Vol. 171, No. 1, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages

August 31, 1990

Molecular

Evolution

and

Zinc

of Leukotriene Hiroyuki

Toh’,

Minami’

Engineering

6-f-3

Furuedai,

2Department

Suita,

Shimizuz

Institute,

Osaka 565 Japan chemistry

of Medicine,

Motif

*

and Takao

Research

of Physiological

Faculty

Binding

A4 Hydrolase

Michiko

‘Protein

Ion

216-221

University

and Nutrition, of Tokyo

Tokyo 113 Japan Received

July

Summary In order

9,

: Leukotriene

to investigate

hydrolase,

In agreement

Leukotrienes

(LTs)

is synthesized

arachiclonic

is an enzyme chemotactic

(1, 2,4,5).

LTA4

(6-8).

placental

libraries,

and the primary

of the enzyme A weak

to investigate

of LTAd

hydrolase

was reported evolution

deduced

0006-291XE’O

between

LTA4

and function

of LTA4 sequence

$1.50 Press, any

form

Inc. reserved.

in LTAd

216

have various

of 5-lipoxygenase

hydrolase. by atomic

physiological

moiety

via LTA4,

(3,4). of LTA4

and closeiy with

a molecular

was cloned

from

human

activity

hydrolase hydrolase, was aligned

(9,lO).

Furthermore, cob

and aminopeptidases the amino

from the Ministry

to the weight

spleen

in Escheriachia

with

to yield

related

enzyme

was determined

a full enzyme

author.

Copyright 8 1990 by Academic All rights of reproduction in

which

of granulocytes

* This work was supported in part by a grant-in-aid Education, Science and Culture of Japan. ’ Corresponding

in zinc binding

as determined

an epoxide

hydrolase

structure

from the cDNA

involved

tree was

k in d s of LTs are synthesized

All

compound

with

of LTAd

Inc.

hydrolyzes

cDNA s of LTA4

was expressed

homology

In order

is a zinc protein

hyd ro 1ase is a cytosolic

- 70,000

significance

N were conserved

acid by the action which

N family.

and a phylogenetic

that three residues

Press,

(1,2).

to the aminopeptidase

were aligned

a class of eicosanoids

functions

is a potent

system

Academic

of 68,000 cDNA

and physiological

LTAdhydrolase

constitute

from

hydrolase

LTBl

evolution

it was found

0 1990

and pathological

immune

belongs

to the family

theobservation,

spectroscopy.

LTAl

hydrolase

of the active sites of aminopeptidases

with

absorption

LTB4.

belonging

From the alignment,

and one residue

activities

A4 (LTA,)

the molecular

the enzymes

constructed.

which

1990

and the (11). (12).

acid sequence

those of aminopepof

Vol.

171, No. 1, 1990

tidases.

According

was found

BIOCHEMICAL

to the alignment,

that the purified

Materials

and

AND BIOPHYSICAL

a phylogenetic

preparation

RESEARCH COMMUNICATIONS

tree was constructed.

of LTAa

hydrolase

contains

Furthermore,

it

a zinc ion.

Methods

LTAh hydrolase was subjected to the computational analysis with the method of sequence comparison of protein primary structures. For the pairwise alignment, a program for weak homology detection was used (13). The sequence data were taken from a protein primary structure data base, PRF (re1.89.9) (14). A multiple alignment was constructed, according to the pairwise alignment made by the homology detection program. The significance of the detected sequence homology was checked by statistical test with 100 randomized sequence pairs (15, 16). A ccording to the alignment, a phylogenetic tree was constructed by the neighbor-joining method (17). The references used in this study were as follows ; ref.9 and ref.10 for human LTA4 hydrolase, ref.18 for human aminopeptidase N, ref.19 for rabbit aminopeptidase N, ref.12 for rat aminopeptidase M, ref.20 for E.coli aminopeptidase N, ref.21 for Bacillus stearothermophilus thermolysin? ref.22 for Bacillus thermoproteolyticus thermolysin, ref.23 for Bacillus subtilis thermolysm, ref.24 for Bacillus amyloliquefaciens thermolysin, ref.25 for Bacillus cereus neutral proteinase, ref.26 for Paeudomanas aeroguinosa elastase, ref.27 for rabbit endopeptidase, ref.28 for rat endopeptidase, ref.29 for rabbit collagenase, ref.30 for human collagenase, human stromelysin and rat stromelysin, ref.31 for human collagenase IV, ref.32 for rat transin 2, ref.33 for human stromelysin 2, and ref.34 for rabbit proactivator. LTAa hydrolase was obtained as described previously (11). The zinc content of LTAd hydrolase was determined with a Hitachi atomic absorption spectrometer model Z-6100. Results

