Neuromedin B-32 and B-30: Two “big” neuromedin B identified in porcine brain and spinal cord

Vol.

130,

July

31,

No. 2, 1985

BIOCHEMICAL

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BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

NEUROMEDIN B-32 Naoto MINAMINO,

Received

AND B-30: TWO "BIG" NEUROMEDIN B IDENTIFIED IN PORCINE BRAIN AND SPINAL CORD

Tetsuji

Department

685-691

SUDOH, Kenji

of Biochemistry, Kiyotake, Miyazaki

KANGAWA and Hisayuki

Miyazaki 889-16,

Medical Japan

MATSUO

College,

May 29, 1985

SUMMARY: In mammalian spinal cord, we have previously discovered "neuromedin B", whose structure is closely related to amphibian bombesin. By utilizing a specific radioimmunoassay for neuromedin B, we have isolated two novel "big" neuromedin B, designated neuromedin B-32 and B-30, from pig brain both of which were identified as N-terminally extended forms and spinal cord, B-32 was determined to of neuromedin B. The amino acid sequence of neuromedin be : Ala-Pro-Leu-Ser-Trp-Asp-Leu-Pro-Glu-Pro-Arg-Ser-Arg-Ala-Gly-Lys-Tle-ArgVal-His-Pro-Arg-Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH , while neuromedin B-30 was found to be an N-terminal two amino acids delete 8 form of neuromedin B-32. Isolation of a family comprising neuromedin B, B-30 and B-32 is indicative of their biosynthetic relationship. 0 1985 Academic Press, Inc. It

has

heen known

pharmacological found

in mammalian

These

facts

only

central

else

that peptide

had been

identification spinal

uterus,

C

amphibian GRP, indicate peptides,

by

(NMC),

bombesin

while

both (7,81.

there

i.e.,

of NMB by using

peptides

are specific

stomach

(1-j).

in mammals

peptide(GRP)

tissues

as

was the

and intestine,

(5,6)

of

is also

until

and

our recent

B and C (7,x). search a

two novel

peptides have

found

new type

for

bioassay

of which NMC is

unidentified for

designated

of bombesin-like

and sensitive

homology

decapeptide These

mammalian

In our further

radioimmunoassay,

in rat

B (NMB) and

sequence peptide.

of endogenous

of

neuromeidn

remarkable

and NMB family.

neuropeptides

the contractility

to be a C-terminal

two families

NMC-GRP family its

neural

spectrum tracts

are acting

in mammalian from

a wide immunoreactivity

and gastrointestinal

gastrin-releasing

systematic

NMB is a quite

that

elicits

However,

utilizing

we have isolated

neuromedin

system

identified

of our

cord

bombesin

bombesin-like

isolated

of neuromedin

In the course porcine

nervous

neuromediators.

bombrsin-like

nothing

amphibian

in mammals and bombesin-like

indicate

endogenous

that

activity

amide

to of

results

bombesin-like investigation

we have

recently

Abbreviations: NMB, neuromedin B; NMR-32, NMB-30, N-terminal extended forms of NMB with 32 and 30 amino acids; NMC, neuromedin C; GRP, gastrin releasing peptide; RIA, radioimmunoassay; ir, immunoreactive; RP-HPLC, reverse phase high performance liquid chromatography; "Ii-, molecular weight: TFA, trifuloroacetic acid. 0006-291X/85 68.5

All

Copyright 0 1985 rights of reproduction

$1.50

bv Academic Press, Inc. in any form reserved.

Vol. 130, No. 2, 1985 postulated

the

been discovered named as brain homology

BIOCHEMICAL existence (9).

In this

neuromedin

and

spinal

of

B-32 cord

and differences

"big"

NMB

paper, (NMB-32)

and

their

between

AND BIOPHYSICAL RESEARCH COMMUNICATIONS in rat

we report and

the

neuromedin

complete "big"

brain,

amino

which

isolation B-30 acid

NMB and GRP will

has never

before

of two "big" (NMB-30) sequences. also

from

NMBs, porcine

Sequence

be discussed.

