Gawk, a novel human pituitary polypeptide: Isolation, immunocytochemical localization and complete amino acid sequence

Vol.

126,

No.

January

1, 1985

16,

BIOCHEMICAL

AND

S.

Benjannet,

R.

Laboratories

Leduc,

of

Clinical

C. and

Biochemical Research 110

December

y.

4,

During (RP-HPLC) gland extracts, polypept ide

POLYPEPTIDE: COMPLETE AMINO

Seidah,

Lazure, N.G. M. Chrktien

Molecular Institute of

Avenue

M.

602-609

ACID SEOUENCE

Marcinkiewicz

Neuroendocrinoloay Montreal

and

Pine

Montreal,

tary

COMMUNICATIONS

Pages

GAWK, A NOVEL HUMAN PITUITARY IMMUNOCYTOCHEMICAL LOCALIZATION AM,

Received

RESEARCH

1985

ISOLATION,

Sunwnar graphy

BIOPHYSICAL

West

Canada

H2W lR7

1984

the course purification

reverse-phase

of of

a

high big

postulated

liquid chromatofrom human pituino resemblance to any of this 74 amino acid

pressure ACTH (1)

a highly purified peptide bearing was isolated. The complete sequence polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation failed to reveal any homology greater than 30%. data matrix, An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland. 0 1985 Academic known

The

In

des.

human,

gonadotropins, related

hormones

neurophysins,

phin

The

in other

organs,

structure,

function

ACTH

originating

is

of and

of

great

mechanism

propressophysin the

raises

active include:

polypeptiprolactin,

pro-opiomelanocortin (Z-4).

in

and

lobe

hypothalamus, of

peptides

interest

and

the

C-terminal new

anterior

B-endorphin in

the

discovery

the

hormone

and

biologically

many

in

growth

neurons

by

contains

present

including

vasopressin, (5-E).

gland

thyrotropin,

peptides

innervated

pituitary

pituitary questions

(POMCJ-

The posterior contains (CPP) gland,

as to their

lobe,

oxytocin, and dynoras

well

as

source,

of action.

MATERIALS

AND

KTHODS

Purification. Whole human pituitaries were extracted as in (9). HPLC column, After crude purification on a (2.5 x 30 cm) Cra semi-preparative the immunoreactive material was rechromatographed on a Waters PBondapak Cl8 (0.78 x 30 cm) equilibrated with 20 X acetonitrile (containing 0.1% trifluoroacetic acid, TFA, v/v). The linear gradient was 20-40X acetonitrile (containing 0.1% TFA) in 40 min at a flow rate of 2 ml/min. Immunoreactive fractions were pooled and repurified using a linear gradient of 30-50% acetonitrile (containing 0.13% heptafluorobutyric acid, liFEA, v/v) at a flow rate of 2 ml/min. 0006-291X/85 Cowright All rights

$1.50

0 1985 by Academic Press, of reproduction in any form

Inc. reserved.

602

Vol.

126,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Sequence analyses. Amino acid analyses were carried out at 108°C in 5.7N HCl containing 0.1% phenol and 0.05% B-mercaptoethanol. The analyzer was an updated Beckman 120C. The primary structure was determined by automated liquid Edman degradation and performed on a Beckman 89OC sequencer using a 0.3M Quadrol program. The sequencer was equipped with a Sequemat P-6 automatic converter and a Sequemat SC-510 controller. The phenylthiohydantoins (PTH) obtained were identified by RP-HPLC (10). Ophthalaldehyde was used to block contaminating sequences when positions of proline residues were known as described previously (11). Double cleavages were done at those proline positions. Proteolytic cleavage was carried out with a IX (w/w) ratio of endoproteinase Lys-C (Boehringer Mannheim) in IO mM Tris-HCl pH 8.0 for 6 hr at 37°C. The fragments produced were purified by RP-HPLC. Imunocytochemistry. Five urn Paraffin sections of human pituitaries preserved in Bouin’s fixative were incubated for 48 hr at 4°C with rabbit antiserum and processed by the PAP method according to Sternberger (12). The optimal concentration of the antiserum diluted in PBS was l/2000. For the control, 200 ~1 aliquots of diluted antiserum (l/1000 in PBS) were pre-absorbed with 4.6 vg of the synthetic antigen (residues 20-38) for 48 hr at 4°C with shaking. These were centrifuged at 12,000 rpm for 5 min and the supernatant was used for incubation. Processing with non-immunized rabbit serum and omission of the first antibody were used to determine non specific background. for 24 hr amino acid

RESULTSAND DISCUSSION complete

The

acids

amino

neither

is

shown

sequence

in

glucosamine

Fig.

nor

of 1.

The

the

polypeptide

polypeptide were

galactosamine

is

which

is

detected

not by

composed

glycosylated, amino

acid

of

since analysis.

