Vol. 169, No. 3, 1990 June 29, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1242-1247

BIOCHEMICAL

NEUTROPHIL CHEMOTACTIC ACTIVITY

OF N-TERMINAL

PEPTIDES

FROM THE CALPAIN

SMALL SUBUNIT 1 Kunimatsu , Xiao Yasumasa Hamada**,

Mitoshi

Department

of Biochemistry and *Department of Otorhinolaryngology, University Medical School, Mizuho-ku, Nagoya 467, Japan

**Department

Received

Jing Ma, Joh Nishimura*, Shunkichi Baba*, Takayuki Shioiri**, and Makoto Sasaki

of Synthetic Nagoya City

May 28,

Organic

University,

Nagoya

Chemistry, Faculty of Pharmaceutical Mizuho-ku, Nagoya 467, Japan

City

Sciences,

1990

On the basis of previous findings that N-acetyl nonapeptide from the human calpain I large subunit has chemotactic activity for neutrophils, a series of peptides with the N-terminal sequence of the calpain small subunit were synthesized and their chemotactic activity was examined. Potent activity was found in N-acetyl tetra, hepta, octa, nona and a larger peptide of 13 residues, although N-acetyl tripeptide showed only weak activity and N-acetyl penta and hexa peptides showed almost no activity. Since the small subunit is identical in calpains I and II, the results indicate that both calpains could be precursor proteins of chemotactic factors for neutrophils. Q1990 Academic Press, Inc.

Calcium

dependent

are widely revealed

distributed

other

7).

These

Wr, with

80K)

micromolar

have

distinct

enzymes

subunit

particular,

almost the

I)

enzyme activity

N-terminal

islet

and cells composed subunit

(9),

porcine sequence acid

cells

contain

contain

both

of a large 30K)

(calpain

with 19 amino

of higher

EC 3.4.22.17) organisms

measurements

(Mr,

and identical

of human identical

and cells

and millimolar

subunits

I and II,

II)

small

subunits

(IO),

rabbit

homologies

sequences

and detection only

calpain

calpains

or catalytic (3).

oow291x/90

should be addressed Nagoya City University

I (2,

I and II subunit

(8). (ii),

requirements The structures and bovine

of about

97X

subunits

(12)

(3). are

to: Mitoshi Kunimatsu, Department of Medical School, Mizuho-ku, Nagoya 467,

$1.50

8 1990 by Academic Press. inc. of reprduction in any fom reserved.

1242

of

The two isoenzymes

calcium

of these

as

homologous.

1Correspondence Biochemistry, Japan.

Copyright All rights

heterodimers or regulatory

(calpain large

are

completely

are

tissues

(calpains

tissues

and Langerhans

mammalian

and a small

small

calpains

methods,

Erythrocytes

6) and most

proteinases

in various

by immunological

mRNA (l-5).

of the

cysteine

In

(5,

Vol.

169, No. 3, 1990 Recently,

BIOCHEMICAL

we found

of human calpain

I,

chemotactic

isolated

human calpain I (14), sequence of the large residue

sequence,

factors

of bacterial In this

acid

acetyl

of

with

the

N-terminus

subunit

is

sequence:

formyl

we synthesized the small

subunit

MATERIALS

sequence

the

of

amino acid a methionine

reported

that the N-

chemotactic

derivatives

with

their

structure

Further,

of the

and its

autodigests

entire

(10).

those

peptides

and examined

the

has been

acetylated

Met-Leu-Phe several

its

the N-terminal was lacking It

resembles

the

N-acetyl

with

(14).

also

Met-Phe-Leu-,

origin,

study,

sequence

small

in

, and determined

When compared

at the

RESEARCH COMMUNICATIONS

neutrophils

this peptide was identical with subunit, except that the peptide

of the

terminal

for

peptide

nonapeptide

and was acetylated

the N-terminus

activity

an active

It was an acetylated (13). Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr.

AND BIOPHYSICAL

(15-20).

