive heart failure. Five patients undergoing electrophysiological Coronary sinus assessment for tachyarrhythmias comprised the second group. either during induced tachyarrhythmia blood was drawn from the latter group, or rapid atria1 pacing. Blood was transferred rapidly to 10 ml tubes containing 15 mg EDTA, 1000 KIU trayslol, and 0.2 mg PMSF, mixed immediately at 4' Plasma was and centrifuged for 10 minutes at 5000 rpm and 4' without delay. brief storage at aspirated immediately and extracted the same day, or after -80'. A total of approximately 700 ml of plasma containing 730 pmol of IR-ANP Two thirds of the IR-ANP came from the tachyarrythmia group. was obtained. The mean concentration of IR-ANF' in coronary sinus plasma was 759 pmol/L in In 4 the heart failure group and 1169 pmol/L in the tachyarrhythmia group. patients simultaneous samples of peripheral vein and coronary sinus plasma In these patients the ratio of mean coronary sinus/peripheral were obtained. plasma IR-ANP concentration was >4. Plasma

Extraction Five

ml aliquots

of

coronary

sinus

plasma 833

were

extracted

on Sep-Pak

(C18)

Vol. 146, No. 2, 1987

BIOCHEMICAL

cartridges as previously under nitrogen they were stored at -80'. A small Affinity

Column

described suspended number of

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(5). After the extracts had been dried in a minimum volume of 4% acetic acid and these extracts were stored in 0.1% TFA.

Purification

The stored extracts were thawed, combined and adjusted to pH 7.4 by addition of 5 ml 1 M Tris followed by 1 M NaOH. After centrifuging at 20,000 g for 40 min at 4', the supernatant was passed slowly through the affinity column. The column was washed with 10 ml of buffer A followed by 10 ml of buffer A without Triton X-100. IR-ANP bound to the column was eluted with 10 ml of 0.1 M Acetic acid, 0.5 M NaCl. All column washings were reprocessed a second time. HPLC Acid eluates from the affinity column (30 ml) were loaded by serial 1.5 ml injections onto a 25 cm Zorbax ODS column pre-equilibrated with 25% solvent B, 75% solvent A (Solvent A = 0.1% TFA, B = 0.1% TFA 60% acetonitrile) at a flow rate of 0.5 ml/minute, and then eluted with this solvent until the W absorbance at 215 nm due to acetic acid returned to baseline. A linear gradient of 25% to 66.6% solvent B was then applied over 37.5 minutes at 1 ml/ minute. Half ml fractions were collected and stored frozen at -80" prior to radioimmunoassay and analytical HPLC. For analytical HPLC, pooled fractions containing IR-ANP were loaded onto a TSK G2000SW size exclusion column and eluted with 0.1% TPA, 20% acetonitrile at 0.5 ml/minute. One ml fractions were collected, and assayed for IR-ANP by RIA. Peptide

Sequence

The peptides in 0.1% TFA, 50% acetonitrile coated, and precycled, TFA treated glass fibre was performed in an Applied Biosystems 470A gas City, CA), with on-line 120A HPLC for detection atives. A custom programme was modified cycle programme for All other cycles of degradation stabilised by derivatization was included as an internal

were loaded onto a Biobrene TM disc. The sequence degradation phase sequencer (ABI, Foster of the PTH amino acid deriv-

used for the peak C sample which incorporated a serine (Applied Biosystems) at cycles 1,5,6,19,25. used a standard programme. Cysteine was not and therefore was not identified. Nor-leucine standard in the S4B solvent for PTH analysis.

RESULTS Purification

- Total

610 pmol.

After

affinity

ANP determined and C Fig

by the

1, upper

respectively. small ated

mid-region

the

however,

large

similar

results

using

was used.

fractions Total

the

IR-ANP

9,

of

mid-region

two antisera, were in peaks

assay.

resolved

(Fig

were

large

hANP (Fig

All

B and C measured 834

with

other

this

(A,B

IR-ANP a number

peaks

of

correl-

A similar by RIA using

1 lower).

three

except that two minor when the carboxy-terminal

detected

IR-

1 upper).

