Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary

Vol.

183,

March

No.

2, 1992

BIOCHEMICAL

AND

ASPARTYL

Department

of

PROTEOLYTIC

VESICLES

Claudia

S. Toomim

Biochemistry,

Received

January

SUMMARY: neural

3,

and Vivian

Uniformed

Services

and

heterologous

intermediate

preproenkephalin with

and aspartyl

and

previously IL

and

NL

precursor

cleaving

characterized

of 4.5;

this

by

granule

granules,

activities.

0 1992 AcademicPress, Inc.

may

and

recognizing precursors

hypothesis

H

Sciences,

amino such

of

peptide

(3-6)

and

that

Abbreviations: PTP, 35S-(Met)-PPE, protease; preprotachykinin.

different

show

of

is

may and

Kcx2-related cells

prohormone thiol protease; 35S-(Met)-preproenkephalin;

449

may

to

cathepsin

like

that

are

function

of

processed

at

several

processing

for

processing

Recent protcases

CGAP, chromaffin 35S-(Met)-R-PPT,

by

proteolytic

peptides

responsible

possess

D

vesicles vesicles,

similarly that

pH

previously

aspartyl

ncurotransmitters.

yeast

neuroendocrine

be

a

important are

acidic enkephalin

secretory

bioactive

an

suggesting

to

secretory and

precursors

sites

with

related in NL

to mature

proteasc’ granules

and

related

pituitary

vesicles

chromaffin activity

(CGAP)

thiol-dependent

hormones mammlian

be

with

precursor

thiol

tachykinin

CGAP

residues,

secretory enkephalin

medulla

of

35S-(Met)-

NL

proteolytic

stimulation

cleavage

substrates, and

vesicles

characterized

‘prohormone

that

prohormone acid

were

a novel

may

precursors

secretory

the

protcase

similar

cellular Different

monobasic

enzymes

carboxypeptidasc

contain

specific

different

Health

cleaved

inhibition activity

results

of prohormone

cells.

aspartyl

A aspartyl

chromaffin

dibasic

pituitary

adrenal

of immunoreactivc

These

conversion

resembled

This

by the presence

upon

in isolated

IL that

from

pepstatin

immunoblots.

released

activity

activity.

neuroendocrine

of the

precursor

also demonstrated

shown

anti-CGAP

The

of bovine

activity

characterized

and

chromaffin

as indicated

University

activities

tachykinin

proteolytic

vesicles

as

Y.H. Hook

35 S-(Met)-R-preprotachykinin.

purified

optimum,

lobes

and

thiol-dependent

a pH optimum

PITUITARY

20814

proteolytic

(IL)

enkephalin

contained

MD

IN

1992

Thiol

(NL)

ACTIVITIES

OF BOVINE

Bethesda.

the

COMMUNICATIONS

Pages 449-455

SECRETORY

(2),

RESEARCH

16, 1992

THIOL

(1).

BIOPHYSICAL

AND

studies

(7-10)

a common

of

support set

of

granule aspartyl 35S-(Met)-R-

0006-291X/92 $1.50 Cop.vright 0 1992 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

Vol.

183,

No.

proteases

2, 1992

for

exopeptidase in

processing involved

secretory

pituitary,

and

of

(ll),

of adrenal novel

have (I),

prohormone

recently

for

thiol

and

enzyme

monobasic

and

PTP’s

concanavalin

A

distinguish

cleaved than

thiol the

the

showed subunit CGAP

could

proendothelin

that

form

cleavage

at

precursor

segments

medullary

be

to the

yeast

secretory

vesicles

This

study,

or

to find

and

substrates We secretory

that

to

suggesting

that

precursor

CGAP

PTP

selectivity, more

cathepsin

be

proteolytic

proteases

activities

form

of 3SS-(Met)-preproenkephalin and

bovine

in

in the

that

kDa

It is possible

as adrenal in

of

and a 30+17

D (2).

similar

(NL) thiol

characterization

such

involved

protease

medullary

site,

in

addition

the

degradation

to of

peptides.

