Amino terminal fragments of human progastrin from gastrinoma

Vol. 123, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 30, 1984

Pages 404-409

AMINO TERMINAL

FRAGMENTS OF HUMAN PROGASTRIN

FROM GASTRINOMA

Joseph R. Reeve, Jr.*, John H. Walsh*, K. Tompkins**, David Hawke*** and John E. Shively***

Ronald

*Center for Ulcer Research and Education, VA Wadsworth Center, Los Angeles, California **Dept. of Surgery, UCLA Medical School, Los Angeles, ***Division of Immunology, City of Hope, Duarte, Received

July

27,

90073 California California

90024

1984

SUMMARY: Two peptides which copurified from a human gastrinoma were found to correspond to the amino acid sequence deduced for the amino terminal portion of human and procine progastrin. The sequence of peptide A is Ser-Trp-Lys-Pro-Arg-Ser-Gln-Gln-Pro-AspAla-Pro-Leu-Gly-Thr-Gly-Ala-Asn-Arg-Asp-Leu-Glu-Leu which is identical to an amino terminal portion of human progastrin. The sequence of peptide.B is identical to that of peptide A except it is missing the first five amino acids. If peptide A corresponds to the amino terminus of progastrin, the signal peptidase cleaves at an Ala-Ser bond.

Gastrin isolated

molecules

from

biologically all

copy

of

cloned More

forms

apparently

the

porcine

nucleotide

recently,

porcine

comprised

for

a

gene was obtained

sequence

for

34 amino

from acid

from

the

of gastrin-34

the

signal

peptide

of

gastrin-like

forms

Component of 0006-291X/84 Copyright All rights

and other

gastrin-34

attached

to

Inc. reserved.

was

one or

been

(2).

to

the

The amino near

amino

were presumed

the

carboxyl coded

to consist

peptides.

presumably connecting

acid

acids

previously

more

amino

has

corresponding

was located

gastrin"(51,

include

A cDNA

determined

(3).

been

14

preprogastrin

connecting

#4

and

antral

58 additional

$1.50 0 1984 by Academic Press, of reproduction in any form

17,

same precursor.

immunoreactivity,

I (4) and as "big-big

These

of the

gastrin

and the

have

(1).

a human tumor

of preprogastrin, terminus

34,

DNA sequence

end

amino

of

sequence

single

lengths

tumors

fragments

mRNA coding and

chain

human gastrin-secreting active

acids,

varying

of

The

of

larger

known consist peptides.

as

Vol. 123, No. 1, 1984

BIOCHEMICAL

Although

of

the

presence

has been deduced

from

peptide

have not

cleavage

tissue

0.5g

amino

porcine

terminal

connecting

and human genes,

been demonstrated

of a gastrinoma

a peripancreatic

lymph syndrome

extracts

of

applied

directly

this

tissue

and

at

280 nm for

and

tyrosine-containing

predominant

peptides

the

directly

sites

of

in mammalian

HPLC

homogenous.

The

carboxyl this

immunoreactive

major

absorbance

of

microsequence

except

this

peak

showed tumor

the

detected

derivatives for

cycles

a patient

high

operation with

eluted

purified (7). in

performance

of the

eluate

peptide

and

the

at of

that

extract

liquid 220

nm

tryptophan

revealed

minor

immunoreactivity

on gradient

analysis were

different

several

gastrin

aliquot

derivatives

with

peak

at

respectively,

along

gastrin

from

Cl8

of total

original

terminal

An

removed

peptides,

of

was obtained

(6) and was quickly frozen. Aqueous 1 were prepared and a portion was

detection

peak

Isocratic

node

to a reverse phase 2 column. Monitoring

chromatography

cycles

the

fragment

Zollinger-Ellison

Two

these

extracts. An

from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

it

peaks

(Fig.1).

was

apparently

contained per

considerably

one

40

mg

pm01

tissue,

later

but

than

the

was subjected

to

HPLC. peak

fraction

Identifiable 23 cycles

were obtained 1 and 4 (Table

amino of the

Edman

in each of 1).

