BIOCHEMICAL

Vol. 168, No. 2, 1990 April 30, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 878-885

STRUCTDRAL EVIDENCE FOR TWO ISOZYMIC FORMS AND THE CARBOBYDRATE ATTACHMENT SITE OF HUMAN GASTRIC CATHEPSIN E S.B.P.Athauda,

Osamu Matsuzaki, Takashi and Kenji Takahashil

Department of Biophysics The University of *Department

of

Kageyama

and Biochemistry, Faculty Tokyo, Hongo, Tokyo 113,

*

of Science, Japan

Primate Research Institute, Biochemistry, University, Inuyama, Aichi 484, Japan

Kyoto

Received March 20, 1990 SUMMARY: The amino acid sequences in the NH2-terminal region and some other parts of human gastric cathepsin E were investigated. The NH2-terminal sequencing revealed that the cathepsin E preparation which had been activated at pH 4.0 contained one major and one minorisozymes in an approximate molar ratio of 3 : sequence of the former was very similar to 1. The NH2-terminal but partly different from that predicted from cDNA sequencing by whereas the latter had an NH2-terminal sequence Azuma et al., identical with the predicted sequence. These results provide structural evidence for the presence of at least two isozymic forms in human gastric cathepsin E. In addition, the site of carbohydrate attachment was elucidated by isolation and analysis of a glycopeptide fraction from an enzymatic digest of cathepsin E. A single carbohydrate chain was deduced to be attached to the asparagine residue at position 34 in the major isozyme and to the corresponding asparagine residue in the minor isozyme. 01990 Academic Press,

Inc.

Human gastric protease proteinase pepsinogens this

cathepsin

present

about

size.

in

E (previously

D-like

(2).

85,000

The enzyme

to

human gastric

apparent

The enzyme

was shown should

to

All rights

0 1990 by Academic Press, of reproduction in any form

Inc. reserved.

two

and different

from

we first

a molecular

subunits

be addressed.

moving an acid

be a glycoprotein,

878

etc.)

and investigated

was shown to have of

as slow is

D. In 1980,

0006-291X/90$1.50 Copyright

(2)

mucosa

homogeneity

and composed

1To whom correspondence

called

proteinase

A and C and cathepsin

proteinase

properties of

(I),

cathepsin

of

purified several weight

an identical containing

2

Vol.

168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

glucosamine

and 8 mannose residues

this

were

enzyme

further

studied

per

RESEARCH COMMUNICATIONS

subunit.

by Samloff

The properties

et

al.

of

(3) and by us

(4). Very

recently,

Azuma et

sequence

of

gastric

analysis

of

cell

line.

residue

cDNA clones

proenzyme

derived amino

determined

acid

sequence

on the

protein

signal

So far,

sequence

the

acid

from

the

adenocarcinoma includes

peptide, however,

and the

site

a 379and three

there

of human gastric

level

amino

E predicted a gastric

sites.

amino acid

reported

from

and a 17-residue

glycosylation on the

(5) have

procathepsin

The predicted

potential report

human

al.

is

cathepsin of

no E as

carbohydrate

attachment. In

the

sequences

present

of

the

presence

cathepsin

4.0. to

which

The major but

sequence

sequence.

These results

presence

addition, in

the

MATERIALS

of

we have

The NH2-terminal

that

hand,

the

with

provide

isozymes-

elucidated

forms

sequence

minor

isozyme

predicted

first

in human gastric

the

site

of

cathepsin

very

similar the

the

evidence cathepsin

carbohydrate

at pH

cDNA

had an NH2from

structural

of

revealed

enzyme

from

acid

parts

in the

predicted

that

the

amino

sequencing

to the mature

from

identical

isozymic

the

and some other

had an NH2-terminal

other

terminal

the

region

and one minor

different On the

investigated

had been activated

isozyme

partly

sequencing.