N-terminal

chemotactic

amino

activity.

AND METHODS

Materials: Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden), chemicals and resins for peptide synthesis from MilligenfBiosearch Division of Millipore (San Rafael, CA, USA), a 48-well micro chemotaxis chamber and polycarbonate membrane filter from Neuro Probe (Bethesda, MD, USA), Diff-Quik from Kokusai Siyaku (Kobe, Japan), Hank's medium from Nissui (Tokyo, Japan), and formyl Met-Leu-Phe (f-MLF) from Peptide Institute (Osaka, Japan). N-acetyl Met-Phe-Leu (AC-MFL), N-acetyl Met-Phe-Leu-Val (AC-MFLV), N-acetyl Met-Phe-LeuVal-Asn (AC-MFLVN), N-acetyl Met-Phe-Leu-Val-Asn-Ser (AC-MFLVNS), N-acetyl MetPhe-Leu-Val-Asn-Ser-Phe (AC-MFLVNSF), and N-acetyl Met-Phe-Leu-Val-Asn-Ser-PheLeu (AC-MFLVNSFL) were purchased from Fujiya (Tokyo, Japan). Preparation of neutrophils: Human neutrophils were isolated from heparinized blood of healthy volunteers by the method of Bovum (21). The cells were more than 98% neutrophils as determined by Diff-Quik-staining and living cells consistently exceeded 90%. Chemotactic assay: The assay of the chemotactic activity for human neutrophils was carried out with the use of a 48-well micro chemotaxis chamber and a polyvinyl pyrrolydone-free polycarbonate membrane filter (3 micron pore size) Neutrophils adhering to the surface of the filter were stained with (22, 23). Diff-Quik, and counted using a color video image processor (Nikon, Tokyo, Japan) at 200 x magnification and chemotactic activity was expressed as the total number of 5 fields. Preparation of calpain derived chemotactic factors: N-acetyl Met-Phe-Leu-Val-Asn-Ser-Phe-Leu-Lys (AC-MFLVNSFLK) and N-acetyl Met-Phe-Leu-Val-Asn-Ser-Phe-Leu-Lys-Gly-Gly-Gly-Gly (AC-MFLVNSFLKGGGG) were synthesized with Fmoc-amino acid and polystyrene resin according to the method of Hudson (24). The latter N-acetyl peptide of 13 amino acid residues was prepared as a peptide containing an oligoglycine sequence of various lengths The peptide chain was elongated using an following the 9th residue of lysine. Model Excell) according to automated peptide synthesizer (Milligen/Biosearch, the standard operation programs. The N-acetyl group of the synthetic peptide was confirmed on the NMR spectrum recorded with a JEOL GSX-400 spectrometer in dg-dimethyl sulfoxide using tetramethylsilane as an internal standard.

RESULTS AND DISCUSSION Chemotactic lengths subunit.

activity

of synthetic As shown

for

peptides in Fig.

human neutrophils with

lA-

the N-terminal

H, dose-dependent

1243

was measured

with

several

sequence

of the

calpain

migrations

of neutrophils

small are

Vol.

BIOCHEMICAL

169, No. 3, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IO

1 A : human PO~Cl~~

rabbit bovine

B :

f-3

20

AC-MFL *

0

0.1

1

IO

50

0.1

CONCENTRATION

1

IO

OF PEPTIDES

50

f-8

AC-MFLVNSFL

* *

AC-MFLVN

1-9

AC-MFLVNSFLK

1-6

AC-MFLVNS

1-13

AC-MFLVNSFLKGGGG

*

200

268

100

: 30 K subunlt

0

2

(PM)

AC-MFLVNSF

1-5

2

1

Q

glyclne-rich blnding

1

l-7

AC-MFLV

Tb.--; H

40

t-4

1 c

30

~FLVNSFLK~GGGGGGGGG;~LGGGLGNVL~GLISGAGGG~G--MFLVNSFt.KGGGGGGGGGGGLGGGLGNVLGGLlSGAGGGGG--MFLVNSFLKGGGGGGGGGGLGGGLGNVLGGLlSGAGGGGGG--MFLVNSFLKGGGGGGGGGGLGGGLGNVLGGLlSGAGGGGGG---