and

From

Discussion

computer

is a member different

assisted

sequence

of aminopeptidase

frorn

comparison,

N family,

evolution

of these enzymes of these enzymes.

from 106 to 326 residue)

and their

was aligned

with

long ( from 180 to 418 residue

residues

long, ( from 1 to 239 residue

( from

180 to 417 residue

75 to 327 residue

).

N has not been determined The C-terminal

significance

and the normalized of LTA4

N-terminal

yet.

Fig.

rabbit

aminopeptidase

M is 7.70 S.D., S.D. To determine

further,

181 - 264 of the alignment)

acid long (

intestine

aminopeptidase

N of 791

aminopeptidase

M of 964 residues

N of 870 residues

of the the rabbit

long

aminopeptidase

of the homologous than the N-terminal

region. half.

The

to be involved in the LTAd hydrolase activity is present 119 in the alignment ) (9, 10). In order to check the the alignment

aminopeptidase

that

the amino

of 611 residues

N of 967

are more conservative

was subjected

of LTAd

the alignment of the pair

to the statistical

The normalized

hydrolase

hydrolase

and E.coli

of the C-terminal of LTAl 217

hydrolase

test

score for the pair

N is 9.45 S.D., that of LTAd

N is 8.29 S.D., and that of LTAd

although

the molecular

aminopeptidase

the alignment

scores were calculated.

and human

is quite

intestine

aminopeptidase

sequence

1 shows

homology,

alignment

hydrolase

), that of rabbit

hydrolase

hydrolase

we compared hydrolase

), that of rat kidney

half of the alignment

of the detected

to investigate

that of human

The

Cys residue which is considered in the aligned region ( position

In order

of LTAa

) and that of E.coli

LTA,

of LTAh

functions,

a region

that

the function

(12).

enzymatic

As a result,

residues

long ( from

although

those of the aminopeptidases

sequences

it was suggested

hydrolase

and

and rat aminopeptidase aminopeptidase

consevative and E.coli

region

N is 2.86 (position

aminopeptidase

N

Vol. 1 LIA ANH AHR AHR ANE

121 lid ANH ANR AM8 ANE

241 LTA ANH ANR AMR AXE

171, No. 1, 1990

.. .. ... .. t ._......, +

.

BIOCHEMICAL

AND BIOPHYSICAL

t .. .. .. .. .t. . .. .. .. .+

+

RESEARCH COMMUNICATIONS

+ .. .. ... +

.t . . ..._. +

S-FETSPRSSALQ-WtTPEQTSCKEHPYt-FSQCQ--AlHCRAlLPCQD-TPS--------------VKLTYTAEVSVPK-SLVAL~SAl-RDG-KTPDPEDPSRKlYKFlQKVP-fPCY SEFECEL-ADDlACFYRSEYnEGIYRRYYbTTQ~Q--AADARKSFPCFD-EPA--------------~KAEF~~TL~HPK-DtTAL-SNUlPKGPSTPLP~DPNWNVTEFHTT-PK~STY -QFQGEL-ADDLACFYRSEYWECnVRKVVATTQ~Q~QAADARKSFPCFD-~PA--------------SKATFN~TllHPR-DYTAl-SNHLPR-SSTALPEDPNWTVTEFHTT-PK~STY SEFQGEL-ADDLAGFYRSEYREGGNKKVVATTQHQ--AADARKSFPCFD-EPA--------------~KASFNlTt~HPN-NLTAL-SN~LPK-DSRTlQEDPSWNVTEFHPT-PK~STY KEKEGALVISN1PE--RFTLKlINEISPAANTALE--G1YQSGDAlCTQCE-AEGFRHITYY~DRPDVtARFTTKiIADKIKYPFtLSNCNRVA-QGEL&NCRHW-V-QWQDPFPK-PCY 00 * 0 0 0 *o 0 0 0 * 8 0

+

+

00

IO.

