Identification of the 14 kDa bile acid transport protein of rat ileal cytosol as gastrotropin

Vol.

177,

No.

June

28,

1991

BIOCHEMICAL

3, 1991

OF THE

Alex

D.

Vodenlich,

Marie

C.

*Department

Jr.,

Lin*,

Research

RESEARCH

J.

COMMUNICATIONS

May 13,

PROTEIN

1147-1154

OF

AS GASTROTROPIN

Gong*l,

Lanzett

Pfizer

Medicine,

ACIDTRANSPORT

BILE

Yong-Zhong

Anthony

Division,

of

14 kDa

ILEAL CYTOSOL

RAT

Received

BIOPHYSICAL

Pages

IDENTIFICATION

Central

AND

Kieran

i , and

Inc.,

Eastern

Pennsylvania Hershey,

F.

Frederick

Geoghegan,

A.

Point

Wilson*1

Road,

State University PA 17033

Groton,

College

CT 06340

of

Medicine,

1991

The 14 kDa bile acid binding protein of rat ileal cytosol (I-BABP), previously shown to be the major intracellular transporter of bile acids in enterocytes, was purified by affinity chromatography and gel electrophoresis. Enzymatic digestion of I-BABP which had been electroblotted to nitrocellulose led to the recovery and sequence analysis of four peptides representing 47 residues of sequence (-35% of the ful I sequence). All the peptide sequences displayed high levels of identity (>6G%) and homology 080%) to the sequences of porcine and canine gastrotropin. This high level of homology together with other features of I-BABP identify it as rat gastrotropin, establishing gastrotropin as the major intracellular bile acid carrier of rat enterocytes. 0 1991 Academic Press, In

The lumen

bile

from

acids the

detergents, of

intestine

operating

entering

the than

bladder

the

in

process

putative basol

bile atera

in

cytosolic kDa

that

fatty respect

(4)

intestinal

bile

a blocked

binding

i ve I y)

(6)

than

that in

in

cytosol

of mixed

stool.

binding

rat

brush rat

is

pattern

the

bile been

(I-BABP)

and

liver obtained

amounts

and

incompletely

from

to

acids. to

identify

(2,3), The

major

a polypeptide

distinct (I-FABP

the

acids

membrane

is

of

this

used

of

from and

the

14 14

kDa

L-FABP,

CNBr-cleaved

1 Present address: Division of Gastroenterology, Medical University Please address Carolina, 171 Ashley Avenue, Charleston, SC 29425. to Dr. Wilson at this address.

1147

part

bile

a complex

border

immunologically

intestine

the

border

transport

i lea I enterocytes.

protein

and

sequence

of

5X of

has

brush

this

passive

of

analogues the

(5)

in

as

the

and

forms

intestinal

Acting across

Typically,

circulation acid

proteins

acid

the

the

food.

absorption

reabsorption

bile

N-terminus

The

less

so

enter

acids

active

enterohepatic with

of

fatty

by

Ileal

proteins .

of escape

(l),

and

ingestion

that

excreted

transport

I membrane

has

acid

the

which

efficiency

are

labeling acid

acids high

humans.

Photoaffinity

the

ileum

gut

sterols

absorption

Bile with

in

acidic

following

jejunum.

proximal

of

the

recovered

1 g/day

understood

a family

facilitate

the are

systems

less

gall

they

membrane

are

I-BABP

of South correspondence

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

177,

No.

could

not

FABP,

be

explained

confirming The

BABP.

in

that

present

by

and were

identity

and

porcine

(7)

acids.

This

and

peptides

canine

(8)

a distinct

by

HPLC

of

I-BABP

allowed

I-BABP

to

be

Supplies. from

Sequence

Boehringer

MO) .

An

Mannheim

affinity

as

AND

METHODS

(9).