*

3

1

5?

unknown

membrane domain

chemotactic releasing

binding

iactol domain

domain

Fig. 1. Chemotactic activity of synthetic peptides with the N-terminal sequence Human neutrophils, 0.5 x IO5 cells/50 ~1, were ofccalpain 30K subunit. added to upper wells of the 48-well micro chemotaxis chamber and incubated at 37'C for 30 min in 5% COP. A. AC-MFL and f-MLF, B. AC-MFLV, C. AC-MFLVN, D. AC-MFLVNS, E. AC-MFLVNSF, F. AC-MFLVNSFL, G. AC-MFLVNSFLK, H. AC-MFLVNSFLKGGGG. Chemotactic activity is represented by the number of migrated neutrophils per 5 microscopic fields. Values are the mean C standard deviation of 8 healthy individuals. synthetic

+, peptides

standard with the

chemotactic N-terminal

factor, sequence

of

formyl Met-Leu-Phe. the calpain 30K

a, subunit.

Fig. 2. Comparative representation of the structures of N-terminal amino acid sequences of calpain 30K subunits, synthetic N-acetyl peptides, and the domain structure of the 30K subunit. A. N-terminal amino acid sequences of human (9), porcine (IO), rabbit (11) and bovine (12) calpain 30K subunits deduced from the cDNA sequences. B. Structures of synthesized N-acetyl peptides. *Chemotactic

peptides. newly

C. Multifunctional

recognized

function

The shadowed

neutrophils.

domain structure of domain 1 is area corresponds

the

of the calpain release to the

30K subunit.

of chemotactic region of the

The

factors chemotactic

for

peptides.

observed

with

several

terminal

peptide

activity

suggests

have

chemotactic

only

(Fig.

octa,

of

activity,

by autodigestion (25).

with

N-terminus

our

of both

with

molecular into

of small

peptides

the

showed is

The fact

it small

sizes

possesses

of

smaller

residues

size

an N-

chemotactic

of the

could

consist

peptides,

activity.

one of the most because

that

10 to 19 residues

to 19th

chemotactic

subunit

of glycine

N-acetyl

nona,

Among the

likely

candidates

substrate

also

peptides produced

specificity

of

has been reported that calpain autolysis begins and large subunits and results in the release of

consideration, subunit

10th

as to the

small

Furthermore,

own findings

product(s)

since

nonapeptide

of the

calpains

lengths.

(AC-MFLVNSFLKGGGG)

N-terminal

In addition, N-acetyl

of fragments

all

and tetrapeptides

synthesized,

the

of different

13 residues

that

2).

hepta

peptides

lOOO-

3000 Mr (12,13).

it

apparent

that

but also

the

is

autodigestion,

1244

Taking not

only

secondary

these

and

the primary and/or

Vol.

169, No. 3, 1990

tertiary

products

of lower

carboxypeptidase however,

only

weak

influenced

itself

their

C-terminus,

hydrophilic

of the

a lysine

whereas

those

of the

other

bacteria;

f-MLF

and its

possess

a lysine

residue

tyrosine

at the

C-terminus

terminus

and lysine,

general

C-terminus

with

acid

than

action

chemotactic

amino

derivatives;

of

activity.

AC-MFL,

peptides

N-formyl

f-MLFK,

such

For example,

chemotactic

at the

acid

with

from

and f-MLFMY

also

phenylalanine

as

at

a

peptide

N-acyl

peptides

of the

amino

terminated f-MLFF

acid

and tyrosine

of N-acyl

most

chemotactic

f-MLFYK,

amino

20).

residues:

or a hydrophobic

inactive

or a hydrophobic (15-18,

acid

residue

lysine.

phenylalanine

characteristic

have

by the

and AC-MFLVN and AC-MFLVNS almost no of N-acetyl peptides appears to be largely

terminated

amino

RESEARCH COMMUNICATIONS

generated

, might

activity

by the nature peptides

sizes

activity,

The chemotactic

active

AND BIOPHYSICAL

molecular

and calpain

showed

activity.

