Amino acid sequence near the amino terminus of porcine pepsin

Vol.

41,

No.

BIOCHEMICAL

3, 1970

AMINO

ACID

AND

SEQUENCE OF

BIOPHYSICAL

NEAR

Received

September

Institute, Copenhagen,

A

IN0

COMMUNICATIONS

TERMINUS

r

PEPSIN

Jordan Genetic

THE

PORCINE

RESEARCH

Tong2

Deportment Copenhagen,

II,

University Denmark

of

29, 1970

Summary: The amino acid sequence near the amino terminus of porcine pepsin wos By diogonal electrophoresis (l), a peptide was isolated containing 9 residues studied. from the amino terminus of pepsin. Structural studies established their sequence to be Ile-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyr. Comparison of this amino-terminal sequence with thot known for rennin showed a definite homology. Alignment of the homologous residues revealed that the sites at which the respective rymogens ore cleaved to form active pepsin and rennin are 2 residues apart. The implications of this finding in the mechanism of activation of zymogens of the acidic proteases ore discussed. The

existence

pepsin,

rennin,

enzymes pepsin

and

descended and

denced to the

of o structural

bovine

gastricsin from

common

is now

a common

rennin,

by comparison

the

of partial

homology

sequence

Asp-Glu-Pro enzyme

‘This

(6).

was

well

ancestral

shared

are

amino

acid

elsewhere

The

partially

disparity

from

during

closely

homologous

in these

by

NIH

evolution.

Porcine

in structure,

as evi-

two

enzymes

are completely

surprising

one

by hydrolysis’of

AM-01

(4). the

In contrast first

dissimilar.

in pepsin

when

Grant

these

however,

whereas

including

Apparently,

molecules,

Research

proteases,

their

in the

(5),

zymogen

3).

protease

sequences

is especially

gastric

(2,

of pepsin and rennin

its respective

supported

certain

established

is Gly-Glu-Val-Ala-Ser

is activated

work

among

in particular,

residues from the amino tennini rennin,

homology

5

In

it is Ile-Gly-

considers

that

each

a peptide

bond

to

107.

n

‘Recipient Oklahoma Oklahoma

of NIH Career Development Medical Research Foundation, City, Oklahoma 73104.

Award 5 K04-AM-38638. University of Oklahoma

697

Permanent Medical

address: Center,

Vol.

41,

No.

3, 1970

form

a new

amino

near

the

amino

limited

terminus tennini

terminus it was

felt

rennin

would

amino

acid

phoresis below)

that

prove

Louis,

Japan.

to a much

One

to be more

RESEARCH

would

thus

homologous,

near

COMMUNICATIONS

expect

the

sequences

as a reflection

(1) was

isolation

Subtilisin

was

Other

because

purchased

purchased used

from

Novo

were

of the

5 residues

of

the

from

sequence

for

with

already

that

to establish

of pepsin.

pepsin

was

chemicals

undertaken

amino-terminal

Crystalline

Thermolysin

in pepsin

terminus

adopted

of specific

Methods:

were

amino

of only

amino-terminal

sequence

studies the

a sequence

longer

of a longer

et al.

MO.

Denmark.

BIOPHYSICAL

enzyme.

enzymes

revealing,

sequence

and

active

has identified

comparison

of Butler

Materials

on pepsin

as opposed

permit

AND

change.

work

(6),

in the

of these

evolutionary Previous

St.

BIOCHEMICAL

The

rennin

amino

(5).

Since

known

in further

method

techniques

the

,for

detail

of diagonal

involved

the electro-

(described

peptides. was

obtained

from

Daiwa

from Kaseai

Terapeutisk highest

Sigma

Chemical

K. C.

Co.,

Laboratorium, purity

Co., Osaka,

Copenhagen,

purchased

from

commercial

sources. The

amino

groups and

on the

protein

groups

of lysine

aminoethyl

maleyl

derivatives

excess

of maleic

anhydride

powder

protein,

and

cystein)

pH was

maintained

according

the

Within

10 minutes

against

0.05

carried

out

at room

M NH40H, by adding

N-ethylmorpholine

(such

to the

were

method was added between

temperature, the

protein

buffer,

amino-terminal

was

with

maleic

of Butler

et al.