1-74 GAWK 20-74 GAWK

25 30 P-+~~~:~~E-L-D-R-:FY-L-N-Y-G-E-E-G-AGAWK 1-74 GAWK 20-74 GAWK 23-74 L-l

GAWK 20-74 GAWK 23-74 L-f L-2 L-3

GAWK

20-74

L-3 L-4 Fig.

40

--P--v---P----P-----P--P--------------F-P-P7-77--

-7-T--------7-r-P-v-P------

45 50 55 P-G-K-W-Q-Q-Q-G-D-L-Q-D-T-K-E-N-R-K-E-A--------7-r-r--v-P-P-P-m-----------r--v W-Y -w-v-v-.-v-P-F7~~~ -w----.-r 65 70 74 R-F-Q-D-K-Q-Y-S-S-H-H-T-A-E -P-w-7-P-v-r-r-------v---------

be

sequenced

was

The

amino

GAWK 23-74. N-terminus and

acid

The

sequence second

603

of was

GAWK l-74. GAWK 20-74

The which

60

first

peptide added 3 more residues at the was seouenced up to residue 72 (see Fia 39). A lonner peptide identified as GAWK l-74 was later isolated and the first 3fl The fragments denoted L-l resirlues were identified by seouencina (Fio. 3A). L-2, L-3 and L-4 were produced by enzymatic cleavaqe of seoment (3 nmol) 20-74 by ehdoproteinase Lys-C. The amino acid composition of these fraoments are shown in Table 1.

to

1.

35

74

Vol.

126,

No.

initial

The

acid

amino

encompassing

chemical

very

amino

limited

anount

in

2.

fication the

min

that

peptide

1).

As

were

computed

acid

sequencing

shown

Fig.

to

be

1

to

and

computed

to

on

the

be

ce,

were

amino

these denoted

to The

fragments, L-4

residues

of

together

Ala-Glu

the

in and

following

was

with

the

the

1

On the

basis

form

protease

as and

results

Fig. confirm

Thr residue 604

carried

out

since

sequence

amino

hand,

locate

to

an

overlap

it

of

as

the

both

the

sequences

74.

proposed of

at The

sequen-

polypeptide

the of

resulting

the

sequence

of

most

1 he fragment

presence

at position

were

starting

1 respectively. the

20-30,

a possible

of

at

region

sequence

of

digestion

well

min

us

this

characterization

by

at 44.4

that

available

out

confirmation

followed

Table

for

identified

thus

led

(positions

yield

longest

compositions,

listed

sequenced

initial

a Lys-C

20-74 acid

allowed

there

respectively.

were

acids

residues

3A)

system

72 (Fig.

other

the

His-Pro-Gln-Gly-Ala-Trp...

nmol

acids,

(Fig.

for

a

puri-

results

yields On

using

position

to

as

Further

previous up

initial

since

and

RP-HPLC

eluting

our

pepti-

material

degradation

extends

and

peak

composition

amino

and

molecule

the

2A).

of

development

more

(Fig.

the

this

and the

by

respectively.

eluting

IO

confirm

20

of

purify

Edman

GAWK

Because

(HFBA)-acetonitrile

Automatic

nmol

by means of

are

was

3

20-38.

peaks

this

of

permitted

isolated

immunoresctive

us to

repetitive

and

amino

was

system

repetitive

sequence

acid

identified

fragments.

and

of

89.1%

us to

acid

position the

part

72

corresponding

95%

The

Phe-Leu-Gly..., two

38,

the

amino

remaining

at

later

3A).

helped peak

28).

allowed

the

of

corresponding

and

also

in

turn,

major

(Fig. peak

starts

NH2-terminal

Figs.

two

residue

an antiserum

region

polypeptide

analyses

of

heptafluorobutyric

eluting

to

(TFA)-acetonitrile

of

novel

this

acid

acids

available,synthesis

immunoreactive

respectively

early

the

the

a 0.13%

identification

50.2

and

nal

single

A

acid

using

in

amino

sequence

material

production

the

four

call

corresponding

This,

trifluoroacetic

0.1%

on

Fig.

to

COMMUNICATIONS

a 28 amino

first

Further

natural

allow

a radioimmunoassay.

shown

us

RESEARCH

determine

The

notation).

of

to

1).

a fragment

of

de was necessary,,to

to

(Fig.

BIOPHYSICAL

us

prompted

acid

synthesis

AND

allowed

23-50

(Gly-Ala-Trp-Lys)

(one-letter

of

sequence

residues

sequence

the

BIOCHEMICAL

1. 1985

of two additio-

72 (Fig.

1).

of

Vol.