.. . ...__+.. . .. . +. .. .. .. .. +. ... .. .. . +.. . .. .. .. +. ... .. ... +. .. .. .. .+..,.._... +. .. .._.. +.__.._... +_..,._.,. + . .._.. + tlALVVGAlES-RQI-----GPRTLYWSEKEQV~KSAYE---FSKT---ES~lKlAEDlGCPYVHGQYDtLVtPPSFPYGG~SNPCl-TFVTPTLLA-GD------KS-tSNVlAH~lSH 1LAFIVSEFDYV-EK-QASNGVLlRiWARPSAiAAGHGD---YAlNVTGPllNFFAGHYDTPYPlPKSDQ~GLPD-FNACA~ENWGLVTYRENSLLFDPLSSSSSNK~RVVTVlAHElAH 1LAYIVSEFTNl-EA-QSPNNVQlRlWARPSAlSEGHGQ---YALNVTGPlLNFFANHYNTPYPLEKSDQlGlPD-FNAGA~ENWGlVTYRESALlFDPlVSS~SNKKRVVTVVAHELAH tLAYIVSEFKYV-EA-VSPNRVQlRlWARPSAlDEGHGD---YALQVTGPlLNFFAQHYNT~YPLEKSDQlAtPD-FNAGAMENWGtVTYRESALVFDPQSSSlSNKERVVTVlAH~lAH LFALVACDFDVtRDTFTTRSG---REVAlElYVDRGNLDRAPWAUTStKNSHKWDEERFGLEYDlDlYMlVAVDF-FN~GANENKGLNlFNSKYVLARTDTATDKDYLDlERVlGHEYFH 0 000 00 0 I 1.00 0 0 0 8 to.., * 0 00 0

0

,oo*,

,........ +.. .. ... .. +. .. .. . SWTGNLVTNKTWDHFWtNECHTVYlK QWFGNLVTIEYYNDLWLNEGFASYVE QWFCNLVTVDWWNDLWLNECFASYVE QWFGNLVTVDWWNDLWLNEGFASYVE NWTGNRVTCPDWPQLSLKECLTVFRD , ,L 0 0 0 * a. 8. *

Fig. 1 Alignment of LTA4 bydrolase and amiuopept~idase N derived from buman and E.coli. Opeu circle indicates the site occupied by chemically similar residues and closed circle indicates the site occupied by identical residues. Abbreviated name of each enzyme is written on the left side of the aligned sequence. The abbreviations are as follows, LTA : llurnau LTAd hydrolase, ANH ; humau aminopeptidase N, ANR ; rabbit aminopeptidase N, AMR ; rat amiuopeptidase M, ANE ; E.coli aminopeptidase N.

was

subjected

hydrolase that

the

evolutionarily

to the and

detected

statistical

test.

E.coli aminopeptidase sequence related

to

normalized

N is 5.66

homology the

The

S.D.

is statistically

aminopeptidase

alignment

N,

These

results

significant, at least,

score of the that

at the

of this

is,

homologous

z* z VIAHEISHSW

(14 residues)

Z LNEGH

VIAIIELAHQW VVAHELAHQW VIAHELAHQW VIGHEYFHNW

(14 (14 (14 (14

residues) residues) residues) residues)

LNEGF LNEGF LNEGF LKEGL

thermolysin G.stearothermophilus theraolysin B.thermoproteolyticus thernolysin B.subtilis thernolysin B.anyloliquefaciens neutral proteinase B.cereus elsstase P.aerguinosa