assayed

almost

the

peaks

showed

and both

99-126

B contained

TFA,

58 and 251 pmol

size

same fractions region

well profile

peak

and relative

47 in peak by the

41-45

contained

was approximately

by RP-HPLC with

in three

One small

time

when the

fraction measured

of

in

extracts

215 nm absorbance

carboxy-terminal

as that

region

RIA eluted

peaks.

peaks

IR-ANP

Sep-Pak followed

The peaks

was apparent to

instance

IR-ANP

pooled

chromatography

panel).

and two the

relationship antiserum

in the

The corresponding

peaks with

IR-ANP

times

In this as much

fractions

gave

peaks

in the antiserum

antiserum

was 138

Vol. 146, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A.U. 0.064

I

;60

=0) .G 6 $ z

-40 -20 -0

A.U. 02

Fr

act [on

number

Figure l.Reverse phase HPLC of purified IR-ANP from coronary sinus plasma. Upper: fractions assayed with antiserum (R27) directed to the mid-region of 99-126 hANP (O-O), overlaid on the trace of 215 run absorbance (lower solid line). Acetonitrile concentration is shown by the upper solid line. Arrows at top indicate elution position of synthetic standards, from left to right 111-126 ANP, 103-123 rANP, [oxidised MetI"] 99-126 hANP, 99-126 hANP, 105-126hANP, BhANP. Lower : same as for upper, except the same fractions were assayed with antiserum to the carboxy-terminal part of 99-126 hANP.

and 265 pmol the

using

peak

of

peaks

these

antisera

showed

a small

two components

C and B gave

oxy-terminal obtained

The discrepancy

two different

B, which

presence ing

respectively.

antiserum using

the

results

was compatible shoulder

in peak

an area result

on the B.

ratio

fraction

results

with right

of

of C:B = 10~7 antiserum

the

but

for

side,

areas

in agreement

did

not

47

agree

across

suggesting under

(C:B = 1O:Z).

47 contained

fraction

W absorbance

hand

Integration

(C:B = 10:5),

R27 mid-region

suggested

between

absorb-

W

with with

Taken

a carboxy-terminal

the the

carb-

results together fragment

of

ANP. Because fractions rising subjected

the

small

(47 & 48)

were

peak

of C.

to

size

To assess exclusion

amount pooled purity,

of material for

analysis, aliquots

chromatography

835

in peak B, both of the peak as were the two fractions comptaken (Fig

from 2).

the In

pooled this

peaks

system

were

IR-ANP

Vol.

146,

No.

BIOCHEMICAL

2, 1987

AND

690K I, l>K

BIOPHYSICAL

6K3K ii.

COMMUNICATIONS

0.18K 1 '3K -peak "... y peak

200

RESEARCH

C B

I-

Fraction

number

Figure 2.- HPLC - size exclusion chromatography (Toy@ soda G2000SW column) of aliquots from peaks B and C of Fig 1. Fractions were assayed for IR-ANP using R27 mid-region antiserum. Closed squares (top) indicate the elution position of standards and their molecular weight. Bovine thyroglobulin Mr 690,000; Cytochrome C Mr 12,000; Aprotinin Mr 6,000; labelled ANP Mr 3209; labelled Angiotensin I Mr 1,298; Tyrosine Mr 191.

from

peaks

elled

B and C each

ANP (Fig

Sequence The

- Peptide peak

major

hANP.

Two

eluted

as a single

peak

sequences

established

(C) contained

unidentified

only

residues

from

Peak

Peak 6,

Peak

E2

Peak

B3

same position

as lab-

in

this

B and C are

which

sequence

shown

was identical

occurred

at

to

the

SLRRSSCFGGRMDRIGAQSGLGCNSFRY

S L R R S S - F G G A M D R I

G A Q S G L G -

N(S)F(R)V

SLRRSS-FGGRHDRIGAQSGLG-NSFRY

FGGRMDRIGAQSGLG-NSFRY (S)(L)(R)(R)(~)(S) Possible

structure

combining

Bz

+ t13 Peptides

1

I SLRRSSC

FGGRMDRIGAQSGLGCNSFRY

Figure 3.- Comparison of amino acid sequences of ANP peptides Blr Bz, Bs) from coronary sinus plasma with the known structure 126 hANP (top). Amino acids that were not positively identified enclosed in brackets. At bottom is a possible peptide structure would give rise to peak Bz and BS sequences. 836

in Fig 99-126

expected

SEQUENCE

hANP C

the

peaks

one sequence,

PEPTIDE

99-126

at

2).