126,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

o FRAUION

1.0

NUMBER

r

FRACTION

NUMBER

Fiq. 2. Qeverse-phase HPLC purification of the polypeotide from whole human pituitary extracts. Chromatooram A depicts the HPLC purification of immunoreactive material previously enriched on a 2.5-cm semi-preparative column (not shown). The column used was a Waters UBondapak Cl8 (0.7R x 30 cm) with 0.1% TFA/acetonitrile as eluent. Immunoreactive fractions (shown as histoorams) were pooled and repurified usino 0.13% HFBA/acetonitrile (chromatooram 8). The dashed lines represent the linear oradient used in each case. The elutions were made at room temperature and monitored by the absorbance at 231-l nm.

A

at the

developed

ty,

Washington,

30%) to pin,

computer

other

proalbumin

encompassing,

data

National

and

search

Biomedical revealed

D.C.) known

bank

proteins

Research

or

fragments

thereof.

of 605

basic

resemble residues.

data

(i.e.

Only the

human

region This

matrix

Universi-

(Georgetown

homologies

precursor a pair

a mutation

Foundation

no significant

a-fetoprotein

interestingly,

using

(13)

greater

than

prosomatotro15-22

comparison

of

GAWK thus

Vol.

126,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CYCLE Nul!8ER

Fig. 3. The phenylthiohydantoin function of seauenator cvcle number obtained native polypept’ide and of (B) fragment 20-72. cycle of the sequence run is depicted in one represents the average decline in yield along repetitive yield of 89.1% (A) and 95% (8).

confirms new

novel

the

family

is

lar,

which

polarity Lys,

the

interesting presence

the

peptide,

letter the

which

Arg

which

to

the

resulting

feature of

represents (141,

His,

of

are represented as a sequencinq of (A) the amino acid at each The solid line notation. sequence, giving an average

the The-deduced

could

represent

entirely

an

proteins.

of One

peptide

character

yields

durinq

20% of is total

acidic

numerous the

expressed number

from

residues

amino

acids

as the

ratio

of

amino

the

sequence

(Fig.

acids,

%) of is

high

this

poly-

Glu in psrticu-

I),

present. (in

of

Also

the

Glu,

Asp,

(46%).

degree Thr ,

of Ser ,

Moreover,

Vol.

126,

No.

BIOCHEMICAL

1, 1985

AND

BIOPHYSICAL

TABLE AMINIl

ACID

COMPOSITION

LYS-C Amino

L-l

AND NATIVE

ENDOPROTEINASF

PDLYPEPTIOE

L-4

L-2

COMMUNICATIONS

1

OF ISOLATED

FRAGMENTS

RESEARCH

Whole

Acids

Found

Molecule 3.2

Asx

(3)

Thr

2.0

(2)

l.n

(1)

-

Ser GlX

3.6

(3)

Pro

i.n

(1)

GUY

3.4

(3)

Ala

1.2

(1)

4.4

(4)

(9)

9

(1)

2.0

(2)

2

1.9

(2)

2.1

(2)

2

2.3

(2)

18.1(1A)

(1) -

Seauence

9.1

1.0

1.3

in

1.0

(1)

18

2.3

(2)

2

7.1

(7)

7

4.5

(5)

5

Val

1.3

(1)

1

Met

0.9

(1)

1

4.4

(4)

4

Ile Leu

2.2

(2)

TV

1.6

(2)

1.0

(I) -

(1)

1.n

Phe LYS

1.2

(1)

1.0

(I)

n.9

(1)

His

1.9

Am

*

Trp

(2)

*

3.0

(3)

3

2.1

(2)

2

5.5

(5)

5

5.0

(5)

5

6.4

(6)

6

*

*

2 74

Total

*

the

Not

determined

presence

of

His-His,

Glu-Glu,

teristic

which

residues, hormones of

the

fragment

paired

some

residues,

Ser-Ser,

Arg-Arg,

at position

or

biologically

major

forms

20-74

is

Gln-Gln,

was mentionned

18-19

I),

to

(Fig.

peptide in

this

be expected

(Fig.

Another the

a feature

study

of

such

as

charac-

a pair

commonly

(15).

of

basic

observed

in

Interestingly,

corresponds cleavage

I),

important

presence

precursors

from

twice

appear

intriguing. is

identified is

which

previously,

active

which

of

one

precisely

after

the

to

pair

of

the

basic

residues. We observed of

the

lpbe.

The

granules of

anterior

the

lobe

observed

dispersed nuclei.

positive

of

the

pituitary,

human

immunoreaction inside

Two

immunoreactive

main

the

areas

in

but the

very

anterior

in contrast

cytoplasm, of

material

localization 607

in cells little

in

on the

lobe

showed

to the

neqative

are evident:

within

sections posterior

dark-brown reaction the

cells

Vol.

126,

No.

1, 1985

BIOCHEMICAL

AND

g**B).