MATERIALS AND METHODS Isolation: (a) Porcine brain. Brain tissues (ca. 20 kg wet weight) excluding cerebellum were collected from 200 pigs at a local slaughter-house. Minced tissues were boiled for 12 min in 60 L of water to inactivate proteases. After cooling, CH COOH was added to a concentration of 1M and extraction was carried out at 4°C a y a Polytron homogenizer. The extracts (62 L), obtained after 30-min' centrifugation at 12,000 x g, was desalted through a Pellicon PCAC membrane (Millipore 00005, Mr 1,000). The Pellicon concentrate was then precipitated with 75% acetone. The supernatant was evaporated to dryness. A half portion of dry materials were dissolved in 1M CH3COOH (400 ml) and adsorbed on a column H -form), of SP-Sephadex C-25 (4.1 x 38 cm, preequilibrated with 1M CH COOH. Successive elutions with 1M CH COOH (600 ml), 2M pyridine (800 ml) 2nd 2M pyridine-CH,COOH (pH 5.0: 800 mY?) yielded three fractions, SP-I, SP-II and SP-III. Thid chromatography was repeated twice. Fractions SP-III obtained by the above chromatographies were combined and lyophilized to give dry materials of 6.8 g, which were used as the starting material for the present purification. One fourth of the starting material was dissolved in 0.5 M CH COOH (200 ml). adsorbed on a RP-column of LCsorb with 300 ml of (300 ml, Chemco), washed dith 0.5M CH COOH (300 ml), and eluted H20:,CH CN:lO% TFA = 40:60:1 (v/v,? was lyophilized and then The eluate subJec 2 ed to successive gel-filtrations on Sephadex G-50 (fine, 4.5 x 142 cm) and on Sephadex G-25 (fine, 4n aliquot of each 4.5 x 140 cm) in 1M CH COOH. fraction was submitted to RIA for NMB. After l$ophilization, fraction B, containing ir-NMB of Mr ca. 3,000 daltons, was separated by C,M-52 ion exchange chromatography (NH+-form, 2.4 x 45 cm, Whatman) with a linear gradient elution containing 10% CH?CN. Two fractions with ir-NMB (pH 8.5) (correspon to RP-HPLC Of HCooNHk!.ing to NMB-32 and -30) thus obta'rned were each subjected on a diphenyl column (Chemcosorb 4-6 x 250 mm, Chemco) with a 7-diphenyl, solvent system of H 0-CH CN-10% TFA. NMB immunoreactive fractions were further Chemco) at separated by RP-HPL8 on 2 Chemcosorb 3 ODS-H column (8.0 x 75 mm, 45°C with a solvent system of H 0-CH?CN-10% TFA. NMB-30 was purified at this stage. Final purification of N6 B-32was performed on the same column at 45°C with a shallow CH CN gradient of H20-CH CN-10% TFA system. The column effluents were mon 2 tored by measuring absor d ante at 210 nm or 280 nm. (b) Spinal cord. The starting material for the present purification was the side fraction obtained in our previous purification of neuromedin B and C. Fraction B (see Fig. 1 of ref. 7) was first subjected to a preparative (7,8). RP-HPLC on a column of TSR gel ODS-12OA (2.0 x 25 cm, Toyosoda). The main irNMB peak on this chromatogram was then separated by cation exchange HPLC on TSK CM-2s~ (4.6 x 250 mm, Toyosoda) with a linear gradient elution of HCOONH (pH 6.5) containing 10% CH CN. TWO peaks of ir-NMB (NMB-30 and NMB-32) thu 4 RP-HPLCs on obtained were each purifi d d to homogeneity by successive Chemcosorb 7-diphenyl (4.6 x 250 mm, Chemco) and on Chemcosorb 30DS-H a solvent system of H,O-CH,CN-10% TFA. (4.6 x 75 mm, Chemco) with RIA for NMB: RIA for NMB was performed as repo&ed dreviously (9), by using specifically the C-terminal the antiserum (#104-6) for NMB, which recognizes part of NMB and crossreacts little with NMC and GRP ( <0.05%). Half-maximum inhibition was observed at 20-30 fmol/tube. All analyses were carried out on a picomole scale. Amino Sequence analyses: acid analyses were performed with an amino acid analyser(Hitachi-835), after hydrolysis of the peptide (ca. 300 pmol) in 3M mercaptoethanesulfonic acid NMB-32 and NMB-30 (200-400 pmol each) were (Pierce) at llO°C for 20 hr. 686