(1).

in small 8 and

pH

portions

9 by the was

lyophilized.

to 8.0,

group

reacted

maleylation

1 mg of subtilisin acetate

as the

Thirty

incubating

E-amino

to form

times

molar

to a 1% solution addition

After

mixture

powder.

dialysis

digestion

protein the

of

of Na2C03

subtilisin

100 mg of maleylated and

the

anhydride

complete. The

and

was

in 5 ml of 0.2 for

16 hours

at 370%. Detailed described

procedures (7,

8).

After

in diagonal proteolysis

electrophoresis and

first-dimensional

698

were

performed electrophoresis,

as previously the

masking

M

Vol.

41,

No. 3,197O

maleyl

group

peptides

was

in the

accomplished pH

BIOCHEMICAL

3.5,

formed

2nd-dimensional

18 hours

Edman

identified

by

the

residue

produced

a pattern

(Fig. NT.This

of pepsin,

as judged

which

above

from

or near

to be lysine-containing

electrophoretic

to the

COMMUNICATIONS

mobility

Removal

was The

vapor

of the

of the maleyl

of a pyridine

involved

group

acetate

was buffer,

A)

peptide

peptides

the

line

were

of the

determined

newly by thin-

residues

of maleyl

major

in the

of the from

digest

spot

to have

change

derived

acids

Carboxyl-terminal

subtilisin

was assumed

neutral

by determination

amino

(10).

in which

its mobility the

dansyl

out

were

(11).

of the

1, Part

carried

sheets

digestion

electrophoresis

lay

(9).

carboxypeptidase

peptide

strips

on polyamide

Diagonal

was

paper

degradation

Results:

line

the

RESEARCH

(1).

sequential

chromatography

BIOPHYSICAL

electrophoresis.

at 60’

amino-terminal

layer

thus altering

by exposing for

The

removed,

AND

second

laying

derived two

porcine off

from

the

dimensions. dimension

carboxyl-terminal

the

region

diagonal

amino The

(L,-L9)

pepsin

terminus

peptide were

of the

assumed molecule

1 10

0 -0

10

20

:, w

30

I 20

CM. FROM Fig.

1.

ORIGIN,

1 st

DIMENSION

Diagrams of pH 6.5 diagonal electrophoresis subtilisin digest of porcine pepsin (A) and fmgment derived from the amino-terminal aminoethylated porcine pepsin (B).

699

I

10

spots

of demaleylated and a cyanogen bromide half of reduced and

Vol.

41,

No.

(12).

3, 1970

Deamidation

spots

that

appeared

(PI-Plo) that

were

sites.

To confirm

were

performed

reduced

was

line.

Numerous

near

the

diagonal

line.

Since

this

derived

(Fig,

analysis,

maleylation fragment

from

the

B).

again

Only

traces

peptides

CB2,

which

containing

from

peptides

these

as the

maior

P, -P4

aminoethyl

cleavage

were

160 (Tang,

laying

off

present.

cystein,

of

about

molecule

spot

autolytic

experiments

contained pepsin

peptide

showed

sites,

bromide

of the

of peptides

(13)

electrophoresis

cyanagen

emerged

few

intensity

et al.

produced

by

half

the

of minor

cleavage

diagonal

produced

for

Rajagopolan autolytic

and

Fragment

NT

spots

peptides

amino-terminal

Peptide

COMMUNICATIONS

to be responsible

many

amino-terminal

obviously

experiments

diagonal

of pepsin.

made

amino

acid Ile,

NT

the

same

digested

Tyr.

Peptide

NT

37”

C for

18 hours

gave

contained

portion the

4 residues,

determined

by the I).

Thus,

Pro-Leu-Glu-Asn-Tyr

peptide

was with

the

mobility the

had

of peptide

sequence

contained

with

and

sequence

peptide must

from

of the

have

the

Peptides

the sequence

peptides

of peptide

only

maior

from

the

with

protein.

The

of which

on paper

I).

By using

the

at different NT

.