E.

of one major

E preparation

we

NH2- terminal

the

human gastric

study,

cDNA for E. In

attachment

enzyme. AND

METHODS

Materials: Human gastric cathepsin E was purified essentially as described previously (2,4) and the major component (SMP-I(4)) was peptides and Staphylococcus aureus V8 peptides were used. Tryptic prepared from the reduced and carboxymethylated (RCm-)protein and fractionated by high performance liquid chromatography on a TSKGel ODS-120T column ( 0.4 x 25 cm) in a manner as described The glycopeptide fraction was previously (6) (data not shown). prepared by digestion of the protein with thermolysin and B. subtilis aminopeptidase followed by chromatography on a Sephadex 879

BIOCHEMICAL

Vol. 166, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

G-25 column (1.6 x 40cm) as described previously (2). Reagents for automated amino acid sequencing were obtained from Applied Biosystems. Reagents for manual Edman degradation were purchased from Wako Pure Chem. Ind.,Ltd. Other reagents used were of the highest grade available. Amino acid sequence analysis: The NH -terminal amino acid si by using a protein sequence of the protein was determine sequencer model 477A/12OA in the presence of pepstatin (about 2fold molar excess). The protein was analyzed before or after treatment at pH 4.0 and purification on pepstatin-Sepharose. In the latter case the enzyme was dialyzed against 0.05 M sodium acetate buffer (pH 4.0)-0.2 M NaCl at 4OC for 3 h and applied to a pepstatin-Sepharose column (1.0 x l.Ocm) equilibrated with the same buffer. After washing the column with same buffer (60 ml), the protein was eluted with 0.05 M Tris-HCl buffer (pH 8.0)-l M NaCl, and further purified by mono Q chromatography (data not After desalting through a Sephadex G-25 column, the shown). protein fraction was submitted to automated sequencing. Peptides were sequenced partly by the sequencer and partly by a modification (6,7) of the manual method (8) of Edman degradation. RESULTS

AND

DISCUSSION

E2-Terminal

amino acid

When the directly

by

amino acid that

the

human gastric the

with

the

pyroglutamyl

residue,

other

after

the

original

of

sequence

could

the of

mature amino

be deduced

an asparagine (Fig.1).

the

The

blocked.

This

is

residue

of

converted

followed

procathepsin

E

mature

at

enzyme revealed

and the

except

for

the

sequence

the

the

corresponding of

880

residues

the

NH2-

suggest before

that

pH 4.0-

converted

pH 4.0. presence

NH2-terminal residue

affinity

obtain

was

a

On the

by

These results

1.

pro-form

acids

into

Edman degradation.

we could

enzyme

in

indicating

was the

this

into

sequence

is

to

preparation

that

autocatalytically

which

resistant

(PTH)-

amount,

glutamine

be easily

as shown in Fig.

enzyme and

sequence

may

on pepstatin-Sepharose,

treatment

major

is

pH 4.0-treatment

sequences

degradation

protein

NH2-terminal

which

was analyzed

no phenylthiohydantoin

the

putative

E (5),

E preparation

in a significant

of

procathepsin

purification

almost

be detected

NH2-terminal

hand,

cathepsin

sequencer,

could

consistent

terminal

sequence

Edman of

one

39 residue at

position

procathepsin 22-37

was further

34, E

Vol.

168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Protein 1

10

Major

:

IQFTExxxMDQMAKEPLINYLDM

Minor

:

7'"' IQFTES?SMDQSAKE--777777777777777 37

cDNA(5)

:

20

777777777777777 10 15

40

50

59

IQFTESCSMDQSAKEPLINYLDM

Protein 21 Major

:

V8-peptide

:

cDNA(5)

:

30

39

EYPGTISIGSPPQ?FTVIF--7777777777777777-?77

Y F G T I S I G S P P Q ? F(T)V 7777777777777777 60

--70

70

EYFGTISIGSPPQNFTVIF---

Fis.1. Comparison of the NH2-terminal amino acid sequences of human gastric cathepsin E with the corresponding sequence of the procathepsin E predicted from its cDNA sequence (5). The protein sequence was obtained with human gastric cathepsin E sample treated at pH 4.0 and then affinity-purified on pepstatinSepharose. The sequence of residues 22 to 37 in the major sequence was further confirmed by sequencing of a V8-peptide. Procathepsin E numbering is used for the sequence predicted from the cDNA sequence. deletion. each cycle of Edman degradation. (?), ambigu%s identificaty&.

confirmed

by isolation

peptide

as shown

similar

to

that

in Fig.

predicted

a few positions. the

al.

(5)

in

the

intermolecular

Further, which

suggested

lacks

presence used.

In of

approximately sequenced

this

ratio

at

this

1 : 3, and the

simultaneously.

This

the

cysteine

position

in

the

sequence 881

of

the

may be

the

procathepsin

also

revealed

major

15

major

of a serine

to

the

Azuma

formation.