Vol.

130,

BIOCHEMICAL

No. 2, 1985

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BIOPHYSICAL

RESEARCH

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with a gas-phase sequencer (model sequence analyses to amino acid Applied Biosystems). The resulting PTH-amino acids were analyzed by with a RP-HPLC at 50°C using an Ultrasphere ODS column (4.6 x 250 mm, Altex) (pH 5.6)) at a three-solvent gradient elution (CH CN, H 0, 40mM sodium acetate flow rate of 1 ml/min to give a codplete2resolution of PTH-amino acids (8,lO). Column effluents were monitored by simultaneous measurements of absorbance at 269 nm and 320 nm to detect dehydro-Ser and dehydro-Thr in addition to normal PTH-amino acids. subjected

47OA,

RESIILTS AND DISCUSSION Isolation

of NMB-32 and NMB-30

From porcine were

monitored

recognized

brain: by a

extracts

L) of brain

to Mr <5,000, (Mr <5,000)

further

preparations

obtained

G-25 to

separated

two

HPLCs.

pigs) fractions

corresponding

stimulant

activities

and strong observed.

of

Fraction

by CM-52 cation and b) of ir-NMB

found

submitted

The bioactive to the

ir-NMB.

second

containing chromatography. in highly

on

gel-filtration that

from native larger

NMB by

Mr ir-NMB, As shown

basic

regions.

was in Fig.

NMB-32 and

.6-

0

20

40 Froctlon

Fig.

60 nur4w

a0

100

CM-cellulose ion exchange chromatography of fraction 1. Sample: The basic peptide fraction of Mr 3,000 obtained brain (410 mg). Flow rate: 35 ml/min. Fraction Column: CV-52 (2.4 x 45 cm, Whatman) pre-equilibrated Solvent system: Linear gradient elution frc'm (A) (A) 1C mM HCOONH (pH 6.5) : CH CN = 90 : (B) 0.5 M HCOONH; (pH 6.5) : CH$N = 90 :

687

to

fractions

NMR-immunoreactivity

to be derived B,

exchange were

acid

20 kg from 200

subjected

peaks

that peptide

of the was

Bs

G-50 to collect were

further

antiserum

B. The basic

C-25 chromatography

Mr peak was verified

exchange

1, two peaks(a

were

yield

in the smaller ion

(ca. Sephadex

neuromedin

"big" an

utilizing

of neuromedin

NMB-immunoreactivity

muscle

thus

on Sephadex RP- and

on

in which

smooth

part

by SP-Sephadex tissues

gel-filtration

purification,

radioimmunoassay

the C-terminal prepared

(62

the first

emerged

sensitive

specifically

fraction(SP-IIT),

several

In the present

B. from porcine

size:

20 ml/tube. with solvent to (B). 10 (V/V) 10 (v/v)

(A).

Vol.