possesses of

as 3 5 S -( Met)-Raspartyl

at a hydrophobic

may

active

detected

47 kDa polypeptide

lobe

and

p1 of 6.0, and binding

Biochemical

precursors

cleavage

become

that

acidic

a major

granule

neural find

the

of

(l),

protease

rcsemblc

processing

if pituitary

vesicles,

within

35S-(Met)-preproenkephalin

residues.

and which

granules,

specificity

site

B

chromaffin

dibasic

in

do not

candidate

enkephalin-containing

demonstrated

an aspartyl

an unusual

sites, that

a

precursor

cleavage

vesicles,

cathepsin

granules

35S-(Met)-R-preprotachykinin.

and

involved

requires

heterologous

chromaffin

(7-9)

is a likely

active

PTP

precursor

activity.

-- that co-purify,

chromaffin

preprotachykinin.

genes

enkephalin PTP

of 33 kDa,

lysosomal

enkephalin

in (2).

secretory

mass

Interestingly,

also contain

(IL)

pituitary

from

granules

intermediate

in

it

model

dibasic

In this

medulla,

chromaffin

appropriate

that it exists in two forms -- a single

two

bovine

optimally to

molecular

at hydrophobic

that

from

related and

appropriate

generates

localized

protease.

cleaving

cleaves

is

precursor

Chromaffin

CGAP

adrenal

are

(8,9),

protease

is

tachykinin

preprotachykinin (CGAP)

that

H, an is located

mammalian

brain

possesses

residues,

processing.

be a novel

it

(Met)enkephalin,

proenkephalin

readily

of

involved

(P’W aspartyl

since

and

it

(PC’s), (8,9),

Carboxypeptidase processing,

including

expression

and characterized D-like

environment,

since

Recently,

protease’

intragranular

may

tissues

in pituitary

purified

processing

dibasic

of prohormone

convertases

found

and a cathepsin

intermediates

steps

COMMUNICATIONS

(10).

‘prohormone

processing

(3-6).

RESEARCH

precursors.

later

neuroendocrine

prohormone

medulla

We

many

has been

BIOPHYSICAL

peptide

of the

pancreas

subtilisin-like

gene

AND

different

in one

vesicles

brain,

encoding Kex2

BIOCHEMICAL

aspartyl

pituitary protcolytic

these

activities

AND

METHODS

may

to those

secretory were

in adrenal

vesicles

characterized and

of with

35S-(Met)-8-

activities

were

be similar

to

present those

in

granules.

MATERIALS

Purification of bovine uituitarv secretory vesicles. Secretory vesicles of bovine intermediate and posterior pituitary were purified as described by Loh et al. (12), and by Russell (13). Intermediate (IL) and neural (NL) lobes from 20 fresh bovine pituitaries (Biological Research and Delivery Service, Gaithersburg, MD.) were dissected and homogenized in 0.25 M sucrose-10 mM Hepes pH 7.0 buffer in a Thomas

450

Vol.

183,

No.

