A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on kupffer cells

Vol. 188, No. 2, 1992 October 30, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 671-677

A PEPTIDE SEQUENCEON CARCINOEMBRYONIC ANTIGEN BINDS TO A 80kD PROTEIN ON KUPFFERCELLS Peter

Anthony

Thomas,

Laboratory

T. Petrick, Carol A. Toth, and Glenn Steele Jr.

of Cancer Biology, Department Hospital, HarvardMedical Molecular

Received

September

2,

Diagnostics

Eben S. Fox,

James J.

of Surgery, The New England School, BostonMA

Inc.,

West Haven,

CT

Elting

Deaconess

06516

1992

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA This peptide sequence is located in the region between the N-terminal sequence. and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases. Q 1992 Academic Press, Inc.

Carcinoembryonic is

used

(1).

to monitor

CEA is

repeating

from but

28 potential of

chain

sequence

C-terminus

for

that

molecules

emerged.

Rodent

cell

members

showed

that

intercellular acts

the

adhesion

lines

molecules

as an intercellular has

been

adhesion

adhesion

suggested

during

that

embryonic

comprehensive

discussion

in a review

by Thomas

CEA

cross

reacting

of

pregnancy

with

(7,8,9).

Rojas

molecule,

et

however,

function

development al

function

and

as in

functions

al

(NCA),

(10) binding

a mediator

the

(PSGs).

family

(6).

molecules

for as

at are

of a family

antigen

cDNAs specific can

tail

glycoproteins

as adhesion

The

there

a member

these NCA

(2,3).

hydrophobic

supergene

proteins

cancers and three

Furthermore

immunoglobulin

and

CEA can

specific

solid

C2 type

a short

non specific

inblood

domain

the

(4,5). CEA is

of the possible et

predicts

of

(2).

transfected

both

domains

glycosylation

larger role

and other

an N-terminal

to secretion

and the

to the

a functional

loop

prior

N-linked (BGPs)

with

gene also

lost

measurement

carcinoma

acids

disulfide

cloned is

belong

Recently

It

like the

colorectal

668 amino

includes

glycoproteins

These

with of

this

sites

molecules

biliary

(CEA) is a glycoproteinwhose

patients

single

immunoglobulin

deduced the

antigen

has

CEA gene family Ca++

showed

independent that

BGP also

was Ca++dependent. of

tumorigenesis of the CEA family

intercellular A more (7). canbe

found

(11). 0006-291X/92 671

$4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

188,

No.

2,

1992

BIOCHEMICAL

Our interest

is in determining

circulation occurs

and we have via

receptor

the

junction

by

In synthetic Kupffer

not

the

present

the

minimum

peptides cell

and

peptide

of clearance

experimental

animals

by

to Kupffer study

from first

binds

loop

covering cells

to confirm

the

cell

digest

in

this

(12,13).

amino

and binding

sequences

that

the A

of CEA and covering

from

cells

of CEA from

and humans

Kupffer

domains

COMMUNICATIONS

other

acid is

106

to

inhibited

regions

of

the

(14). attempted

required

as an 80kD surface

the

a pepsin

to Kupffer

we have

sequences

RESEARCH

the mechanism

kD) isolated

Glycopeptides

bind

BIOPHYSICAL

endocytosis

N-terminal This

CEA (17). did

identify

the

150.

intact

molecule

(5.5

of

in both

mediated

deglycosylatedpeptide approximately

shown

AND

for

identity

to binding.

map

this

binding

Furthermore

of the CEA binding

protein

site

and

we have

used

on the

rat

protein.

MATERIALS

and METHODS

Glvcooroteins. CEA was purified from a single colorectal carcinoma hepatic metastasis, as The preparation was characterized by sodium dodecyl previously described (13). sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography analysis, and activity in commercial CEA assay systems. Normal Cross Reacting Antigen (NCA) was purified from colorectal cancer hepatic metastases as described previously (13), and subjected to the same criteria of purity. The MW of the purified NCA was approximately 55,000 by SDSPAGE and HPLC. Protein