130,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a

0.2

a 000 500 0 30

20

40 T lme

Fig.

the

0.10

40

respectively.

each peak was submitted

column(Fig.

immunoreactive

pure

30 T I me

from peak a and b,

cartridge,

2a

peptides

RP-HPLC on a Chemcosorb state

20

lmln)

purified

c-18

diphenyl

60

50

60

(mm)

2. First RP-HPLC cf ir-NMB fractions. Sample: (a) Fraction a (#71-74) in Fig. 1 (2/S portion). (b) Fraction b (#75-77) in Fig. 1 (l/Z portion). Column : Chemcosorb 7-diphenyl (4.6 x 250 mm, Chemco). Flow rate: 1.5 mljmin. Solvent system: Linear gradient elution from (A) to (B) for 120 min. H2° : CH3CN : 10% TFA = (A) 90 : 10 : 1, (B) 40 : 60 : 1 (v/v,.

NMB-30 were Sep-pak

50

and

2b)

obtained

to

above

3 ODS-H column.

give were

After

desalting

with

to RP-HPLC on a Chemcosorb one

main

each further

ir-NMB to

Two

peak.

purified

NMB-30 was purified

7-

by another a homogeneous

Final purification of NMB-32 was accomplished by RP-HPLC on 3b). same column as above but with a shallow gradient of acetonitrile to afford (Fig.

NMB-32 as a single

peak

(data

not

shown).

.._._____...--~-__________.... ---I; I a

b

II;; - 40

.._.-- ---

~~_~.___.___...---.-~--

0.2

JO

- 20

- 10

0.1

I

20

Fig.

30 Tl

40 m e (mlnl

50

(

10

20 Tl me

30 (mln)

40

3. Second RP-HPLC of ir-NMB fractions. Sample: (a) ir-NMB fraction eluted at 50-52 min in Fig. 2a. (b) ir-NMB fraction eluted at 50-52 min in Fig. 2b. Flow rate: 2.0 ml/min. Column: Chemcosorb 30X-H (8.0 x 75 mm, Chemco). Solvent system: Linear gradient elution from A:B = SO:20 to A:B 0:lOO Solvent 4 and B were as in Tie:. 2. for (a) 128 min and (b) 192 mjn.

BIOCHEMICAL

Vol. 130, No. 2, 1985 From porcine Mr>3,000

spinal

obtained

the

starting

the

first

material

in the

preparative

B,

containing

purification

present

RP-HPLC,

the main peak of ir-NMB 1. Each peak of ir-NMB

basic

peptides

of

spinal

cord,

is

1 of ref.

7).

from porcine

purification

one main

yielded

(see

ir-NMB

fraction

ir-NMB

fraction

Amino acid

eluted eluted

sequence

Fig.

On at 142 min,

peak was observed

found

brain.

acid

starting

cords

of NMB-32

gas-phase

sequencer. yield

brain isolated

a gas-phase Sequence

based

except

neuromedin specificity of NMB, "big"

because

from

NMB which

as shown in Fig.4.

*NMB-32: -25(3), Phe 1.1@(l), NMB-30: Gly 3.12(3), Phe 1.09(l),

strictly

about

in

a

a

high

assigned

of NMB-30 from was submitted

amounts

of

generated,

identified.

Identity

those

from brain

these

results the

and spinal

were

NMB-32 and B-30 have been

of

was confirmed

combined C-terminal

cord

to

peptides.

acids

all

recognizes

at an

with

case of NMB-32 and NMB-30 recovered

with

from brain

comprising

relationship,

Asp Ala Trp Asp Ala Trp

cord

acids

by using

proceeded Sequence

of

and '240

brain,

and PTH-amino

definitely

to be

with

the

structure unambiguously identified

as

B.

of a family

biosynthetic

were

Thus,

the

In

per

The yields

was definitely

very limited

Considering

NMB-32 and NMB-30 both

Isolation

of the

spinal

NMB-32

peptide

estimated

performed

from

and His-30.

in good yields

comparison.

neuromedin

purified of

residues

of PTH-amino were

at each cycle

Thr-28

residues,

and B-30

of RIA for

identified

the

and NMB-30

300 pmol(NMB-32)

analyses

the purified

performed

two Trp brains.