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

After centrifugation of homogenates (3000 x g, 30 sets.) B t&on/glass homogenizer. the pellet was rchomogenized and recentrifuged the supernatant was kept on ice, and the resultant supernatant was combined with the first (3000 x g, 30 sets.), This combined supernatant fraction was centrifuged at 4000 x supernatant fraction. g for 1.5 minutes, the resultant supernatant was collected and centrifuged at 26,000 x g for 15 minutes; the pellet was the crude vesicle fraction. This crude vesicular fraction was resuspended in 5 ml 0.25 M sucrose-10 mM Hcpes pH 7.0 buffer and layered onto a discontinuous mctrizamide gradient consisting of 5 ml each of 0.28, 0.25, 0.20, 0.15, 0.10, and 0.05 M metrizamide in 0.25 M sucrose/l0 mM Hepes buffer. The gradient was centrifuged at 104,000 x g for 60 minutes, and the vesiclecontaining fractions were collected according to Loh et al. (12) and Russell (13). These vesicles were then subjected to a second identical metrizamide gradient to insure removal of lysosomal material. Vesicles were lyscd by freeze-thawing and protein content was dctcrmincd by the Lowry method (14). Production of Assav of 35S-(Mct)-PPE and 35S-(Met)-PPT clcavinrr activities. 35S-(Mct)-prcproenkephalin ( 35S-(Met)-PPE) and 35S-(Met)-13proteolytic substratcs preprotachykinin (35S-(Met)-p-PPT) from the rat PPE and human p-PPT cDNA’s was achieved by in vitro transcription and translation as described previously (1,2). Cleavage of these precursors by lysed NL and IL secretory vesicles (20-25 ug protein) was assayed by measuring the production of trichloroacctic acid soluble radioactivity, as described previously (1.2). 35 S-(Met)-PPE or 35S-(Mct)-PPT (20,000 to 50,000 cpm) were incubated with enzyme samples at pH 3.0 to 8.0 (final buffer concentrations wcrc 100 mM Na-citrate pH 3.0-5.5; 100 mM potassium-phosphate, pH 6.0-7.0; and 100 mM Tris-HCl pH 7.5-8.0) in a total volume of 20 ul at 37O C for 9 or 2 hours, respectively. All assays were performed in duplicate. Production of anti-chromaffin eranule aruartvl urotease serum and immunoblots. Chromaffin granule aspartyl protcase (CGAP) was purified from bovine adrenal medulla by concanavalin A-Sepharose, Sephacryl S200, and chromatofocusing as dcscribcd previously (2). Balb/c mice were injected intramuscularly and subcutaneously with purified CGAP (lo-20 ug in Freund’s adjuvant) three times at two week intervals. The antiserum was tested in immunoblot analysis with purified CGAP, chromaffin granules, and NL and IL secretory vesicles. After SDS-PAGE and clcctropborctic transfer to nitroccllulosc, immunoblots were pcrformcd according to the manuIacturcr’s protocol (Biorad).

RESULTS Heterologous

precursor

(35S-(Met)-PPE) characterize those

in

showed (IL)

and

proteolytic

activities

adrenal

medulla

chromaffin

granules.

thiol-dependent

neural

Dithiothreitol

lobe

by complete

indicate

that and

pituitary

cleaving

activity

proteolytic

inhibition

of

little soybean

acitivity

Lack

trypsin

pituitary

vesicles

vesicles,

with

the

of inhibition

inhibitor

reducing

activity

indicated 4.51

was

by

165-183% cleaving

or effects

was

to with

(Table

I) lobe

stimulation

dithiothreitol. over

and NL vesicles, inhibition

used

intermediate

agent

to

in IL

by EDTA

studies in

as demonstrated

Partial

activity

secretory

present

and 35S-(Met)-PPT

inhibitor.

inhibitor

were

was

was also present

metalloproteolytic

(35S-(Met)-R-PPT) Protease

cleaving

35S-(Met)-PPE protease

of

activity

secretory

35S-(Mct)-PPE

pepstatin A, an aspartyl chymostatin and leupeptin. PMSF

proteolytic

(NL)

increased

Aspartyl

of 35S-(Met)-preproenkephalin

35S-(Met)-R-preprotachykinin

compare

of 3 5 S-(Met)-PPE (100%).

in the form

and that

and

substrates

controls as shown

activities

by

achieved

by

by CaC12 and CdCl2

present. Lack of inhibition that serine proteases were

by not

Vol.

183,

No.

BIOCHEMICAL

2, 1992

Table

I.