700

was

established

of

12.5%.

residue of SO:1

at pH 6.5.

and

and

the

from dansyl

Peptide acids

at

Peptide

derived

degradation

The steps

yield

ratio

to be Ile-Giy-Afp-Gb-Pro. * Le,v-G$-Afn-Txr.

terminus

carboxyl-terminal

apparently

Edman

amino

present

Its amino-terminal

electrophoresis Ile,

peptide

a molar

at a substrate-enzyme

an amino-terminal

sequence

derived

(Table

whole

bands

established

the

9 residues

thermolysin

NT.

of the

NT,

100 mg of pepsin,

it to contain

as that

two

5 residues

amino-terminal

purified

showed

was

that

patterns,

was

analysis was

procedures,

it evident

electrophoretic

Peptide

residue,

(Table

contains

RESEARCH

(4).

These

NTT

presumed

to be the

1, Part

were

is known

pepsin

pepsin.

results).

diagonal

in both

diagonal

aminoethylated

unpublished

AT -A6

the

on a purified

and

residues,

below

assumed

was

BIOPHYSICAL

heating

crystalline

were

AND

during

found

commercial

PT-PI0

The

BIOCHEMICAL

amides

of Edman

degradation

to be:

Ile-Gly-Asp-Glu-

NT2 were

1. 2. 3. 4.

-0.47

o.094

NT2-1

NT2-2

:

(9

f&d

Orange

Red

Red

-

(S)

Red

Red

- Ninhydrin Color

Orange

Orange

Cadmium

1.00

1.07

in Edman degradation. peptide is considered

1.08

Glu 2.04

Amino

1.22

Asp 1.97

Characteristics of Region of Pepsin

The electrophoretic mobilities are relative to that of Asp ( = -1 .O). The sign (S) designates that color development was slow. the number of residues removed The numbers after the - sign indicate the Slight mobility is associated with an asparagine amino terminus;

-0.69

Degradation3

NT,-3

Edman

-0.38

-0.84

-0.73

in pH 6.5’ 2nd Dim.

-1.05

from

-1.05

Electr. Mobility 1st Dim.

NT,-2

Peptides

NT2

NT’

NT

Peptide

I

Amino Acid Composition and Peptides from Amino-Terminal

TABLE

neutral.

-

1.00

1.10 -

GIY 1.00

Composition mtio)

Pro 1.01

Acid (molar

-

0.96

Ile 0.98

1.01

-

Leu 1.18

0.70

-

TY~ 0.96

Vol.

41,

No.

3, 1970

Discussion: porcine first

Peptide pepsin,

contains aligned, respective

in the

peptide and

more

zymogens. portion

Perlmann

of pepsinogen

(14)

is a Glu-lie Glu-Val

amino

terminus

of the

difference

hydrolysis

of an X-Gly of pepsin

that

The

However, bond

and

two rennin

the

residues

Pepsin:

-Ala-Glu m-m -

which

in proteolysis

very

1 2 3 456789 Ile-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyrt m-w -mm ---

two

2.

activation

is a closely

is accompanied

The

9

finding sites

10 -

by since

on the

11 -

--

Homology in amino acid sequence of the amino-terminal regions in The solid underlines indicate bovine rennin (5) and porcine pepsin. the residues which are identical; the broken underlines indicate the residues whose difference could be the result of a single-step mutation. The numbering of amino acid residues, in both cases, starts at the amino terminus of the enzymes. The two residues on the left of amino terminus of pepsin ( -Ala-Glu) are the last two residues in the portion of 41 residues which are cleaved off during the activation of pepsinogen (14) -

702

that

residues

Activation Cleavage in Zymogen

Fig.

amino-

activation

during

surprising

8 -

last

in rennin

similar.