Polyvinyfpyrrolidone-40 HPLC

Jackson

were

was

HPLC

Purification affinity

of

described

(6),

replacing

by S in

Bedford,

MA)

the

the

Aebersold

et

cut

into

strips

The

tube

was

M acetic

al.

then

containing M sodium buffer

for in

during by

into

100-200 phosphate,

From

bands

to

smaller

was 0.1 M NaHCO

3,

at

37oC

pH 8.2,

with

(10

dry

1.2

ml

For

V8

5% acetonitrile. containing 1148

across

with

0.1%

(Millipore red and

from

Corp., band

the

amount

of

method

of

a protein-free

digestion. performed

by

the

nitrocellulose ml

of

blots

PVP-40

protease

The

tube. dissolved

PVP-40

to digests, For

5X acetonitrile.

in

to was

nitrocellulose

protease

were

microcentrifuge

0.5%

the

Excess

x 1 ml).

as

to

excised

1.5

of

by

loaded

stained

x 1 m) and transferred

buffer.

also

cytosol

a single

was

were

Cl8

electrophoresis

iQ water

digestion.

water

containing

i teal

gels

a blank

adsorption

(1

A Vydac

electroblotted then

a single

enzymatic with

CA).

column

amount in

from

into

prevent

digestion

pH 7.8,

use

excised

pooled

pieces

of

for

and

(Pittsburgh,

City,

rat

blot,

BABP

Burdick

CA).

Mill

each

kDa

Digestions

30 min

order

washing

gl

cut

I-&W.

and

were with

Louis,

described

(Milwaukee,

Fisher

gel

an equivalent

then

subsequent

extensive cut

rms)

blots

(St as

Biosystems.

were

obtained

from

Standard-size

washed

14

I-BABP

x 15

incubated acid

dried.

of

(10). (2

air

was

by

from

step.

The 3 min,

determined;

blot

digestion

nitrocellulose removed

and

isolated

fatty

function

Sigma

Aldrich

(Hesperia,

material

for

from

Iysylglycocholate-Sepharose

exchange

methods.

was

stained

.hzynatic

was a

were

prepared

(Foster

Applied

Group

affinity-purified

taken

of

from

as of

obtained

SDS-polyacrylamide

anion

acid

2 min,

nitrocellulose

also

on

from

supplied

Separations I-BABP

standard

to

S was

Biosystems

affinity-purified

1X acetic for

corresponding

were

DE-52 with

nitrocellulose

were

the

well

similarities

water

acetonitrile Applied

from

V8 protease

purchased grade

sequence

isolated

was

HPLC

I-

gastrotropin.

Ponceau

was

micropreparative

final

width

Ponceau

0.01

from

performed

with

the full

part

grade

acid

I-BA3P.

chromatography

their

HPLC

from

rat

of

resulting

as

other

aureus

S.

IN).

from

cartridges

purchased

protein

lysylglycocholate-Sepharose

composed

MI),

OD-300

column

of

trifluoroacetic

Aquapore

and

(PVP-40)

solvents

(Muskegon, and

trypsin

two

digested

>60%

chenodeoxycholate

identified

(Indianapolis,

column

WI) . PA),

grade

bind

the

possessed

kDa

with

MATERIALS

was of

analyzed

together

the

analysis

electrophoresis

a 14 can

of

sequence

fractionation

gastrotropin,

homology,

sequences

acid

gel

COMMUNICATIONS

(6).

amino and

that

acid

polypeptide

regions

ilea

of

RESEARCH

amino

extensive

to

level

known

isolated The

homology

high

structure,

is

the

BIOPHYSICAL

chromatography

sequenced. >80%

AND

of

describes

purified

enzymatically,

terms

I-BABP

report

I-BABP

digests

and

BIOCHEMICAL

3, 1991

tryptic

a new

then strips

tube this digests,

was

0.1 the

Vol.

177,

No.

3,

1991

H&C.

Peptide

treatment

were

performed

on

pump

and

by

unit hand.

mixtures

fractions

were

selected

used

in

sequencer were

in

was

with

1

shows

1,

(Figure column

to

Data

lane)

region

nitrocellulose with

in

Ponceau the

the

same

low

levels

severely

S.

release

were

of

eluted

(Figure

1,

protein.