Effect

of

AND BIOPHYSICAL

Protease

Inhibitors

Enkeuhalin NL 100% 97 165 117 99 112 101 94

Control PHMB DTT, 10 mM EDTA, 1 mM CuSO4.4 mM CaC12, 1 mM CdCI2, 1 mM Soybean trypsin inhibitor, 50 pM Iodoacetate, 1 mM Pepstatin A, 60 pM Leupeptin, 1 mM Chymostatin, 50 pM PMSF, 0.1 mM al-antitrypsin, 3.6 pM al -antichymotrypsin, 4.0

and

Activators

Precursor IL 100% 116 183 118 100 106 118 123

96 8 70 88 99 83 99

pM

RESEARCH COMMUNICATIONS

Tachvkinin NL 100% 62 102 84 133 140 83 99

119 10 70 111 100 116 91

Precursor IL 100% 85 120 94 125 134 103 102

97 6 81 61 82 44 81

100 17 62 69 102 32 76

Lysed pituitary sccrctory vcsiclcs from NL (neural lobe) and IL (intermediate lobe) were prcincubatcd with inhibitors or activators in the abscncc of substrate on ice for were initiated with the addition of 35S 15 minutes, and the protcolytic reactions 35S-(Met)-p-preprotachykinin. Values are expressed as (Met)-prcproenkephalin or percent of control (100%) without inhibitors or activators, calculated as the average from two or three dctcrminations. Standard deviation for all assays was less than 20%. Abbreviations for inhibitors are: PHMB, p-hydroxymercuribenzoate; DTT, dithiothreitol; EDTA, ethylencdiaminetetraacetic acid; PMSF, phcny1mcthy1sulfonyIfluoridc.

detected.

These

protease

cleaving

activities

in

and

aspartyl

radiolabeled (Fig.

pituitary

proteases

Conversion

of

optima

the

products at pH

for

profiles IL

and

characterized was

1). Conversion

optimal

inhibitor

optimal

5.0

with

cleavage

most

at pH

used

in

protease

similar

granules. not

major

CGAP

obtained below

per

probably

the detection

The

NL

occuring

amounts

and

to 1.75

of the CGAP

mg NL

vesicle

immunoblot 452

band

contain

an

medullary

subunit

staining

protein

assay.

(CGAP)

pH

aspartyl

the 47 relative

with

(40 gg obtained

IL

was

chromaffin

53 and 47 kDa

obtained

also

The

the 17 kDa subunit

of this

4.0-5.5 was

autoradiography,

protease

recognized

possessed

a 30 kDa

pH

4.0-6.0.

and

vesicles adrenal

CGAP;

soluble

(data not shown).

aspartyl

antiserum

medulla

of IL vesicles

pH

radioactivity

bovine

(TCA)

between

thiol (1,2).

radioactivity

between

granule

vesicles

activity

by SDS-PAGE

soluble

similar

granules acid

soluble

secretory

of low

secretory

amount

much

that the

adrenal

bands,

low

compared limits

2) showed because

immunoreactive gland,

from

of bovine

polypeptides.

was also present.

pituitary

(Fig.

subunits

detected,

and 30 kDa

if

purified

resemble

chromaffin

to acid

to chromaffin

find

that.

Immunoblots

kDa polypeptide was

to to

with

as assessed of TCA

vesicles

35S-(Met)-R-PPT-

to trichloroacetic

precursor

of the activity

generated

immunoblots

5.0,

and

medulla

precursor

of the precursors,

antiserum

secretory

adrenal

of the tachykinin

was the same as that for production Mouse

NL

in

enkephalin

of 35S-(Met)-PPE-

of CGAP to the 47 species

lower

as

intensity

vesicle per

and 30

protein

gland)

was

Vol.

183,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

PPT

PPE

5,

.b

2

3

4

5

PH

6

7

8

PH

Figure 1. pH Dcoendcnce of Proteolvtic Activities in Bovine Pituitarv Secretory Vesicles. Panel a: Cleavage of 35S-(Met)-preproenkephalin by proteolytic activity in NL (0) and IL cm) sccrctory vesicles was assessed by measuring the production of trichloroacetic acid (TCA) soluble radioactivity, expressed as cpm (x 10e3) generated per assay tube. Panel b: Cleavage of 35S-(Met)-B-prcprotachykinin by proteolytic activity in NL (0) and IL sccrctory vesicles (m) was asscsscd by production of TCA soluble radioactivity.