acid

the

Arranging

PTH

reaction. first the

20 amino

lThe 0.5 g frozen tissue specimen was minced with a scalpel and quickly immersed in 5 ml boiling 0.1 M ammonium bicarbonate centrifuged, the and boiled for 5 min. The extract was cooled, supernatant saved, and the pellet reextracted in 5 ml 2% aqueous centrifugation, the two trifluoroacetic acid at 4 for 6 h. After supernatants were combined. 2Aliquots of 1 ml of combined supernatant were applied to Vydac Cl8 reverse phase HPLC column equilibrated with trifluoracetic acid (Solvant A). The column was rinsed with same solution for 10 min., then eluted with linear gradient acetonitrile containing 0 t0 100% buffer B (50% trifluroacetic acid).

the 0.1% the from 0.1%

BIOCHEMICAL

Vol. 123, No. 1, 1984

l-4

-

1.2

-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,’

,’

- 80

I’ I’

- 70

,I’ 1.0

-

I'

0.8 -

- 60

/' 1

50

-

0

i m

- 4o

- 30 ap 20

‘la,;

10 I’

co

0.3 t

I

20

40

60

Effluent

80

100

(ml)

Figure 1: Elution profile of an aqueous extract of Deripancreatic lvmvh node tumor removed from a vatient with thz iollinger-Ellisonkyndrome. One ml (corresponiing to O.lg of tumor) of extract was loaded directly onto a Vydac Cl8 reverse phase high pressure liquid chromatography column. The eluate was monitored at 220 nm and 280 nm.

acids

according

abundant

residue

to yield

at each

step,

a member

of peptide

A and the

peptide

B, resulted

in two peptides

with

the

peptide

B contained

five

acids

at

In

order

necessary of

the

addition

to

to change

predicted determined

the

amino

alignment

the

assignment

Edman degradation

(cycles

to serine,

significance sequences

achieve

less

of for

this

gene

shown in based 6,

A

and

B

product.

a combination

8,

is

of

9,

not

correspond

406

results

two

the

more

a member

same sequence its

amino Table at

12

and

known.

except

it

was

five

steps

18).

In

one. The

exactly peptide

of

terminus. 1,

in cycle

The the

other

on yield

was identified

observation

peptides

human from

alanine

calling

and

The entire to

the

sequences

shown in Table

1 and

BIOCHEMICAL

Vol. 123, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Microsequence Analysis

of HumanTumor Progastrinl

Peptide A (Primarvm2 pm01 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Ser Trp LYS Pro Arg Ser Gln Gln Pro5 Asp Ala Pro Leu GUY Thr Gly Ala Asn Arg Asp Leu Glu Leu

Peptide B i5Y.esuem2 pm01

-

(425) (394) (725) (3121 (149) (100) (203) (141) (116) ( 81) (235) ( 72) (196) ( 96) ( 50) ( 87) (145) ( 39) ( 44) ( 28) ( 90) ( 36) ( 60)

(Ser) 3 Gln Gln (Pro14 Asp Ala Pro Leu GUY Thr Glv Ala Asn Arg Asp Leu Glu Leu Pro (Trp)

(249) (253) (108) (270) (107) (157) (127) ( 80) (111) (255) ( 77) ( 69) ( 40) ( 70) ( 651 ( 40) ( 31) ( 25)

1

Approximately 5 micrograms (2 nmolsl of sample were applied to a spinning cup microsequencer. The yield of peptide A was 36% and peptide B 11%. It should be noted that Ser gives rise to multiple PTH amino acid derivatives in Edman chemistry; quantitive yields for only one of the derivatives is given here. 1

The secondary sequence is for the most part a subset (from 6 on) of the primary sequence. Yields in pmols are shown in parentheses. Assignments to primary or secondary sequence are based on relative yields assuming a relative ratio of 2:l and by analogies between the two sequences. 3 225 pmol of PTH-Ala was also observed, but Ser has been assigned due to the identity seen with the rest of peptide A. The sum of yield of PTH-Ser for peptide A and B is shown. 4 Only the sum of the yield of PTH-Pro for peptide A and B is shown. 5 PTH-Gln was also observed, but is probably a carry-over from the previous two cycles.

maximal

homology

between

are shown in Figure The present calculated

amino in

and with

the

human gastrin

gene

2.

terminal

extremely from

chains

the

fragments high

of

concentration

absorbance 407

human in

at 280 nm and

progastrin this the

tumor. amino

were We acid

BIOCHEMICAL

Vol. 123, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 10 15 SWKPRSQQPDAPLGTGANRDLELPWL SWEPRSQQPDAPLGTGANRDLEL SQQPDAPLGTGANRDLELPW SWKPgFQLQDASSGEGANRQgEEBEL