Either

present

experiments.

or the

or

of

legends. a peptide

lane).

HPLC on

peak

precycled

(11).

as

described

by

SDS-polyacrylamide

This

fraction

In

character

factors

of

peptides acid

to

have

staining

can

result

available

from

however,

generated sequence

been

kDa to

by

cases,

the

affinity 14

electroblotted

those

amino

appeared

the

visualized

unfavorable

of the

in

protein

percentage

(10). sample

was and

nitrocellulose-blotted

solution

48 noted

material

a high

to

cytosolic

was

digestion,

of

physical

the

band

to

these

unit.

Biosystems

lysylglycocholate-Sepharose

amounting

the

of

assessed from

digestion

in

Applied

sequenced

analyzed

as

a single

enzymatic

opportunities both

and

I-BABP

the

middle for

performed protein

figure

HPLC

then

derived

from

electrophoresis,

peptides

restrict

the

AND DISCUSSION

material

to

Enzymatic

digestion

at

can of

work

the in

KDa tt f-

116 94 67

t

k-z+.

Purification -PO yacrylamide gel

electrophoresis: enterocyte purified

cytosolic by affinity

140A

collected

presence

an

on-line and

acquired

of

bound

preparation

of

were

in

the

using

120A ~1,

30-60

purification

was

subjected

polypeptide

in

the

When

left

and

given

was

a Model

fractions

with

performed

a Model

volume

filters.

Figure

are

included

analysis.

equipped

electrophoresis.

Peak

enzymatic Chromatography

which

consistent

RESULTS

gel

HPLC.

Biosystems

experiments

spectra

COMMUNICATIONS

following

microbore

detector.

Sequencing

reduced

polybrene-treated

Applied

individual

sequence

RESEARCH

nitrocellulose or

diode-array

sequencing.

470A

from

from

absorption

for

Peptide

1OOOS

BIOPHYSICAL

narrowbore

system

a Model

with

fractions

by

a modular

AND

eluted

fractionated

Columns

Peak

Model

BIOCHEMICAL

43

e---

30

t

20

of intestinal electrophoresis.

bile acid binding protein as assessed by The following were subjected to weight protein standards (right hand lane); the ileal 200 cg of protein (left hand lane); and I-BABP

molecular fraction, chromatography

(see

1149

text),

20

pg

of

protein

(middle

lane).

the

Vol.

177,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.05

O.&l*

2 F: 5

0.03V8-II

e 3 E

V8-I 0.02

-

* *

*

3 $

O.Ol-

pL&dL&+

0.00

A 0

I

I 20

10

I 30

I 40

I 50

t

(mid

TIME

Narrowbore reversed-phase HPLC fractionation of V8 protease digest of W-F nl rote lulose-blotted I-8A8P. Solvent A was 0.1% trifluoroacetic acid in water, and solvent 8 was 0.03% trifluoroacetic acid in 70% acetonitrile (v/v). The entire volume (105 ~1) of condensed eluate from an on-membrane digest was applied to an Aquapore OD-300 column (2.1 x 100 mn; 7 p packing; Cl8), and fractionated using a 60 min linear gradient of O-100% solvent 8 delivered at a flow rate of Absorbance at 220 nm was monitored and peaks were collected by E;J.i ml/mln.

V8

protease

subsequent

HPLC

standard

elution

yielded

sequence

sequence.

tenth.

The of

of

two

(ISTICDVTYE)

C-terminal

Glu

V8 protease. revealed

high

protease

fragment

(residues

that

with

this

alignment in

a

the

protein

later

either

pig

for

or

Almost there

relative

dog

gastrotropin. is

mass

identification I-BABP

of became

sequences. of

two

deferred sequences

and

was

a 14

I-BABP

as

available, Peak

in

until

data

present

peak

obtained (see

with

all

the

substitutions

ileum

are

gastrotropin. data 2)

were gave

fraction.

compared a mixed Interpretation

subsequently

permitted

be I ow) .