Modification. CXA (100 ug) was radiolabeled with 1 mCi Na lz51, (17 Ci/mg) (New England Nuclear, Boston, MA), using the chloramine T procedure (15). The labeled CEAhad a specific radioactivity of -6 mCi/mg. CEA (lmg) was also conjugated to fluorescein isothiocyanate (20 ug) overnight at 4°C at neutral pH. The conjugate was separated from unreacted fluorescein by chromatography on Sephadex G-25. CEA was conjugated to (sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl1,3'dithiopropionate (SASD) (Pierce, Rockford, IL U.S.A.) using a modification of the manufacturers instructions. SASD (1.5pg) was radioiodinated with Na'=I (1mCi) by the Chloramine T procedure. After stopping the iodination reaction CEA (1OOpg) was added and the pH adjusted to 8.4 with a O.lM borate buffer. The reaction was carried out for 30 min. at ambient temperature. The conjugate was purified by chromatography on Sephadex G-25. All procedures were carried out in the dark. Both CEAandNCAwere conjugatedwithbiotinusing 3(N-maleimidopropionyl)biocytin. Previous studies had shown that intact disulfide bridges were not needed for CEA binding to Kupffer cells (13). Svnthetic

Peotides. Peptides were synthesizedonanAppliedBiosystems 430Apeptide synthesizer usingt-BOG chemistry and cleaved from the resin using anhydrous HF. Purification was performed by reverse phase HPLC using two solvent systems. Solvent system 1 was A:50 mM ammonium acetate in water and B:50 mM ammonium acetate in 75% acetonitrile/water. Solvent system 2 was A:O.l% TFA in water and B:O.l% TFA in 90% acetonitrile/water. A linear gradient of 1% B/minute was used for both solvent systems. All peptides were analyzed for amino acid composition. Isolation gm.)

of Rat Kuoffer Cells. Kupffer cells were isolated collagenase perfusion by

from of

fasting male Sprague the liver followed 672

Dawley rats (-300 by differential

Vol.

188, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

centrifugation and purification on a metrizamide gradient, as described previously (13). Further purification of the cells was achieved by allowing them to attach overnight to plastic tissue culture dishes. The cells were >95% viable by trypan blue exclusion. The preparation was greater than 85% Kupffer cells based on staining for endogenous peroxidase and their ability to phagocytose 1.1~ latex beads. LiPand

Binding Assays. '?I-Peptides (1Opg) or lZ51-CEA were incubated up to 45 min. with rat Kupffer cells in triplicate using a modification of the method of Stahl et al (16). Free ligandwas separated from cell bound ligand by centrifuging at 11,000 rpmthrough an oil phase consisting of dibutylphthalate:dioctylphthalate 3:l (17) as described previously (13). Affinitv

chromatograohv of Kuoffer cell surface oroteins. Isolated rat Kupffer cells were surface labeled with '%I using the lactoperoxidase procedure. The labeled cells were dissolved in 0.5% TritonXlOO in O.lM Tris buffered saline pH 7.4 (TBS) containing 5mM PMSF and lpg/ml leupeptin, and centrifuged in an Eppendorf microfuge for 10 mins to remove insoluble material. The supernatant containing the labelled cell surface proteins was chromatographed on a column of immobilized CEA (2 mg CEA/ml of wet CNBr activated Sepharose) in 0.1% Triton X-100, TBS, with 10 mM CaC12 at 4OC. The CFA/Sepharose was washed well with the above buffer. Bound material was eluted from the CEA/Sepharose by 0.1% Triton X100, in TBS containing 10 mM EDTA. The eluted radioactive fractions were pooled concentrated and examined on 10% SDS-PAGE. Three major proteins were present (18). Because it was likely that at least one of these proteins was a galactose recognizing lectin the eluted radiolabeled proteins were rechromatographed on a asialo fetuin Sepharose 4B column. The eluted proteins were dialyzed against the 0.1% Triton X100, TBS, 1OmM CaCl, buffer overnight at 4°C and subjected to chromatography on immobilized asialofetuin Sepharose. The unbound protein fraction was examined by SDS-PAGE as above. The SDS gels were examined by coomassie blue staining and exposure to Kodak Xomat X-ray film at -7O'C.

Crosslinking of CEA and Peotides to Isolated Kuoffer Cells. Kupffer cells (1 X 107) in O.lM phosphate buffered saline (PBS) pH 7.4 were reacted with 5pg of labelled conjugate (approximately 2pCi) for 30 min. at 37"C, and crosslinked with short wavelength UV light for 10 min. The cells were washed 3X in PBS and extracted and reduced with 0.5% SDS, 5% 2-mercaptoethanol in O.lM tris buffered saline pH 7.4 (TBS) with 1mM PMSF and lpg/ml leupeptin. The extracts were dialyzed overnight against 0.5% SDS in TBS and concentrated using a Centriconmicroconcentrator. The samples were examined by SDS-PAGE on 10% gels and crosslinked proteins were visualized by autoradiography. Lieand