VVAHELTHAV VVAIIELTHAV VTAHEHTIIGV VTAHEf4THGV VIGHELTHAV VAAHEVSHGF

(15 (15 (15 (15 (15 (15

residues) residues) residues) residues) residues) residues)

INEAI INEAI LNESF LNESF LNEAI HNEAF

endopcptidase endopeptidase

rabbit rat

VIGHElTHGF VIGHEITHGF

collagenase collagenase collagenase traosin 2 stromelysin stromelysin stromelysin proactivator

rabbit human IV human rat hunan 2 human rat rabbit

VAAHELGHSL VAAHELGHSL VAAHEFGILAM VAAHELGHSL VAAHEIGIISL VAAHELGHSL VAAllELCllSL VAAlfELCHSL

ieukotriene aninopeptidase aminopeptidase aminopeptidase aminopeptidase

A.q hydrolase N N M N

human rabbit rat E.coli

region

calculation LTA4

LTAl suggest

hydrolase region.

2 Alignment of the motif region. Fig. ‘Z’ indicates the site, which . . ._ . . is cousdered to act as ligaud of ciuc ion. ‘*’ indicates the site, which is considered to be active site for peptidase activity. R. e f erences of the sequence data are listed under ‘Materials and Methods’. Numbers in the parenthesis indicate the leugths of the regious conuecting two motifs.

218

is

*

Vol.

171, No. 1, 1990

BIOCHEMICAL

In the aminopeptidase alignment region

N, a highly

) is considered contains

to constitute

a sequence

of zinc-metalloproteinases aminopeptida.se motif Two

(12,18-34).

Among

of the motif.

N (12,18-20)

and LTAd

in the other

By atomic Furthermore,

suggests absorption

that

procedures

LTAd

and quantitative

are on going

to elucidate

whether

the inherited

structural

homology

of peptidase(s)

is contaminated

immunobistochemical

zyme is rich in the epithelial hydrolase

may explain,

gastrointestinal with

tract.

the experimental

the purified

of LTA4

residue

contains

activity.

More

elsewhere. of LTAJ

possible

a zinc ion.

hydrolase

of sequence

aminopeptidase

hydrolase that

Phylogenetic

tree

(36).

significance

analysis

activity

of the enzyme

described

N ( rabbit

M ( rat

LTA4

( human

hydrolase

219

is due to

that

The peptidase

N ( human

of aminopeptidase

studies

a small

has demonstrated

aminopeptidase

minopeptidase

detailed

Further

amount

kidney

)

1

intestine

N ( E.coli

N family

intestine

spleen

)

)

)

and

LTA,

the enof LTAl in the

above are consistent

investigations.

-

3

hydrolase.

enzyme.

aminopeptidase

Fig.

(see Fig.1 and Fig.2).

hydrolase

It is still

in

has not been identified

in the LTAh

activity

in

In the thermolysin,

in aminopeptidases

LTA4

the physiological

-

Glu

is conserved

has a peptidase

cells of small intestine the results

extensively.

and a Glu residue

data will be published

in the purified

the sequence

motif

and collagenase

of the enzyme.

at least partly, Thus,

hydrolase.

corresponding

or not the peptidase

study

all contain

in the sequence

hydrolase

motif

(35) ( see Fig.2 ). As shown

a zinc ion may be present

spectroscopy,

sequence

the

of zinc ion, and an invariant

of peptides

endopeptidase

because

has been characterized

Glu residue

hyd ro 1ase, although

the recombinant

experimental

The

thermolysin

Corresponding

(15,16),

classes of zinc-metalloproteinases,

is also found in the LTAl

two classes of proteinase,

observation

domain

231 - 258 of the

is a ubiquitous

act as ligands

sites for hydrolysis motif

( position

and collagenase,

that a zinc ion binds two His residues

the downstream

These

Th ere are four

in the motif

Fig.1 and Fig..2, the sequence

region

which

endopeptidase,

RESEARCH COMMUNICATIONS

protease

VXXHEXXH,

these proteinases,

His residues

is one of the active

it is known

conservative a functional

(see Fig.2).

N, thermolysin,

invariant

residue

motif,

AND BIOPHYSICAL

hydrolase.