the recovery

acid

for

all

C-terminal

B-32

on

degradation

except

cord, were

by chromatographic

peak from

NMB-32

including

to be about

in the same way as NMB-32.

sequencer,

several

case

and NMB-30 from

footnote):

kg of pig

Sequence

Edman

spinal

analyses

acids

18.5

deduced

cycle.

residue

from

a single

HPLC

of NMB-32 and NMB-30 were

and the PTH-amino

was analysed

(see

and 280 pmol of NMB-30,

repetitive

up to C-terminal

data

from

were

repectively,

Edman degradation

350 pmol

analyses

The yields

pmol

spinal

pmol(NMB-30),

to

exchange

to Fig.

RP-HPLC as in the

NMB-32 was purified on cation

of 32 and 30 amino

respectively.

initial

by repeated

similar

of NMB-32 and NMB-30

and 560

750 pm01 peptides from

in a pattern

later.

to consist

molecule,

peaks

purified

earlier

Based on the amino were

two ir-NMB

was further

of NMB-32 and NMB-30 from ir-NMB

their

previous

U-25 (lo), which with minor ones corresponding to GRP and neuromedin Cation exchange HPLC on a TSK CM-2SW column of at 138 min and 131 min.

along eluted

the

Fraction

cord:

in our

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.98(2), 2.78(3), 1.51(2), 1.95(2), 1.80(2), 1.58(Z),

Thr Val Lys Thr Val Lys

NMB, as

NMB-30 and NMB-32 is

observed

1.00(l), 1.00(l), 1.26(l), 0.81(l), 1.00(l), 1.18(l),

S er Met His Ser Met His 689

in another

2.16(2), O.81(1), 1.91(2), 2.12(2), 0.87(l), 1.93(2),

GIU

Ile Arg Glu Ile Arg

indicative

mammalian

1.16(l), 0.96(l), 3.74(d). 1.18(l), 0.96(l), 3.97(d).

family

Pro 3.84(d), Leu 2.72(3), Pro 3.30(3), Leu 2.75(3),

of of

Vol. 130, No. 2, 1985

BIOCHEMICAL

1

Neuromedin

B-30

Fig.

from is

NMB

series

found

and

NMC

may

of

human

signal

in

is

homology

observed

fact aligned

protein,

and

physiological

our

previous

for

two

even

(9)

(NMB they

and

are

context, the

to

note

hand, NMB-.?(I

Furthermore, uterus NMR-,?O

was

that

Reeve tetrapeptide

our

isolation

is

almost NMB-32

similar may

to serve

not

have

isolated

of

constant

in

NMB-30,

both

NMB. only

brain

precursors

690

and

y , it to

is

most

NMB but

In

(14).

spinal

also

this

On of

cord

the

NMB-3” ‘. (5:4).

effects

likely

the

dipeptidyl

ratio

stimulant

potent

of

CRPC5-271,

GRP

relative

in

family,

(13).

P

own

out

cleavage X-Pro

of

human its

precursors

intestine

form the

(4:3)

have

to

be

distribution

peptide

substance

deleted

of

distinct

canine

that

According1 as

of

to

pointed

a possible

from

indicated

of

the

known

of we have

similar case

is

of

bombesin-like

enzyme the

Ala-Pro-Val-Ser yields

and

that

an

GRP

:J-terminus

that

existence

way.

a remarkable despite

differences

mammalian

in

al.

The

NMC groups

requires

by

the

Pro-

N-terminal

mRN.4 structure

the

a Pro-

the

four to

Since

a

processing.

residues,

at

NMB-.?O

reported et

in

a similar to

that

addition

ln

by

for of

in

we have

preceded

indicate

GRP.

far.

processing also

sequence

exist

possible in

in

NMB-3’2

sequence

that

N-terminal

other

processed

Ala-Pro

so

NW3 and

NMC groups)

from

aminopeptidase

to

the

facts

VMB-30. a similar

which

signal

than

and

that

is

C-terminal

sequence

unknown

suggest

unit,

GYP,

longer

signal

of

suggesting

(hANPs),

similar

twelve

presumed is

site,

for

Pro-Arg,

Interestingly,

These

and

of

residues

which

A conversion N-terminal

NMB-,i2

As

after

NMB-3” I‘-

cleavage the

NMC.

place in

C,

hormone

( 11 ).