Human gene Peptide A Peptide B Porcine gene

20

25

Figure 2: Sequence of the peptides isolated from the gastrinoma tumor with similar regions porcine compared of human and progastrin as determined cDNA techniques (2,3). The underlined residues of the porcine gene indicates the differences between hog and human progastrin.

analysis

that

progastrin

each

fragment,

immunoreactive

concentration

antrum

(8). presence

producing

peptide

whether progastrin identification

require

in the

the

is

a of

undemonstrated gastrin

from

progastrin.

pressure

It

leader

Analysis

of

liquid

sequence

of

this

a single

peptide

on a different gene

normal

to

gastric

gastrinfraction of

determine

fragments but

of

specific

probably

indicates

cleavage

human preprogastrin

by a

followed

a

will

previously

characterized

possible

points are

single

human

a gastrin

step

analysis

and secreting

messenger

gene of

the

of

tumor RNA.

are Recent

high

analysis

product. cDNA

at

separated

by microsequence

about

408

times

antibodies.

extract

and a single

needed

tissues

recently

gastrin

The

identification

terminal

peptides

the

chromatography

are

amino

in

these

tumor

the

than

S-10

a minor

phenomenon,

indicates

results

with

studies

also

a

with

favored

specific

of

new information

performed

compared

of

with

has produced of

of

antrum

of

gene. the

population

fragments

product

which

human

pure

pm01

tumor.

of normal

tumor-specific

Characterization

same

in extracts

storage

radioimmunoassay

the

higher

at

Further

these

times

were present

in gastric

not

1000

components

gastrinoma

fragment.

or

25 in

relatively

a

contained

concentration

than

cells

tissue

components

of

cells

gastrin

this

of

immunoreactive

higher

The

a

gastrin

gastrin-like

of

milligram

The analysis

compatable studies

of

Vol. 123, No. 1, 1984

cDNA and

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sequences products

importance

and peptide and of the

of direct

interpretation

the

independent

biological

similar

portion

peptides which

data

that

the

of naturally

terminal

cleavage

calcitonin

precursor

cannot

be obtained

in normal

on preprogastrin through

have shown

the

peptides

in

(g-13). of gastrin

in

signal

have for

This

tissue. for

could

recently

(14).

is processed

occuring

site

portion

precursors

occurring

as was found

activity

events

enkephalin

determinations

how preprogastrin

may reflect

indicate

amino

of the

gene products

structure

of the

shows

calcitonin

analysis

of gene

Furthermore,

products

pair

tumor

a of

tissue

The results peptidase,

cDNA studies.

ERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Gregory, R.A., (1979) Bioorganic Chemistry 8, 497. Yoo, O.J., Powell, C.T., Agarwal, K.L., (1982) Proc. Natl. Acad. Sci. USA 79, 1049. Rehfeld, J.F., (1983) Proc. Natl. Acad. Sci. USA 70 May, 2866-2869. Rehfeld, J.F. and Stadil, F. (1973) Gut 14, 369. Yalow, R.S. and Berson, S.A. ((1972) Biochem. Biophys. Res. Comm.

48,

391.

Jensen, R.T., Gardner, J.D., Raufman, J.P., Pandol, S.J., Doppman, J.L. and Collen, M.J. (1983) Ann. Int. Med. 98, 59. Shively, J.E. (1981) Meth. Enzymol. 79, 31. Malmstrom, J. Stadil, F. and Rehfeld, J.F. (1976) Gastroenterology 70, 697-703. Gubler, U., Seeburg, P., Hoffman, B.J., Gage, L.P. and Udenfriend, S. (1982) Nature 295, 206-208. M. Teranishi, Y., Takahashi, H., Toyosato, M., Notake, Noda, M., Nakanishi, S. and Numa S. (1982) Nature, 297, 431-434. Kakidani, H. Furutani, Y., Takahasi, H., Noda, M., Morimoto, T., Asai, M., Inayama, S. Nakanishi, S. and Numa, Y ., Hirose, S.

(1982)

Nature

298,

245-248.

Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R.M. (1982) Nature 298, 2240-244. Jacobs, J.W., Goodman, R.H., Chin, W.W., Dee, P.C., Habener, J.F. ((1981) Science 213, 457-459. MacIntyre, I. Hilyard, C.J., Murphy, P.K., Reynolds, J.J., Gaines, Das R.E. and Craig, R.K. (1982) Nature 300, 460-462.

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