Vol.

BIOCHEMICAL

171, No. 1, 1990

A phylogenetic

tree was constructed,

tree shows the early

divergence

the investigations

means

that

function.

The

suggests activity

that

sequence LTAd

and peptidase

by gene sharing,

Acknowledgment

hydrolase

RESEARCH COMMUNICATIONS

to the alignment

( see Fig.3

from aminopeptidase

family,

Last year, a novel evolutionary

of crystallins,

a gene acquires

according

of LTAd

the age has not been determined. from

AND BIOPHYSICAL

which

a novel

homology,

function together

is called

gene sharing

without

duplication

with

hydrolase

have

two

activity.

LTA4

hydrolase

strategy

different

(37).

although

was proposed Gene

sharing

and keeps its original

the recent

experimental

enzymatic

activities,

may have evolved

). The

investigation, LTAd

hydrolase

from aminopeptidase

N

or vice versa.

.

We thank

Dr.Yoshihiro

Urade

for stimulating

discussion

advice.

References 1 Samuelsson,B., Dahldn,S.-E., Lindgren, J.A.., Rouzer,C.A. and Serhan,C.N. (1987) Science, 237, 1171-1176. 2 Shimizu,T. and Wolfe,L.S. (1990) J. Neurochem. 55, l-15. 3 Samuelsson,B. and Funk,C.D. (1989) J.Biol.Chent. 264, 19469-19472. 4 Shimizu,T. (1988) Int.J.Biochem. 20, 661-666. 5 Needleman,P., Turk,J., Jakscih,B.A., Morrison,A.R. and Lefkowith,J.B. (1986) Annu. Rev. Biochem. 55, 69-102. 6 Ridmark,O., Shimizu,T., and J%nvall,H. and Samuelsson,B. (1984) J. Biol. Chem. 259, 12339-12345. 7 Evans,J.F., Dupuis,P. and Ford-Hutchinson,A.W. (1985) Biochim. Biophys. Acta 840, 43-50. 8 Bito,H., Ohishi,N., Miki,I., Minami,M., Tanabe,T., Shimizu,T. and Seyama,Y. (1989) J.Biochem. 105, 261-264. 9 Funk,C.D., RQdmark,O., Fu,J.Y., Matsumoto,T., J&nvall,H., Shimizu,T. and Samuelsson,B. (1987) Proc. Natl. Acad. Sci. USA 84,6677-6681. 10 Minami,M., Ohno,S., Kawasaki,H., Ridmark,O., Samuelsson,B., Jiirnvall,H., Shimizu,T., Seyama,Y. and Suzuki,K. (1987) J. Biol. Chem. 202, 13873-13876. 11 Minami,M., Minami,Y., Emori,Y., Kawasaki,H., Ohno,S., Suzuki,K., Ohishi,N., Shimizu,T. and Seyama,Y. (1988) FEBS Lett. 229, 279-282. 12 Malfroy,B., Kado-Fong,H., Gros,C., Giros,B., Schwartz,J.-C. and Hellmis,R. (1989) Biochem.Biophys.Res.Commun. 161, 236-241. 13 Nishikawa K., Nakashima H., Kanehisa M. and Ooi T. (1987) Protein Sequences and Data Analysis 1, 107-116. 14 Seto,Y., Ihara,S., Kohtsuki,S., Ooi,T. and Sakakibara,S. (1988) In: Lesk AM (ed) Computational Molecular Biology ~~27-37 Oxford University Press, Oxford. 15 Scwartz,R.M. and Dayhoff,M.O. (1978) Matrices for Detecting Distant Relationships in Atlas of Protein Sequence and Structure Vol.5 Suppl.3 M.O.Dayhoff Ed., ~~353-358. The National Biomedical Research Foundation. 16 Toh,H., Hayashida,H. and Miyata,T. (1983) Nature 305, 827-829. 17 Saitou,N. and Nei,M. (1987) Mol.Biol.Evol. 4, 496-425, 18 Olsen, J ., Cowell,G .M., Kflnigsh0fer,E., Danielsen,E.M., Meller, J., Laustsen,L., Hansen,O.C., Welinder,K.G., Engberg,J., Hunziker,W., Spiess,M., Sjcstram,H. and Norkn,O. (1988) FEBS Lett. 238, 307-314. 19 Nor&O., Dabelsteen,E., [email protected],P.E., Olsen,J., Sj&trijm,H., Hansen,G.H. (1989) FEBS Lett. 259, 107-112. 220