DISCUSSION and

Pituitary

adrenal

processing,

may

contain

monobasic

amino

acid

enkcphalin

precursor The

(1,2).

as a novel

sites.

Bovine

cleaving

activity

‘prohormone

thiol

vesicles

of

secretory

protcascs

responsible

(CGAP),

resembling

pituitary

of

similar

proteases

protease granules

medulla

adrenal

cathepsin

1

and

(l),

chromaffin precursor

were and

study,

neural

(NL)

of prohormone at dibasic granules

cleaving

purified

and

a chromaffin

In this

contain

site

precursors

tachykinin

D (2).

also

processing

activities

(PTP)

(IL)

a major

medullary

and these

protease’

medulla,

for

adrenal

for

intermediate

vesicles,

and

contain activities

characterized

granule

aspartyl

we show that secretory lobes, like chromaffin

dithiothreitol-stimulated

enkephalin

2

-66kDa -+53

kDa -45

“wmc47

-14 Figure

2.

Immunoblot

eVesicles (12% polyacrylamidc) membranes. Anti-CGAP CGAP in chromaffin f.rg protein, lane 2).

Analvsis

of

Chromaffin

Vesicle

Granule

fractions

and were electrophoretically antiserum (final dilution 1:500) granules (60 ug protein, lane 1)

453

Asoartvl

Protease

in

were subjected to SDS-PAGE transferred was used

to for

nitrocellulose immunoblots

of

and in NL secretory vesicles (60

Vol. 183, No.

2, 1992

precursor

cleaving

cleaving of

activity

activities

pituitary

with

Further the

amount of

allowed

in

of

specific

immunoblot

possible

band

that

CGAP

was

not

detected

was detected

in NL

the

characterization

NL

will

kDa

proopiomelanocortin

(POMC)

in

have

been

(16) and

53 kDa

CGAP

immunoreactive

often

possess

find

if

a high

of the related

aspartyl

Purification yet been presence

of

homology

pituitary

aspartyl

protcascs

exist

in

in adequate 33 kDa

quantities

thiol

protease

possessed

CGAP

53

CGAP

the and

CGAP

characterized.

Thus, and

CGAP. pituitary

may

be related. kDa

production

that

not

may

kDa

process in

known

if the

be related

to the

mechanistic study

will

sccrctory

of

class possible

bc important

to

vesicles.

chromaffin

granules

of antibodies. may

Further 53

provasopressin

future

vesicles

of It is

of

CGAP

from

protease’

amounts

granuIes.

of the aspartyl

(17).

in pituitary

lower

It is

proteasc(s)

kDa

the

and

(15)

protease

for

vesicles

that

ncurocndocrine

thiol

levels

lobe

Proteases

immunoreactive of its low

70

aspartyl

the

(CGAP)

because

approximately

and

cleaving

of the ‘prohormone

achieved of the

purified

However,

CGAP

isoform

medullary

of

polypeptide.

degree

70 kDa

those

because

protease

to chromaffin

an

intermediate

provasopressin

to

feasible

gland.

granules,

hypothesis

adrenal

proteases

POMC

be

the

not

Interestingly,

compared

may test

aspartyl

70 kDa

similarities

vesicles

and

vesicles

similar

aspartyl

secretory

kDa.

in chromaffin

to

each

possessed

pituitary

30

band

precursor

secretory

be

was

was not detected

NL

that

may

granule

granules

and

necessary

polypeptide

Pituitary pituitary

53

be

immunoreactive

posterior

bands. 47,

that

from

the 17 kDa band 53,

suggest

proteases

Chromaffin

30 kDa

tachykinin

pituitary obtained

of

COMMUNICATIONS

(1,2).

chromaffin

to the 47 and band

proteases

vesicles

relative

immunorcactive

aspartyl granules

data

to the

of 47 and 30 kDa; bands

These

isolated

analyses.

immunoreactive

optima.