BlottinP Assavs. Isolated rat Kupffer cells were extracted with O.lM TBS pH 7.4 containing 1% Triton X-100 and inhibitors of proteolysis (1mM PMSF, lpg/ml leupeptin and Extracts were separated on 10% SDS-PAGE with aprotinin). biotinylated MW standards and the proteins transferred to nitrocellulose. The membrane was blocked with 10% BSA for 1 hour at 37°C and washed in 20mM TBS with 1OmM Ca++ and with the biotinylated CEA or NCA (2pg/ml) for Mg++. The membrane was incubated 1 hour at 37'C and washed 3 times (10 min.) in the buffer at 25V. The membrane was incubated with avidin/alkaline phosphatase in TBS for 10 min. and developed 5-bromo-4-chloro-3-indolyl phosphate and tetranitro blue with a solution of tetrazolium. RESULTS and DISCUSSION To identify four

pentadeca-peptides

the

site with

on CEA recognized 5 amino

acid 673

by the Kupffer overlaps

based

cell on the

we synthesized sequence

of the

Vol.

BIOCHEMICAL

188, No. 2, 1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

105

115 125 135 110 120 130 140 I I 1 I 1 1 I 1 RVYPELPKPSISSNNSKPVEDAVAFTCEPETQDATYLW

100 1 5.5kD+c-75

-

C-76'

-

c-77'

-

C-78

TGQFRVYPELPKPSI PKPSISSNNSKPVED KPVEDKDAVAFTCEP FTCEPETQDATYLWW

-

C-85

YPELPK

-

Fig.1.

Structure of the 5.5kD pepsin peptide and the 5 synthetic peptides. from Thomas and Toth (14). * Peptides C-76 and C-77 were synthesized with an extra tyrosine residue at the C-terminus to allow them to be radioiodinated. All peptides were labeled with "'1 Data

l

using the radioactivity

previously

identified

and a hexameric Kupffer C-75

acid

cells. and

were

cells

FITC

101-115

acids

not

CEA related

also

showed

(PDLPK,

precursor

(PELPK)

substitutions

site

and could

with

only

the for

be inhibited

the the

by

one of

isolated

rat

by unlabeled (Fig.

2B).

isolated

rat

These Kupffer

binding

site

to

amino

expected

homology

with

sequence

(RVYPELPKPSI)

human

(19)

peptides

only

proteolytic

with

human

enzyme,

complement

1 (PDLPR)

(19).

Cls These

changes. PELPK in complement

YPELPK (C-85)

to determine

was rapidly

endocytosed

peptide the

of

the

of the sequence

by both

acid

that

C-76

collagenase

conservative

This

15 amino shows

has the

with

a specific

be inhibited

however

123-127)

hexapeptide

CEA.

could

sequence

human

to

and endocytosed

localizes

homology

of the appearance

we synthesized

the binding

This

acids

and

represent

Because Cls,

amino

(20)

2A

immunofluorescence This

of

labeled

The four Fig.

C-75

NCA, and BPGs,

degree

was

pentadeca-peptide

peptides.

CEA sequence.

a high

prostromelysin

lz51 labeled using

glycoproteins

(14). 1.

was bound

overlapping

confirmed

in the

the

of

(1OOpg)

in Fig.

(C-75)

by the

labelled

peptide

peptide

shown

peptides

further

with

binding

are

The binding but

CF,A

results

5.5kD

peptide

15 amino

these

Each 4mCi/mg.

Chloramine T procedure. of approximately

larger

peptide

C-75

if

subcomponent this

was also

by Kupffer

cells

and by CEA itself

(Fig.

2C). Because development

of

interested cells. which

mixture molecular

hepatic

Previous of

on asialo weight

three

using major

Ca++ and,

of CEAbinding

from

the binding

studies

require

implications

metastases

in identifying

the presence of

of the possible

colorectal

site

affinity CEA binding

for

fetuin

Sepharose proteins.

tumors peptides

chromatography

with

proteins removed This 674

both resulted

an

we were

CEA-Sepharose cell

(18). Chromatography the higher (170kD) in

in the

on isolatedKupffer

on the Kupffer

binding

cells

(21,22,23)

these

or Mg++ for

(35kD)

to Kupffer

almost

showed

surface

all

of this and lower homogeneous

Vol.

188,

No.

2,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

15

COMMUNICATIONS

45

TIME (minutes)

B

16

6

CCdd

Control

C-76

Fip;.

CEA

c-75

2.