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Bally,M.., Foglino,M., Bruschi,M., Murgier,M. and Lazdunski,A. (1986) Eur.J.Biochem. 155, 565-569. Takagi,M., Imanaka,T. and Aiba,S. (1985) J.Bocteriol. 163, 824-831. Titan&K., Hermodson,M.A., Ericsson,L.H., Walsh,K.A. and Neurath,H. (1972) Nature New Biol. 238, 35-37. Yang, M.Y., Ferrari,E. and Henner,D.J. (1984) J.Bacteriol. 160, 15-21. Vasantha,N., Thompson,L.D., Rhodes,C., Banner,C., Nagle,J. and Filpula,D. (1984) J.Bacteriol. 150, 811-819. Sidler,W., Niederer,E., Suter,F. and Zuber,H. (1986) Biol. Chem.Hoppe-Seyler 30’7, 643-657. Fukushima,J., Yamamoto,S., Morihara,K., Atsumi,Y., Takeuchi,H., Kawamoto,S. and Okuda,K. (1989) J.Bacteriol. 1’71, 1698-1704. Devault,A., Lazure,C., Nault,C., Le Moual,H., Seidah,N.G., Chretien,M., Kahn,P., Powell,J., Mallet,J., Beaumont,A., Roques,B.P., Crine,P. and Boileau,G. (1987) EMBO J. 0, 1317-1322. MaIfroy,B., Schofield,P.R., Kuang,W.J., Seeburg,P.H., Mason,A.J. and Henze1,W.J. (1987) B’ tot h em.Biophys.Res. Commun. 144, 59-66. Fini,M.E., Plucinska,I.M., Mayer,A.S., Gross,R.H. and Brinckerhoff,C.E. (1987) Biochemistry 26, 6156-6165. Whitham,S.E., Murphy,G., Angel,P., Rahmsdorf,H.J., Smith,B.J., Lyons,A., Harris,T.J.R., Reynolds,J.J., Herrlich,P. and Docherty,A.J.P. (1986) Biochem. J. 240, 913-916. Collier,I.E., Wilhelm,S.M., Eisen,A.Z., Marmer,B.L., Grant,G.A., Seltzer,J.L., Kronberger,A., He,C., Bauer,E.A. and Goldberg,G.I. (1988) J.BioJ.Chem. 203, 6579-6587. Breathnach,R., Matrisian,L.M., Gesnel,M.C., Staub,A. and Leroy,P. (1987) Nucleic Acids Res. 15, 1139-1151. Muller,D., Quantin,B., Gesnel,M.C., Millon-Collard,R., Abecassis,J. and Breathnach,R. (1988) Bi0chem.J. 253,187-192. Fini,M.E., Karmilowicz,M.J., Ruby,P.L., Beeman,A.M., Borges,K.A. and Brinckerhoff,C.E. (1987) Arthritis Rheum. 30, 1254-1264. Matthews,B.W., Jansonius,J.N., Colman,P.M., Schoenborn,B.P. and Dupourque,D. (1972) Nature New Biol. 238,37-41. Ohishi,N., Minami,M., Kobayashi,J., Seyama,Y., Hata,J., Yotsumoto,H., Takaku,F. and Shimizu,T. (1990) J.Biol.Chem. 205, 7520-7525. Piatigorsky,J. and Wistow,G.J. (1989) Cell 57, 197-199.

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AND BIOPHYSICAL

221

RESEARCH COMMUNICATIONS

Molecular evolution and zinc ion binding motif of leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase belongs to the aminopeptidase N family. In order to investigate the molecular evolution and physiological significance...
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