9 instead

enzyme

NH2-terminal

deletion

Since

mature

sequence

at

may be involved

bond

degradation

very

differs

residue(s)

in

a V8-

is

42-44).

residue

position

Edman

of

is

disulfide

corresponding

sequence

but

bond formation. other

of

obtained

E residues

intermolecular

addition,

thus

difference

cysteine

a methionine

the

analysis

cDNA sequence,

(procathepsin

residue,

one minor

The molar

the

disulfide

the

we found

sequence.

that

this

for

occupies

The sequence

from

sequence

et

responsible

1.

sequence

The most notable

Ser-CyS-Ser

sequence

and partial

to

the

preparation sequence

residues

appeared

E

was

could

be identical

be

Vol.

BIOCHEMICAL

168, No. 2, 1990

with

that

results

predicted

provide

two

isozymes,

are

products

al.

(5)

a gastric

in

of

different

the

on the

Amino acid

In addition

sequence

to the

protein.

in the

to obtain

For example,

latter

seems to

residues

based

proteinases.

but

proline,

COOB-terminal

of

peptides

coincide

indeed

and 372-379 predicted

residues

of

two

peptide 80-88,

were

This

is

et al.

E.

about

can be assumed

with

The those

of

and

site

882

is

not

to

of

had

the

arginine

or

from

the

these

124-138,

with

the

respectively,

three 258-268,

respectively,

sequence

acid

aspartic

to be derived

3%. Further,

360-367,

the

The

peptide

residues

coincide

of

aspartic

among

which

and the

to

acid

respectively.

peptide),

VS-peptides,

amino

E

sequenced

sequences

procathepsin

confirmed

the

regions

tryptic

of

the

RCm-cathepsin

homology one

protein.

297-310,

pro-

1, some of

from

active

COOH-terminal

(5) of

other

the

hand,

COOH-terminal

sequence

sequences tryptic

(the

the

long.

were partially

sequence

so it

the

cathepsin

GCQAIVDTGTS---,

other

VGLAPAVP, the

This

and cancer

that

COOH-terminal

one of

the

On the

sequence, lysine

and

on

of rat

et

may be

made by Yonezawa

information

two V8-peptides

contain

normal

36 residues

peptides

and the

be TGSLSGIIGADQVSV---

which

genes

shown in Fig.

tryptic

interior

E,

regions

V8-peptide

and the

least

mucosa.

indicate

E (5) is

NH2-terminal

sequenced

sequences

results

at

corresponds

two

the

These

by Azuma

line

the

in

recently

V8-peptides also

that

present

of other

of

in the human gastric

prediction

sequences

presence

cell

proportion

the

1).

of human cathepsin

possibility

procathepsin

from

(9) based

were

isozyme

a different

the

the

adenocarcinoma

minor

In addition,

sequence

for

(Fig.

The cDNA obtained

genes.

an interesting

cells.

other

evidence

different

to the

expressed

cDNA sequence

and hence isozymogens,

from

suggests

the

structural

of

apparently

from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the of

the

partial another

sequences in

of the

Vol.

168,

No.

predicted

sequence

therefore,

(5)

protein

differences

on this

of

the

NH2-terminal to

COMMUNICATIONS

far

the

sequence

necessary

as

examined,

sequence

of

except

for

Further

draw

the the

protein

a definite

conclusion

attachment

glycopeptide

80 % from

a thermolysin-aminopeptidase

chromatography The of

mol

acid

peptide)

Gly

0.78,

24h).

In

addition,

after

7.5h),

glucosamine

(1.80

amino

acid

Ile

after

mannose

chromatography

sequence

degradation

peptides,

: 2. Their

deduced

analysis revealed

major

as of

the

I and

II,

sequences

as

shown

were

below. I:

column

as

mol

(8

peptide an

per

0.95,

of

mol

llO°C,

HCl,

100°C,

found

The

by mannual

approximate a mixture

degradation,

by

peptide)

(2).

was

Pro

HCl,

was

fraction

as

Edman

N

peptide)

mol

E

described

Glu

(1

of

cathepsin

(6N

fraction

analyzed

Upon

of

a yield

glycopeptide

1.03,

previously

peptide

in

Ser

the

hydrolysis

described

this

that

of

hydrolysis

acid per

in

digest

of

1.00,

acid

mild

and

G-25

Asp

mol

isolated

composition

was

0.63

analysis

was

a Sephadex

amino

1.88,

gas-liquid

fraction

on

(2).

per

acid

so

regions.