(peak C, of 99are which

3.

Vol. 146, No. 2, 1987 positions

of cysteine,

expected the

serine

length

the

hANP

other

(Bz)

reactivity

This

with

106-126

data

indicate

If added of

105-106

the to

rated

areas

of Peak which

from

the

gave

sequence

in peak

99-105

through (assayed (Fig

high

with

This

antiserum)

so,

which

eluted

in

the

based

position

cycles residues

at cycle

in the

a peptide

the

homologous

99-105

ANP and 106are

supported

by the

of

8

same

two peptides all

hANP

six

in yield

of

that

is

on integ-

first

observed

further

47.

yield,

and arginine

with

showed

then

two RIA's.

HPLC fraction

the

a drop

these

is

hANP region.

hANP : 106-126

serine

presence

structure

105-111

and 10:4

was not

3 where

The cross-

on initial

over

consistent

in Fig

chromatography,

by either

the

30 pmol).

of

99-126

yields

Even

structure

presented bond.

exclusion

of

B (which

with and the

in the

2 RIA's

of

homology

by R27 antiserum,

B, based

content

were

sequencer),

the

assay, of

but of

sequences

complete

fragment

ratio

sequencer. in peak

A peptide

a disulphide size

the

the

be identified,

approximately

the

yield,

identification

the

recognition

Analysis

by the on the

is

from

hANP in peak

C) was consistent

ANP.

showed

carboxy-terminal

hANP sequence

126 hANP sequences using

the

UV peaks.

low yields

99-126

(Bi)

between

low

Two distinct

15 pmol

for

C we establish

from

not

R27 recognises

required

99-126

B was complicated

in the with

data

29.

yield

differences

of

in peak

using

cycle

antiserum

is

Due to

positive

as a carboxy-terminal

the

proportion

that

10:3

at

hANP (initial

bond

hANP.

by the

One peptide

that

RESEARCH COMMUNICATIONS

25 and 21 could

approximately

behave

reconcile

99-126

cycles

3).

yield

hANP would

would

with

was established

B (Fig

(initial

an intact

106-126

AND BIOPHYSICAL

28 and no residue

in peak

99-126

at

peptide

at cycle

identified

by homology

and arginine

of

tyrosine

If

BIOCHEMICAL

IR-ANP

labelled

linked results

in peak 99-126

B hANP

2).

DISCUSSION We chose

to

reasons. substantially close

to

levels that

the

source formed

of

IR-ANP

compared of

most

rat of

the

than of

ANP secretion in the

samples,

purified

found C).

plasma amino

ANP from of

peripheral

elsewhere in our

the

126 hANP (Peak ed from

and purify

concentration

to peripheral

We have

dence

the higher

metabolites sinus

extract

First,

that the major This peptide (3,4) terminal

but

unlike deleted

coronary

sinus

in coronary levels

(10).

reduces

the

influence In view

circulation. in at

of

represents

findings

peptide, 837

in is

high coronary

likely

hormone.

in human plasma major component

in the 103-126

sampling

gradient it

two is

of possible of the very

secreted

IR-ANP to the

for

plasma

Second,

4 patients,

newly

component of is equivalent the

a high

least

plasma

sinus

venous

and evidence

plasma IR-ANP

IR-ANP

rat,

ANP.

is 99extract-

we found no eviInstead we ident-

Vol.

146,

ified

No.

106-126

in peaks remain

BIOCHEMICAL

hANP as a minor

B and C.

Where

or monobasic

(12)

and 99-126 unusual.

unlikely

Peptide (ll),

cathepsin

residues

are

to be an artifact specific

unable

to demonstrate

thetic

99-126 not

This

shown).

ilarly,

good yields

the

suggests

99-126

was no evidence

that

the

the

during

the

has undergone

we have

shown

total

of

to di-

103-126

rANP

of a Cys-Phe

methods

would

context

amounts

bond

adjacent

to

The peptide

here.

not

of peak

presence

been

B when synextraction

of

purification

is have

we have

of Sep-Pak

99-126

hANP

artifacts.