of purified

RESEARCH

A-inhibited

antiserum

bands

the 30 kDa

and

BIOPHYSICAL

pepstatin

chromaffin

characterization

low

production

pH

thiol

identified

AND

and

acidic

possess

previously

of

BIOCHEMICAL

has not

Therefore,

be assessed

the

in future

studies. In summary, medulla

possess

we find similar

dithiothreitol-stimulated inhibited adrenal

aspartyl

The

vesicles It function(s).

participation provasopressin,

presence suggests will be

of PTP

and

vesicles

proteolytic

precursor

cleaving

cleaving

activity. (CGAP)

vesicles of

of pituitary

aspartyl

protease

secretory

medullary

protease(

and

precursor

granule

pituitary

thiol enkephalin

tachykinin

chromaffin

that secretory

these

both thiol

prodynorphin,

in and

processing others. 454

activity, that

contain and

in

and adrenal

as and

detected pepstatin

analyses posterior

cathepsin

aspartyl

pituitary

and NL)

activities

Immunoblot showed

that they may have a role important in future in vitro CGAP

(IL

proteolytic

A-

with

anti-

pituitary

and

D-like

aspartyl

activities

pituitary secretory studies to assess

prohormones

by

including

in

vesicle possible POMC,

Vol.

183,

No.

BIOCHEMICAL

2, 1992

Acknowledgments: Dr.

H.-U.

This

work

The

opinions

The

Affolter

(Univ.

was supported as official

University

of the

thank

of Zurich)

for

by a grant from

contained

construed

authors

AND

herein views

Health

BIOPHYSICAL

G. Hubbard

expertise

in

NINDS,

NIH

are the private of

the

Dept.

RESEARCH

for

technical

generating

the

Defense

assistance antiCGAP

and an intramural

ones of the authors of

COMMUNICATIONS

or

the

and

and serum.

USUHS

grant.

are not

to be

Uniformed

Services

Sciences.

REFERENCES 1.

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Krieger, T.J. and Hook, V.Y.H. (1991) J. Biol. Chem., 266, 8476-8383. Krieger, T.J. and Hook, V.Y.H. (1991) Biochemistry, submitted. Fricker, L.D. and Snyder, S.H. (1983) J. Biol. Chem., 258, 10950-10955. Hook, V.Y.H. and Loh, Y.P. (1984) Proc. Natl. Acad. Sci. USA 81, 2776-2780. Lynch, D.R., Brass, K.M., Hutton, J.C., and Snyder, S.H. (1990) J. Neurosci. 10, 28502860. Davidson, H.W. and Hutton, J.C. (1987) Biochem. J. 245, 575-582. Bresnahan, P.A., Leduc, R., Thomas, L. Thorner, J., Gibson, H.L., Brake, A.J., Barr, P.J., and Thomas, G. (1990) J. Cell Biol. 111, 2851-2859. Smeekens, S.P., Avruch, AS., Lamendola, J., Chan S.J., and Steiner, D.F. (1991) Proc. Natl. Acad. Sci. USA 88, 340-344. Seidah, N.G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubicn, G., Mattei, M.G., Lazure, C., Mbikay, M., and Chretien, M. (1991) Mol. Endocrinol. 5, 111-122. Christie, D.L., Batchelor, D.C., and Palmer, D.J. (1991) J. Biol. Chem. 266, 1567915683. Julius, D., Brake, A., Blair, L., Kunisawa, R., Thomer, J. (1984) Cell 37, 1075-1098. Loh, P.Y., Tam, W., and Russell, J.T. (1984) J. Biol. Chem., 8238-8245. Russell, J.T. (1981) Anal. Biochem. 113, 229-238. Lowry, O.H., Roscbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Loh, Y.P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205. Parish, D.C., Tuteja, R., Altstcin, M., Gainer, H., and Loh, Y.P. (1986) J. Biol. Chem. Barrett, A.J. (1986) In: Proteinase Inhibitors (A.J. Barrett and G. Salvesen, Bds.), pp. 13-16, Elsevier Science Publishers, Amsterdam.

45.5