A. Uptake of the 15 amino acid 's51-Peptides

-l

, C-75,

-v

C-76,

- v

C-77,

(1Opg) by isolated

- 0

rat Kupffer

cells.

C-78.

B. Inhibition of Kupffer cell uptake of peptide C-75. "sI C-J5 uptake was measured in the presence of an equimolar concentration of CEA (100 fold excess by weight) and a 100 fold molar excess of peptides C-75 or C-76. C. Inhibition of Kupffer cell uptake of peptide C-85. '=I C-85 uptake was measured in the presence of an equimolar concentration of CEA and a 100 fold molar excess of unlabeled peptide C-75. All determinations were carried out in triplicate, the error bars represent one standard deviation.

radiolabelled

protein

responsible

for

reducible

of

(SASD) The crosslinker

peptide

produced

as well

as the

80kD

specific

crosslinker

dithiopropionate reduction.

the

by pepsin unreactive

(Fig.

3).

binding

To confirm

of these

peptides

that

sulfosuccinimidyl

2-(p-azidosalicylamido)

which

labels

specifically

was labeled digestion peptide

with

C-76.

In

Fig.

80kD protein

was

ethyl-1,3'-

the binding

'*sI and coupled

of CEA (17)

the

we used a photoactivatable

and to the

protein

following

to CEA, to the reactive

4A an autoradiogram

5.5kD

C-75 peptide of

a 10%

Vol.

188, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

200 116 97 67

kD kD kD kD

4 + + +

45 kD +

Fig. 3. Electrophoresis of the BOkD protein isolated from rat Kupffer cells by chromatography on CKA Sepharose and asialo fetuin Sepharose. Kupffer cells were surface labeled with "'1 and bound to a CBA-Sepharose column. The eluted proteins were rechromatographed on a asialofetuin column and in the text. the unbound fraction examined by SDS-PAGE on 10% gels as described Fig. 4. Cross-linking of Kupffer Cells with CKA and Peptides crosslinked to isolated rat Kupffer cells and Conjugates with 'e51-SASDwere the

labelled

and run on 10% SDS-PAGE gels as described in the an SDS-PAGE of the extracted proteins after Only a single protein at 80kD was crosslinked. crosslinking B. Cells were crosslinked with "'1-SASD-5.5kD peptide complex. The left hand track of the 10% SDS-PAGE in the figure shows that this complex crosslinks a 80kD protein and some minor lower MW components. The right hand track is an identical experiment performed in the presence of a 100 fold excess by weight (2.5 molar Cells were excess) of CPA. c. crosslinked with '=I-SASD-C-75 or C-76. Crosslinkingwith C-75 is on the left track while C-76 is on the right. Only C-75 crosslinked the 80kD protein while no crosslinking of any protein was seen with C-76.

methods.

proteins extracted The figure shows 'e51-SASD-CKA. with

A.

Fig. 5. Binding of Biotinylated CBA and NCA to Extracts of Rat Kupffer Cells Kupffer cell proteins were extracted with detergent (1% Triton X-100) separated by 10% SDS-PAGE and biotinylated CPA or

SDS-PAGE shows the cells. presence of

a 2.5

same

crosslinking

of

from

Kupffer

three

peptide of

CEA binding that

sequences

similar

with

detected.

not

shown).

the

proteins to

major

fold is

by of

Cat+

shown

in

previously

CEA residues

by affinity andcomplement

108-112 676

raises

the

of

protein

4C

results

SDS-PAGE,

of

Further cells when

rat

transferred

both

probes

and Mg++ this

subcomponent

to only

a

binding

107-112 protein

chromatography interesting

in the

on Kupffer

the

the

absence

Fig.

amino acid residues a 80kD Kupffer cell surface

collagenase

Kupffer

Crosslinking

and

implicates

CEA by

rat

by weight).

CEA or NCA. With

absence

with

of crosslinking

5 shows

separated

overlayed

crosslinking

complex

CEA binding Figure

the

was

and isolated

absence

complex

biotinylated

found

stromelysin,

CEA (100

were In

data of

blot protein.

4B shows

C-75-SASD

experiments.

is

and endocytosis

of

C-76-SASD

extracts

our

and the

peptide

identity

conclusion

observation

excess

blotting

80kD (data

binding

the

Figure

peptide

the

and probed of

In

molar

the

crosslinked.

pepsin

by

detergent

nitrocellulose abolished

fold

ligand

cell

protein

5.5kD

The a 80kD

the CEA-SASD complex

80kD is

protein with

confirmation comes

of

by the

of

the

to nitrocellulose. which bind to

of

reactionbetween

One protein

same protein

transferred NCA both

is

in the one of (18).