sequencing

of

from

ANP

significance

cleave

In this

resulted

deamidation

peptide

(14) acted

of

the

adjacent

cleavage

unexpected

result

of

biological

cleaved

same conditions

the

COMMUNICATIONS

production

but

bond.

hANP peptide of

the

significant

that not

of methionine the

in

purification

of

20-30%

often

to have

to the

is

are rat,

Cys-Phe

presence

derivative

that

there

however

the

and its

the

unlikely

hANP was exposed

cleaved

Also

at

to

formed

and thermolysin

as our mild

cleavage

and its possibility

in

RESEARCH

amounting is

as occurs

G (13)

but

BIOPHYSICAL

hormones

rANP respectively

caused

(data

peptide

residues

Both

phenylalanine

AND

component

this

to be determined.

basic is

2, 1987

Sim-

peak

B excludes

methionine

oxidation.

in the

sequence

some form

of post

data.

It

the

is possible

translational

modif-

ication. In

summary,

human heart detected

but

ANP) remains

is

99-126 its

hANP.

formation

In

that

the

major

addition,

and linkage

form

a smaller to the

of

ANP secreted

amount

of

carboxyterminal

by the

106-126

hANP was

peptide

(99-105

to be clarified.

ACKNOWLEDGEMENTS We thank assays work

Steven

and Trish was funded

Fisher

Riddell in part

and Helena and Susan

by the

Duff

Isaacs

National

for

assistance

for

secretarial

Heart

Foundation

with

radioimmuno-

assistance.

This

of New Zealand.

Postscript. Stephenson and Kenny (Biochem J 1987 243, 183-187) have recently reported that an endopeptidase in kidney microvillar membranes cleaves the disulphide linked ring of hANP at three different sites, including the Cys-Phe bond. Amino acid analysis of the major product showed it to be identical to hANP except that one (unidentified) peptide bond within the ring was cleaved.

REFERENCES 1. 2. 3.

Thiabult, G., Garcia, R., Gutkowska, J., Bilodeau, J., Lazure, N.G., Chretien, M., Genest, J., and Cantin, M. (1987) Biochem 272. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T., and Matsuo, Biochem. Biophys. Res. Commun. 129, 248-255. Schwartz, D., Geller, D-M., Manning, P.T., Siegel, N.R., Fok, C.E., and Needleman, P. (1985) Science. 229, 397-400. 838

C., Seidah, J. 241, 265H. K.F.,

(1985) Smith,

Vol. 146, No. 2, 1987 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Thiabult, G., Lazure, C., Schiffrin, E.L., Gutkowska, J., Chartier, L., Garcia, R., Seidah, N.G., Chretien, M., Genest, J., and Cantin, M. (1985) Biochem. Biophys. Res. Commun. 130, 981-986. Yandle, T.G., Espiner, E.A., Nicholls, M.G.,and Duff, H. (1986) J. Clin. Endocrinol. Metab. 63, 72-79. Yamaji, T., Ishibashi, M., and Takaku, F. (1985) J. Clin. Invest. 76, 1705 1709. Miyata, A., Toshimori, T., Hashiguchi, T., Kangawa, K., and Matsuo, H. (1987) Biochem. Biophys. Res. Commun. 142, 461-467. Forssmann, K., Hock, D., Herbst, F., Schulz-Knappe, P., Talartschik, J., Scheler, F., and Forssmann, W.G. (1986) Kiln. Wochenschr. 64, 1276-1280. Hodgkinson, S.C., and Lowry, P.J. (1982) Biochem. J. 205, 535-541. Crozier, I-G., Nicholls, M.G., Ikram, H., Espiner, E.A., Yandle, T.G., and Jans, S. (1986) Hypertension 8, suppl 2, 11-15. Schwartz, T.W., (1986) FEBS Lett. 200, l-10. Murthy, K-K., Thiabult, G., Garcia, R., Gutkowska, J., Genest, J., and Cantin, M. (1986) Biochem. J. 240, 461-469. Barret, A-J., and McDonald, J.K. (1980) Mamalion Proteases, Vol. 1, Endopeptidases, Academic Press New York. Heinrikson, R.L., (1977) In Methods in Enzymology (S.P. Colowick and N.O. Kaplan, eds.) Vol.XLVII, Pt.E, 175-189.

839