(C) is antigen.

with

within

gland. part

the

to

gland

six

different

are of

needed

secretory

one type

background types

determine

to

cells,

is

in a marked (Fig.

located

mainly

of

antiserum

glandular

Since

whether

this

as was demonstrated

part in

of

the

with

4A),

pituitary

further

peptide

synthetic rela-

contains

at

immunocytochemical

occurs

by Nakane

the

ventral

of immunoreaction the

cells,

(Fig.

anterior

reduction

4C).

stalk

in

(16)

for

known

cstego-

LH and FSH in

of gonadotrophs. back-translation

Nucleotide

reveals code.

of

COMMUNICATIONS

and the

whole

the

pre-adsorption

The

results

lobe

in

immunoresctivity

48).

20-38)

posterior

the

dispersed

major

(Fig.

the

near

sparsely

non-specific

studies ries

However,

(residues

least

sinusoids

cells

the

antigen

tive

the

the

of

of

done by pre-incubating A x 250. B, C x 400.

associated and

Dilution

RESEARCH

in the anterior lobe of the human the antiserum was l/2000 in PBS. Control antiserum (l/1000 in PBS) with synthetic

Immunocytochemistry

pituitary

BIOPHYSICAL

that

A

the

chemically

segment synthesized

44-47

of

exhibits probe

the the

protein least

corresponding 608

sequence

degeneracy to

this

in

region

of the

will

GAWK mRNA be

Vol.

126,

used

to

the

mRNA

No.

BIOCHEMICAL

1, 1985

detect

its

should

precursor

mRNA allow

form,

in

extracts

us

to

of

answer

in situ

and

AND

BIOPHYSICAL

tissues.

different

regarding

questions

hybridization

RESEARCH

should confirm

COMMUNICATIONS

Eventual

cloning

the structure sites

its

of

of

its

of synthe-

sis. ACKNOWLEOGMNTS We thank Dr. Serge St-Pierre for synthesis of peptide 20-38. We are grateful to D. Marcil and A. Sarcone for typing this report. C.L. is a "chercheur-boursier" from the FRSQ. R.L. is a FRSQ pre-doctoral fellow. This work was supported by a Program Grant (PG-2) from the Medical Research Council of

Canada.

REFERENCES 1.

Yalow, 439-445.

R.D.,

Serson,

and

S.A.

Biochem.

(1971)

Biophys.

Res.

Commun.

44,

C.H. (1975) In: Hormonal Proteins and Peptides (C.H. Li, ed.) Vol. 3, 'I-40, Academic Press, New York. and Papkoff, H. (1974) In: Handbook of Physiolooy (E. Nobil 3. Sairam, M.R., and W.H. Sawyer, eds.) Vol. 4, p. 111, American Physioloqical Society, Washinoton, D.C. 4. Boileau, G., Seidah, N.G., and ChrBtien, M. (1981) In: Hormonal Proteins and Peotides (C.H. Li, ed.) Vol. ID, pp. 65-87, Academic Press, New York. 5. Rrownstein, M.J., Russell, J.T., and Gainer, H. (1980) Science 207, 3732.

Li, pp.

37P. 6.

Seidah, Commun.

N.G., IOfl,

Ben,lannet, 901-907.

7. Watson, S.J., 8. Watson, S.J., 9. IO. 11.

Natl. Acad. Rondeau, N.,

(submitted Lazure, C., (1983) Can. Lazure, C., M .,

and

Sternberqer, York.

13.

Dayhoff, Seouence

16.

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ChrBtien, Bioloqy Rasel. Nakane,

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and Chrgtien, Ghazarossian, V.E.,

N.G.,

7R,,

1260-1263. S., Thbherqe,

to Anal. Biochem.) Seidah, N.G., Chrktien, 3. Biochem. Cell Biol. Seidah, N.G., ChrBtien,

M.D. and

Washinqton, 14. 15.

Seidah, Akil, H.,

Gene&,

12.

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J. L.A.

(1984) (1979)

FEBS

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Lett.

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M. M.

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Seidah,

M., Lallier, 61, 287-292. M., Thibault, 172, AO-B6.

Immunocytochemistry,

et al. (19Bl) Protein Sequence Structure, National Biomedical DC (search of June 20, 1984).

Riophys.

Biochem.

N.G., R.,

A.

and and

John

217, B53-854. (19Rl) Proc.

ChrBtien,

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G., Garcia, Wiley

R., and

Data Base, Atlas of Research Foundation,

Y.,

Res

M. S.

Cantin, Sons,

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Watanabe, T., and Wada, H. (1983) J. Biochem. 94, 1269-127R. M., Roileau, G., Lazure, C., and Seidah, N.G. (19B4) In: Cell of the Secretory Process (M. Cantin, ed.) pp. 214-246, Karger, P.K.

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18,

9-20.

New