BIOCHEMICAL

and

Met at the N-

C-terminus

may be a

in bacteria

and higher

organisms. The optimal required that

of the

suggesting its

concentrations

to give

standard that

local

CDCF is

To determine gradient)

concentrations MFLVNSFLK

is

Table

dependent

1.

(l-

Checkerboard

53 166 375 483

5 53.1* + 102.6 f 103.7 t 130.4

(CDCF) higher

(f-MLF)

than

(Fig.

migrations

of f-MLF.

l), unless

One would

factors

taking

expect

into

CDCF (AC-MFLVNSFLK) depends

cell

locomotion

"checkerboard

wells.

is

Neutrophil

that

analysis"

50 )IM) was examined. migration

* f f f

14.4 69.6

92.1 142.1

*Migrated cell number per 5 fields i standard by duplicate determinations. Human neutrophils, were added to upper wells with or without incubated at 37°C for 30 min in 5% CO2.

1245

44.5 79.8 82.2 129.0

deviation

1.0 AC-MFLVNSFLK

increasing

induced

In addition,

76 f 133 f 297 i 401 *

is

in each

with

by Acsignificant

for neutrophil

AC-MFLVNSFLK in upper well (Of) 10 1 53 160 347 450

the

of various

As shown

observed migration

gradient.

is

on a concentration

analysis of AC-MFLVNSFLK peptide chemotactic activity

0

(GO

for that

random

on a concentration

AC-MFLVNSFLK in lower well

0 1 10 50

but

gradient),

lower

that

chemotactic

migration

1, increasing

of CDCF in

origin neutrophil

than

movement

(activated

of Table

factors

considerably

defence. cell

of AC-MFLVNSFLK

column

higher

neutrophil

(directed

of a concentration

concentrations

of bacterial

to foreign

of self

or chemokinesis

independent vertical

10 times

whether

of chemotaxis

chemotactic were

in stimulating

sensitive

mechanisms

derived migration

factor

ineffective is

to be more

consideration result

chemotactic

concentration

neutrophils

of calpain

the maximum neutrophil

50 66 110 174 265

+ + + ?

37.4 61.4 50.8 73.7

of 7 subjects x lo5 cells/50 peptide and

~1,

Vol.

migration

is

and 50 PM),

seen along suggesting

Calpains three

(Fig.

2) (3). 2 is

factors

calpains

mammalian This

multifunctional

of domain

chemotactic

scalds,

and above

I and II

cells

are

may account bruises

are

and their injured, for

calcium

binding

intracellular small

proteases are

could

aseptically

subunits

consist

membrane

respectively,

present

study, of domain ubiquitously

common to both

be a direct induced

(10

response.

a glycine-rich

function

subunits

any of these

small

domain,

In the

to be another

inflammation

and chemical

and their

1 and 3 are

unknown.

CDCF concentrations

as a chemokinetic

Domains

still

RESEARCH COMMUNICATIONS

at higher

as well proteins

was found

tissues,

the diagonal

a chemotactic

and a calmodulin-like

function

tissue

are

domains

domain

Since

BJOCHEMICAL AND BIOPHYSICAL

169, No. 3, 1990

target by cell

binding

while the

the

release

1 (Fig.

of

of 2).

distributed calpains, for damage,

in when

neutrophils. as seen

in

injuries.

ACKNOWLEDGMENTS This research was supported by grants-from and Culture of Japan (RG 62570138 and 01570165).