1150

the

in

the

arisen in

from

the

Swiss-Prot V8

gastrotropin

(8)

Table

la,

a longer

at

the

intestine with

more

sequence

with

the

sequence

showed which

were

did

of

the

isolated

species it

not

pig

match

and

dog.

the information

about

gastrotropin

indicating of

in

peptide the

which

consistent As

give

Follow-up

for

of

in

a predicted

(see

V8-I

protein

the

having

between in

to

PM-Ile

those with

by

of

PM-Clu

canine

that

positions

cytosolic

rat

(Figure

the

merged

peaks

unambiguous

pmol

similar

two

an

of

peptide

for

more

and

failed

26

with

ilea

gave

of

gastrotropin.

reported

two

but

V8-I

4 pmol

homology

porcine

is

in

these

V8-II

peptides

kDa

and

of

even

only

presence

the

sequence

identity

sequence

ISTIGDVTYE

Gastrotropin Its

of

this

which

analyzed

level to

rat

nitrocellulose in

peak

the

with

of

2)

were

signal

consistent

a region

and

at in

111-120)

experiment).

conservative,

detected

levels

partial

peaks

ten

the

(Figure

analyses,

decline was

contains

result

other

Comparison

from

map

sequence

a gradual

database

comparison

Three

from

released

a peptide

successful

with

protein

material

yielded

information. the

cycle

action

electroblotted

fractionation

sequence

first

of

methods

Of

ten-residue the

digestion

this

resolution

the mixed of

presence

sequence the

two

was

Vol.

177,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

Table Alignment

of

I-8AEP

(a) peptides Pig

V8-I

GT

Dog GT

Pig

V8-III

major

GT

Dog CT

Pig

T-I

V8-III

GT

resolved

Sequence

from

CT

indicate

to

by

from

LVEISTIGDVTYER,

the

to

(Figure

*

1

*

assignment

it

them. round

(GT)

*

*

*

*

*

*

could

be

made)

*

*

*

I

*

I

I

1

*

*

+

of

1

1

*

1

1

*

bars

*

the

*

1

Only tryptic

sequence

*

*

recovered pieces

appeared The

pieces

of

incubation

that were

of

from

washed V8

derived recovered

a single

peptide

was

W-1 rat

trypsin

amounts to

remove

1151

the

and

experiment.

(peak

added

T-I)

la).

No

considerable

protein

provide

ilea

further (Table

trypsin, To

rat digest

sequence

retained of

ten the

sequence

gastrotropin

with

protease.

from from

with

this

appreciable

with

replacements.

products

peptide

incubated

sequence

conservative

peptide,

I-BABP

were

major

I-BABP and

This

that

V8-III

indicate

3).

concept

nitrocellulose

S color,

a further

Dog Gastrotropins

the V8-I sequence)

subtraction

trypsin

experiment.

peptides

the

attached

and

nitrocellulose-blotted

encompassed

tryptic As

by

vertical

with

HPLC

this

supporting

further

of

digestion

fractionated

sequence

identity;

sample

subjected

gained

VB-II

-Arg-Val-Ser-Lys-Lys-Leu-Ala * * * * -Arg-Val-Ser-Lys-Arg-Val-Ala * * * * -Arg-Val-Ser-Lys-Lys-Leu-Ala

A further

to

no

(d)

Asterisks

Ponceau

indicates

Dog GT

Dog CT

was

Pig

sequence

I-BABP

were

(Xaa

-Leu-AIa-Leu-Pro-Ser-Asp-Ala-Ile-Asp-Lys* * * * Leu-Gly-Leu-Pro-Gly-Asp-Val-Ile-Glu-Arg * * + * * -Leu-Gly-Leu-Pro-Ser-Asp-Ala-Ile-Glu-Arg-