GRP.

processing

manner.

specific

with

C-terminal tak?

NMB unit

ncuromedin

smallest

in

is

its

polypeptides

that

the

compared

must

a typical

similar

noted

functions

groups

though

five

NMB-,32

paper

a

sequence

after

precursor

a be

the

is

and

the

both

in

directly cl?),

15

NMB-30

the

not

molecule

should

NMB-32

preproGRP

in

be

conserved

that

and

natriuretic

precursor

it

GRP

of

GRP

proceed

must

Additionally,

and

processing

is

a-hANP,

the

NMB-32

N-terminus

atria1

structure

residues

NYB,

between

identified,

.Arg

of

structure

is

recently

to

the

Pro-Arg

relationship

Arg

sequences

NMB

to

the

10

comprising

I’ big”

adjacent

Al though

to

acid

Amino

peptides,

conversion which

5

:

4.

bombesin-like

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on

rat

that

NMB-02

and

have

their

own

Vol.

130,

No. 2, 1985

physiologic their

functions.

pharmacological

BIOCHEMICAL

We are

AND

BIOPHYSICAL

now at the

and physiological

starting

RESEARCH

point

for

COMMUNICATIONS

investigation

functions.

ACKNOWLEDGSMENTS: We sincerely thank Miss Masuyo Hiranaga of our Department for her technical assistance. This work was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture of Jaoan. REFERENCES 1. Erspamer, V. (1980) in "Gastrointestinal Hormones", Ed. by Glass, G.B.J. (Raven, New York) pp. 342-361. 2. Tache, Y. & Brown, M. (1982) Trends in NeuroSci., 3, 431-433. 3. Walsh, J.H., Wong, H.C. & Pockray, G.J. (1979) Fed. Proc., 38, 2:115-2319. 4. Moody, T.W., Thoa, N.B.. O'Donohue, T.L. & Jacobowitz, D.M.71981) Life Sci., 29, 2273-2279. 5. McDonald, T.J., JBrnvall, H., Nilsson. G., Vagne, M.. Ghatei, M., Bloom, S.R. & Mutt, V. (1979) Biochem. Biophys. Res. Commun., 90, 227-233. h. McDonald, T.J., .JBrnvall, H., Ghatei, M., Bloom, S.R. & Mutt, V. (1980) FEBS lett., 122, 45-48. 7. Minamino, N., Kangawa, K. & Matsuo, H. (1983) Biochem. Biophys. Res. Commun., 3, 541-548. 8. Minamino, N., Kangawa? K. & Matsuo, H. (1984) Biochem. Biophys. Rcs. Commun., 9, 14-20. 9. Mjnamino, N., Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 3, 425-932. 10. Minamino, 11. Kangawa, K. & Matsuo, H. submitted to this journal. 11. Oikawa, S., Imai, M., Ueno, A., Tanaka, S., Noguchi, T., Nakazato, H., Kangawa, K., Fukuda, A. & Matsuo, H. (1984) Nature, Coq, 724-726. 12. Spindal, E.P., Chin, W.W., Price, J., Ree, L.H., Besser, G. & J.F. (1984) Proc. Nat]. Acad. Sci. USA., e, 5699-j703. Habener, 13. Kato, T., Nagatsu, T., Fukasawa, K., Harada, M., Nagatsu, I. & Sakakibara, S. (1978) Biochim. Biophys. Acta, 525, 417-422. 14. Reeve, J.R., Walsh, J.H., Chew, P., Clark, B.,xwke, D. & Shively, J.E. (1983) J. Biol. Chem., 3, 5582-5588.

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of