Ministry

of Education,

Science

REFERENCES 1. Murachi, T. (1983) Trends Biochem. Sci. 8, 167-169. 2. Pontremoli, S., and Melloni, E. (1986) Ann. Rev. Biochem., Annual Reviews 55, 455-481. Inc., 3. SuzukirK., Ohno, S., Emori, Y., Imajoh, S., and Kawasaki, H. (1987) Prog. Clin. Biochem. Med. Springer-Verlag Berlin Heidelberg, 5, 43-65. 4. Suzuki, K., Ohno, S., Imajoh, S., Emori, Y., and Kawasaki, H. (1985) Biomed. Res. 6, 323-327. 5. Murachi, T. (1989) Biochem. Int. 18, 263-294. 6. Kitahara, A., Ohtsuki, H., Kirihata, Y., Yamagata, Y., Takano, E., Kannagi, R ., and Murachi, T. (1985) FEBS Lett. l&, 120-124. 7. Takano, E., Park, Y., Kitahara, A., Yamagata, Y., Kannagi, R., and Murachi, T. (1988) Biochem. Int. 16, 391-395. 8. Miyake, S., Emori, Y., az Suzuki, K. (1986) Nucleic Acids Res. z, 88058817. 9. Ohno, S., Emori, Y., and Suzuki, K. (1986) Nucleic Acid Res. 14, 5559. 10. Sakihama, T., Kakidani, H., Zenita, K., Yumoto, N., Kikuchi, T., Sasaki, T., Kannagi, R., Nakanishi, S., Ohmori, M., Takio, K., Titani, K., and Murachi, T. (1985) Proc. Natl. Acad. Sci. USA. 82, 6075-6079. 11. Emori, Y., Kawasaki, H., Imajoh,S., Kawashima, S., and Suzuki, K. (1986) J. Biol. Chem. 261, 9472-9476. 12. McClelland, P., Lash, J. A., and Hathaway, D. R. (1989) J. Biol. Chem. 264, 17428-17431. 13. Kunimatsu, M., Higashiyama, S., Sato, K., Ohkubo, I and Sasaki, M. (1989) Biochem. Biophys. Res. Comm. 164, 875-882. 14. Aoki, K., Imajoh, S., Ohno, STEmori, Y., Koike, M., Kosaki, G., and Suzuki, K. (1986) FEBS Lett. 205, 313-317. 15. Showell, H. J., Richard, J. F.,Zigmond, S. H., Schiffman, E., Aswanikumar, S ., Corcoran, B., and Becker, E. L. (1976) J. Exp. Med. 143, 1154-1169. 16. Marasco, W. A., Phan, S. H., Krutzsch, H., Showell, H. J., Feltner, D. E., Nairn, R., Becker, E. L., and Ward, P. A. (1984) J. Biol. Chem. 259, 54305439. 17. Freer R. J., Day, A. R., Schiffman, E., Aswanikumar, S., Showell, H. J., and Becker, E. L. (1980) Biochemistry 2, 2404-2410. 1246

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BIOCHEMICAL

AND BIOPHYSICAL

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18. Marasco, W. A., Fantone, J. C., Freer, R. J. and Ward, P. A. (1983) Am. J. Pathol. 111, 273-281. 19. Toniolo, C., Bonora, G. M., Showell, H., Freer, R. J., and Becker, E. L. (1984) Biochemistry 23, 698-704. 20. Rot, A. Henderson, LFE., Copeland, T. D., and Leonard, E. J. (1987) Proc. Natl. Acad. Sci. USA 84, 7967-7971. 21. Boyum, A. (1968) Stand. J. Clin. Lab. Invest. 1, 77-89. 22. Falk, W., Goodwin, R. H., and Leonard, E. J. (1980) J. Immunol. Meth. 33, 239-247. 23. Harvath, L., Falk, W., and Leonard, E. J. (1980) J. Immunol. Meth. 22, 239247. 24. Hudson, D. (1988) J. Org. Chem. 53, 617-624. 25. Sasaki, T., Kikuchi, T., Yumoto,3., Yoshimura, N., and Murachi, T. (1984) 3. Biol. Chem. 259, 12489-12494.

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