Pig

of

T-I encompassed

(note:

sequence

miw

I-!3ABP

was

Sequences

-GIy-GIy-Lys-VaI-VaI-VaI-Asn-Ser-Pro-Asn-Tyr-His-His-Thr-AIa-GIu* * * 1 * 1 * * Gly-Gly-Lys-VaI-VaI-Ala-Asp-Phe-Pro-Asn-Tyr-Xaa-GIn-Thr-Ser-Glu * * * 1 * 1 * * + * -GIy-GIy-Lys-IIe-VaI-VaI-Asp-Phe-Pro-Asn-Tyr-Hit-GIn-Thr-Ser-GIu-

I -BABP

(c)

and

with

COMMUNICATIONS

1

-Leu-VaI-Clu-Val-Ser-Thr-VaI-Gly-Gly-VaI-Ser-Tyr-GIu-Arg* * * 1 * * 1 * Leu-Val-Glu-Iie-Ser-Thr-Ile-Gly-Asp-Val-Thr-Tyr-Glu-Arg * * * * * * * * -Leu-VaI-Glu-Ile-Ser-Thr-IIe-GIy-Asn-VaI-VaI-Tyr-GIu-Arg-

I-BABP

(b)

Peptides

RESEARCH

remained and

then

subjected

variation

from

the

Vol.

177.

No.

3, 1991

BIOCHEMICAL

AND

I

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

T-l

TIME (min) Narrowbore

HPLC fractionation of tryptic !3%&%uI0se-bI0tted I-w Solvent A was 0.115% trifluoroacetic water, and solvent 6 was O.lX’trifluoroacetic acid in 70% acetonitrile The entire volume (105 ~1) of condensed eluate from an on-membrane was applied to a Vydac Cl8 column (2.1 x 100 mn), and fractionated linear

gradient

Absorbance

first

of

at

attempt

see

legend

off the

was

0.06

did

not peak

-

with

4)

was

established, was

more

absorbing products

also

B delivered and peaks

at were

V8 protease,

used

liberate

a strongly

interfering

solvent

digestion

digestion

material

O-100%

nm was monitored

Figure

This

washing which

at to

membrane.

220

reversed-phase

were but recovered

in

an

a flow collected

a much

effort

to

Y

more

but

than

before,

that

dominated

fractionated PVP-40

was

from

a

a blank

T

at

4).

likely

ml/min.

process

the

(1’2;

the cost

of

chromatogram The

candidate

digest

a 60 min

from

the

(Figure

digest

using

strenuous peptides

peptides

tryptic

of 0.15 by hand.

elute

component

in

identity as

performed

the

of

this

large

with

T

0.05 ‘ii = R 5

rate

digest of acid in (v/v).

0.04

V8-Ill

0.03

2 i!? 2 0.02 2 0.01

!;r

0.00 3b

lb

w

do

t 3

5b

TIME (min) se

the digestion 40% acetonitrile Conditions except that of 0.1 ml/min.

Microbore treatment period,

reversed-phase HPLC fractionation previously exposed to trypsin peptides were eluted by treating in ammonium acetate buffer, pH 8.9, used in this chromatography were identical a 1.0 x 100 w Aquapore 00-300 Cl8 column

1152

of

material Figure 3). the nitrocellulose at 5OoC for 3 to those used was used with

(see

gained Following

from with

h. in Figure 2, a flow rate

Vol.

177,

No.

3, 1991

nitrocellulose of

the

from

cycle closely

yielded

which

gastrotropins

sequence

with

to

the at

This

tryptic

a predicted

conservative.

blot.

peaks

sequence

could

be made).

COMMUNICATIONS

Despite

were

loss

recovered

This

of

of pig

this

represents

I-BABP

and

part

sequenced.