A single

(mol

In

between

predicted

is

RESEARCH

E.

detected

the

carbohydrate

previously

3

procathepsin was

however,

BIOPHYSICAL

point.

The site

two

AND

and in

sequencing,

over

of

no difference

actual

by

BIOCHEMICAL

2, 1990

by amino Edman

a mixture

of

molar

ratio

of

and

could

be

each

residue

Ile-Gly-Ser-Pro-Pro-Gln-Asx 7777777

II:

Gly-Ser-Pro-Pro-Gln-Asx 777777

could

be

identified

terminal

residue.

residue.

However,

by

dansylation

residue

is

as No it

PTH-amino

could

followed thought

a PTH-amino

to

acid acid

be identified by

acid

be originally 883

except

was as

for

obtained

the

COOH-

from

this

dansyl-aspartic

hydrolysis. an asparagine

acid

Therefore, to

this which

the

BIOCHEMICAL

Vol. 168, No. 2, 1990

carbohydrate

chain

coincide

with

cathepsin

the

the

position

34

(procathepsin position

in

described

glycosylation obtained

The minor

since from

consistent

the

same

the

site

0-glycosylation

fact

the is

also

in

the

only I

thought

the

the

results

residue

at

sequence

asparagine

at

one

site

result

the

carbohydrate

of was

is also

potential

73) and two E

as

1)

fraction

This

at

of

corresponding

to be the

are

residue of

73 in

(Fig.

the

procathepsin

site

monkey pepsinogen

at

E residue

same aspargine

major

Edman degradation

VB-peptide

there

the

the

68) to

why the

isozymes.

that

(procathepsin

34 in

glycopeptide

of both

sites

Interestingly,

a single

to

E isozyme

in the

is also

glycopeptides

asparagine

asparagine

isozyme only

be the

the

the

Therefore,

explains

as

of

67 (or

(5).

to

This

well

mixture

with

glycosylation

Japanese

as

29)

residues

be identified

protein

in the

(or

cathepsin

73).

not

above.

position

major

E residue

mature

or

site

the

34 could

28

E sequence

glycosylation

RESEARCH COMMUNICATIONS

The sequences

sequence

procathepsin

establish

position

bound. residues

E isozyme

predicted

the

is

AND BIOPHYSICAL

N-

potential

sequence

(5).

corresponding attachment

in

(10).

ACKNOWLEDGMENTS

We are

most

Nagoya)

for

We also

thank

of

Medicine,

Hideshi

Inoue

the

grateful

to Dr.

generous

Dr.

supply

Takayuki

The University for

their

help

Masanori of

specimens

Takahashi, of

Ukai

Tokyo),

and useful

Dr.

of

(Ukai

human stomachs.

Masao Ichinose Dr.

Hospital,

Masao Tanji

(Faculty and Dr.

suggestions.

REFERENCES

1.

2. 3. 4.

Samloff, I.M. (1969) Gastroenterol. 57, 659-669. Kageyama, T., and Takahashi, K. (1980) J. Biochem. 87, 725735. Samloff, I. M., Taggart, R. T., Shiraishi, T., Branch, T., Reid, W. A., Heath, R., Lewis, R. W., Valler, M. J., and Kay, J. (1987) Gastroenterol. 93, 77-84. Matsuzaki, O., and Takahashi, K. (1988) Biomed. Res. 9, 515523. 884

Vol. 168, No. 2, 1990

5. 6. 7.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Azuma, T., Pals, G., Mohandas, T. K., Couvreur, J. M., Taggart, R. T. (1989) J. Biol. Chem. 264, 16748-16753. Takahashi, K. (1987) J. Biol. Chem. 262, 1468-1478. van Eerd, J. -P., and Takahashi, K. (1976) Biochemistry

and 15,

1171-1180. 8.

9.

Edman, P., and Henschen, A. (1975) in Protein Sequence Determination (Needleman, S. B., ed.) 2nd Ed, pp.232-279, Springer, New York. Yonezawa, S., Takahashi, T., Ichinose, M., Miki, K., Tanaka, J and Gasa, S. (1990) Biochem. Biophys. Res. Commun. 166, 10;2-1

10.

038.

Kageyama, T., and Takahashi, Commun. 74, 789-795.

K.

885

(1977)

Biochem.

Biophys.

Res.