7 -

the

the

41 in pepsinagen).

of pro-rennin

are

for

residues

during

the

is broken

a somewhat

with

homologous

into

40 and

(6)

of

It is interesting

sites

position

activation away,

2.

the

extends

bond

Activation Cleavage in Zymogen I 1 2 3 4 5 6 Gly-Glu-Val-Ala-Ser-Val-Pro-Leu-Thr-Asn-Tyr---------

Rennin:

in Fig.

in cleavage

corresponding the

a homology

residue

peptide

residues

by Stepanov

when

homology

COMMUNICATIONS

9 amino-terminal reported

at the

the

RESEARCH

established,

( -Ala-Glu,

bond.

bond.

now

that

as shown

However,

showed

with

the

clear,

of pepsinagen

analogous

specificities

(5) becomes

as a result

from

agrees

9 residues

residues

BIOPHYSICAL

derived

of NT with

evidently

of the

Ong

sequence

of rennin two

AND

is apparently

However,

sequence

rennin

NT

The

5 residues.

terminal

are

BIOCHEMICAL

the B-chain

BIOCHEMICAL

Vol. 41, No. 3,197O

of oxidized tical. the

Were bonds

limited the

insulin

alternative hydrolyzed

for

the

specificity

pro-rennin

hydrolyzed

during

conformation

zymogen

zymogens

may

of the requires

activation, On

be independent

thus

producing

when

one

the

presence

(17,

18),

of

cleavage

sites

would

other

hand,

it seems

hydrolytic change

an internal

considers

of no preformed

iden-

the

by

second,

nearly hydrolysis

two

of pepsinogen and

are for

of be

5,

(16)

responsible

be triggered

in the activation enzyme

the

might

plausible

Glu-lie

rennin

site

a pK of about

and rennin

(15)

and

of the zymogen.

is especially bond

pepsin

of pepsin

of a specific

with

by

activity

above

of these

of a group

are

hydrolytic

Cleavage

the

and

the

activation

enzymes.

This

the

discussed by

state

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

be

possible

that

activities of the

of the

ionization

rearrangement other

factors:

is relatively

of bonds. first,

that

unfavorable

the activation

of pepsinogen

enzyme.

Acknowledgement: The author wishes to thank Professor Institute, II, Univenity of Copenhagen, for the helpful of facilities during the course of this work.

Bent Foltmann, discussions and

for

Genetic the use

REFERENCES

(1) (2) (3) (4) (5) (6)

(12) (13) (14) (15)

(16) (17)

(18)

Butler, P.J.G., Harris, J.I ., Hartley, B. S., and Leberman, R., 112 : 679 (1969). mmann, 8. and Hartley, B.S., Biochem. J ., 104 : 1064 (1967). Huang, W.Y.,and Tang, J., J. Biol. Chem., 245: 2189 (1970). Tang, J. and Hartley, B.S., Biochem. J., 1183611 (1970). Trans. Roy. Sot. LondorB 257 : 147 (1970). Foltmann, B., Phil.

Biochem.,

Stepanov, V.M., Abstr. Fed. Europ. Biochem. Sot., 5th Meeting, No. 1089, (1968). Brown, J. R. and Hartley, B.S., Biochem. J., 101 : 214 (1966). Tang, J. and Hartley, B.S., Biochem. J., 102793 (1967). Gray, W.R., Methods in Enzymology, Vol .x p. 469, Ed. Hirs, C.H .W., N.Y. Acad. Press, Inc. (1967). Woods, K.R. and Wang, K.T., Biochim. Biophys. Acta, 133 : 369 (1967). Ambler, R. P., Methods in Enzymology, Vol. II, p. 417,x by Hirs, C.H.W., N.Y. Acad. Press, Inc. (1967). Dopheide, T.A.A ., Moore, S., and Stein, W.H., J. Biol . Chem., -242 : 1833 (1967). Rajagopalan, T.G., Stein, W.H ., and Moore, S., J. Biol. Chem., 241 : 4940 (1966). Ong, E.B. and Perlmann, G.E., J. Biol. Chem., 243 : 6104 (1968). Sanger, F. and Tuppy, H., Biochem. J., 49 : 481 (11). Foltmann, B., Compt. Rend. Trav. Lab. Czlsberg, 35 : 143 (1966). Tang, J., Nature, 199: 1094 (1963). 7 Fruton, J.S., Symposium on Structure and Function Relationships of Proteolytic Enzymes, p. 222, Ed. Desnuelle, P., Neurath, H. and Ottesen, M., Munksgaard, Copenhagen, Denmark (1970). 703

J.,