(X

fragment

fragments

of

and

GGKWADFPNYXQTSE.

protease

sequence

the

level

with

peptide

lc) .

only

In

5M,

There

80%

Peak major

the

sequence

a

was

dog

of

identity

level

the

five

was

and

the

in

identity

amino

resolved

could

ending

of

minor

also

LGLPGDVIER,

Arg

the

with

a distinct

sequence)

by

case, three

V8-III,

the

(preceded this

but

was

from

of

peptide,

tryptic

was

the

resolved

V8

major

50%

n (Tab I e

gastrotropin

of

RESEARCH

lb).

to

(present

gastrotropi

BIOPHYSICAL

major

predicted

(Table addition

data.

of

the

no assignment

homologous

In

part

a number

V8-III

for

AND

a protein-free

chromatogram, Peak

the

BIOCHEMICAL

Lys)

with

acid

from

be

aligned

of

pig

pig

substitutions

homologous

region

were

of

dog

gastrotropin. As peptides

major

To

identity

be and

pig

substitutions approximate is This

M,. of

14

that

to

(6) bind

polypeptide

kDa

ileal is

represents and .

While

organic has

it anions

an

been

is

Glu, this

pig Id).

I-BABP

high

not

this in

Indeed,

have

levels

been

of

present, As

rat

gastrotropin,

of with

noted it

(14),

important

end

of

rat

very

As

not

(Table of

the

the

I-BABP

shares

collected

gastrotropin.

(7,13,14)

has

protein

conservative. with

from

I-BABP.

residues

a convergence

function

did

region

identity

origin

rat

of

dog

resolve

RVSKRVA.

but

exhibited

exception

to

two

information

I-BABP

47

Where

and

I-BABP

the to

the

possible

C-terminal

sequences

without

structure

transport

capacity

virtually

strong

C-terminus

of

gastrotropins.

demonstration

gastrotropin acid

dog

the

amounting

of

sequence of

the

one

V8-III

heptapeptide

fragment

their

the

digest

with

peptides

exception,

are

the

was

became

out

represented

a related

and

it

sequence,

left

it

V8-III

subtracting

successfully

four

Without to

evidence

that

with

fraction

a V8 protease

aligned

summarize,

sequenced.

V8-II by

from

probable could

gastrotropin

mixed

from

procedure

obtained

appeared

peptide

the

This

was

gained

information

mixture.

peptide

this

in

sequence

V8-II

its

sequence

present

further

it

the

earlier

has

function

molecular functional

not in

the

that been

studies

of

studies

of

gastrotropin

apparent

has

until

enterohepatic

bile

bile

now

the that

acid

circulation.

REFERENCES

:: 3. 4. 5. 6.

Wilson, F. A. (1981) Am. J. Physiol. 241, G83-G92. Burckhardt, G., Kramer, W., Kurz, G., and Wilson, F A. J. Biol. Chem. 258, 3618-3622. G., Wilson, F. A., and Kurz G. Kramer, W., Burckhardt, J. Biol. Chem. 258, 3623-3627. Lin, M. C., Weinberg! S. L., Kramer, W., Burckhardt G., (1988 J. Membrane BIoI. 106, l-11. Lin, 1 C., Kramer, W., and Wi Ison, F. A. (1990) J. Biol.

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COMMUNICATIONS

D.

P.,

and

EndocrinoiAgy 123: 25781$58:. ’ ” Von Dippe, P., Ananthanarayanan, M., Drain, P., and Levy, D. (1986) Biochim. Biophys. Acta 862, 352-360. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. H. (1987) Proc. Natl. Acad. Sci. USA 84, 6970-6974. Ceoghegan, K. F. (1990) Technique 2, 43-49. Parekh, B. S., Mehta, H. B., West, M. D., and Montelaro, R. C. (1985) Anal. Biochem. 148, 87-92. Wider, M. D., Snow, J. W., Dass, C., and Desiderio, D. M. (1988) Walz, D., J. Biol. Chem. 263, 14189-14195. Nothwehr, S. F., Lucey, M., Sacchetini, J. C., DelValle, J., Gantz, I., Banaszak, L. J., Naud, M., Gordon, J. I., and Yamada, T. (1990) J. Biol. Chem. 264, 20248-20254.

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