The

Cls contain possibilities

Vol.

188, No. 2, 1992

regarding

their

peptides

that

determine,

BIOCHEMICAL

AND BIOPHYSICAL

with

cells.

interaction bind

if,

to this

like

Kupffer

80kD protein

We will

as inhibitors

CEA, theywillenhance

RESEARCH COMMUNICATIONS now be

of CEA uptake

metastases

able

to

use

in vivo,

fromcolorectalcancer

to cell

lines. ACKNOWLEDGMENTS This from

the

Diabetes

investigation National

was

Cancer

and Digestive

supported

Institute,

and Kidney

by

grants

and DK44305 Diseases,

United

numbers from

the

States

CA44585 National Public

and CA44704 Institute

Health

of

Service.

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23.

Zamcheck, N., Steele, G.D., Thomas, P. and Mayer, R.J. (1986) In: The Manual of Clinical Immunology, (Rose, Freidman and Fahey, eds.) Am. Sot. Microbial. pp. 802-809. Oikawa, S., Nakazato, H. and Kosaki, G. (1987) Biochem. Biophys. Res. Commun. 142, 511-518. Kamarck, M., Elting, J., Hart, J., Gobel, S., Rae, P.M.M., Nortdurft, M.A., Nedwin, J. and Barnett, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5350-5354. Hefta, S.A., Hefta, L.J.F., Lee, T., Paxton, R. and Shively, J.E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4648-4652. Sack, T.L., Gum, J.R., Low, M.G. and Kim, Y.S. (1988) J. Clin. Invest. 82, 586-593. Williams, A.F. and Barclay, A.N. (1988) Annu. Rev. Immunol. 6, 381-405. Benchimol, S., Fuks, A., Jothy, S., Beauchemin N., Shirota, K. and Stanners, C.P. (1989) Cell 57, 327-334. Oikawa, S., Inuzuka, C., Kuroki, M., Matsuoka, Y., Kosaki, G. and Nakazato, H. (1989) Biochem. Biophys. Res. Commun. 164, 39-45. C.P. (1990) Cell GrowthDiffer. 1, 209-215. Zhou, H., Fuks, A. and Stanners, C.P. (1990) Cell growth Differ. 1, 527Rojas, M., Fuks, A. and Stanners, 533. Thomas, P., Toth, C.A., Saini, K.S., Jessup, J.M. and Steele, G. (1990) Biochim Biophys Acta 1032, 177-189. Toth, C.A., Thomas, P., Broitman, S.A. and Zamcheck, N. (1982) Biochem. J. 204, 377-381. S.A. and Zamcheck, N. (1985) Cancer Res. Toth, C.A., Thomas, P., Broitman, 45, 392-397. Biophys. Res. Commun. (1990) 170, 391Thomas, P. and Toth, C.A. Biochem. 396. Greenwood, F.C., Hunter, W.M. andGlover, J.S. (1963) Biochem. J. 89: 114123. Stahl, P., Rodman, J., Miller, M. and Schlesinger P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1399-1403. Maynard, Y. and Baenziger, J. (1981) J. Biol. Chem. 15, 8063-8068. G. (1991) Carcinoembryonic Thomas, P., Toth, C.A., Fox, E.S. and Steele, antigen binding proteins from Kupffer cells. In: Cells of the Hepatic Rijswijk, The Netherlands, Sinusoid Vol 3, The Kupffer Cell Foundation, 512-514. Wilhelm. S.M., Collier, I.E., Kronberger, A., Eisen, A.Z., Marmer, B.L. E.A. and Goldberg, G.I. (1987) Proc. Natl. Acad. Sci. Grant, G.A., Bauer, U.S.A. 84, 6725-6729. Tosi, M., Duponchel, C., Meo, T. and Julier, C. (1987) Biochemistry 26; 8516-8524. Jessup,J.M. and Thomas, P.(1989)Cancer and Metastasis Reviews. 8,263-280. Hostetter, R.B., Augustus, L.B., Mankarious, R., Chi, K., Fan, D., Toth, C.A.,Thomas,P.andJessup, J.M. (1990) J. Natl. Cancer Inst. 82, 380-385. Wagner, H.E., Thomas, P., Wolf, B.C., Zamcheck, N., Jessup, J.M. and Steele, G.D. (1990) Invasion and Metastasis 10, 253-266. 677