Báo cáo khoa học: Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus docx

Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit.. The tyrosinase

Purification, characterization and molecular cloning of tyrosinase

Tetsushi Naraoka1,2, Hidemitsu Uchisawa1, Haruhide Mori2, Hajime Matsue3, Seiya Chiba2

and Atsuo Kimura2

1

Aomori Industrial Research Center, Aomori;2Division of Applied Bioscience, Graduate School of Agriculture,

Hokkaido University, Sapporo;3Aomori Universityof Health and Welfare, Aomori, Japan

Tyrosinase (monophenol,L-DOPA:oxygen oxidoreductase)

was isolated from the ink of the squid, Illex argentinus

Squid tyrosinase, termed ST94, was found to occur as a

covalently linked homodimeric protein with a molecular

mass of 140.2 kDa containing two copper atoms per a

subunit The tyrosinase activity of ST94 was enhanced by

proteolysis with trypsin to form a protein, termed ST94t,

with a molecular mass of 127.6 kDa The amino acid

sequence of the subunit was deduced from N-terminal amino

acid sequencing and cDNA cloning, indicating that the

subunit of ST94 is synthesized as a premature protein with

625 amino acid residues and an 18-residue signal sequence

region is eliminated to form the mature subunit comprised of

607 amino acid residues with a deduced molecular mass of

68 993 Da ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme

Keywords: Illex argentinus; tyrosinase; copper protein; mel-anogenesis; cephalopod

Tyrosinase (monophenol,L-DOPA:oxygen oxidoreductase)

is one of the copper-containing phenoloxidases that are

widely distributed in nature The enzyme is known to be a

key enzyme in the melanogenic pathway that catalyzes the

initial rate-determining reaction, the oxygenation of

mono-phenols to o-dimono-phenols (monophenolase activity), as well as

the oxidation of o-diphenols to corresponding o-quinones

(o-diphenolase activity)[1,2] Type 3 copper proteins,

including tyrosinases, arthropod phenoloxidases and

hemo-cyanins, have been isolated from many organisms The

evolutional relationships of the structures have also been

elucidated on the basis of the amino acid sequences

conserved around two copper-binding sites that form an

oxygen-binding active center [3–6] These proteins are classified into a superfamily and are of interest from the viewpoint of their molecular evolution [7–9]

These copper proteins are particularly important in arthropods Arthropod phenoloxidases [10–13], which are given the same EC number as tyrosinase, are known to be involved in the host defense system termed the prophenol-oxidase cascade as a terminally active molecule in the system [14–17] Hemocyanins are macromolecules that function

as oxygen carriers in the hemolymph of arthropods and mollusks [7] Hemocyanins are also found to exhibit phenoloxidase activity [6,18,19], which is amplified after certain treatments such as proteolysis or exposure to detergents, or by interactions with specific proteins [6,8,20–22] This activation suggests that these hemocyanins may have roles as phenoloxidases in some important biological events

Among the mollusks, the emission of ink for defense against predators is a well-known characteristic behavior of most cephalopods, which indicates their high capacity for melanogenesis We reported previously that a fraction from the ink of the squid, Illex argentinus, in which the illexin-peptidoglycan (IPG)possessing a novel mucopoly-saccharide structure and tyrosinase were contained, showed anti-tumor activity against Meth A fibrosarcoma in BALB/

c mice [23–26] The anti-tumor activity was thought to be expressed through immunostimulation because the fraction had macrophage-stimulating activity [23] Consistent with these observations is the clinical use of hemocyanin from the keyhole limpet (a marine gastropod, Megathura crenulata)

as an immunotherapeutic agent for the treatment of bladder carcinoma [27], and other observations suggesting that

Correspondence to T Naraoka, Aomori Industrial Research Center,

4-11-6 Daini-tonyamachi, Aomori 030–0113, Japan.

Fax: + 81 17 7399613, Tel.: + 81 17 7399676,

E-mail: naraoka@aomori-tech.go.jp

Abbreviations: DHPPA, 3,4-dihydroxyphenylpropionic acid; DOPA,

3,4-dihydroxyphenylalanine; IPG, illexin-peptidoglycan; KLH,

keyhole limpet hemocyanin; MBTH, 3-methyl-2-benzothiazolinone

hydrazone; PPAE, prophenoloxidase-activating enzyme;

pro-PPAE, zymogen of PPAE; ST94, tyrosinase from Illex argentinus;

ST94t, proteolyte of ST94 with trypsin.

Enzymes: tyrosinase (EC 1.14.18.1); trypsin (EC 3.4.21.4).

Note: After submission and during the review of this article, the cDNA

sequence of tyrosinase from Sepia officinalis has been opened in

DDBJ/EMBL/GeneBank databases on 5 July 2003 (Accession no.

AJ297474)by Lieb, B., Erteld, D., Poli, A., Palumbo, A & Markl, J.

(Received 11 May 2003, revised 12 August 2003,

accepted 18 August 2003)

molluscan tyrosinases also are biologically and biomedically

significant Tyrosinase activity has been demonstrated in the

inks of some other cephalopods [28] Recently, there have

been substantial advances in elucidating the mechanism of

ink production in Sepia officinalis [29–33] For cephalopod

tyrosinases, however, only limited information is available

on their characteristics This seems to have been due to the

difficulty of isolating the tyrosinases that occur in ink, which

show extremely complex polymorphism (as observed at

least in ink of I argentinus [26]) In other mollusks, although

tyrosinases have been isolated from a bivalve [34] and a

gastropod [35], there have been no reports on their amino

acid sequences

In a previous paper, we reported a protein that occurred

in the ink of I argentinus with weak tyrosinase activity,

which migrated as a 94-kDa protein on polyacrylamide gel

electrophoresis under native condition [26] The protein,

termed ST94, was assumed to be a partially activated

tyrosinase, one of the abundant proteins in the ink In this

paper, we describe the purification, proteolytic activation,

some enzymatic properties and molecular cloning of the

squid tyrosinase ST94 This is the first report on the primary

structure of a molluscan tyrosinase (see Footnote on

p 4026), which contributes to evolutional studies on type

3 copper proteins

Materials and methods

Materials

L-3,4-Dihydroxyphenylalanine (L-DOPA),D

-3,4-dihydroxy-phenylalanine (D-DOPA), dopamine, pyrocatechol,L

-tyro-sine, D-tyrosine, tyramine, 3,4-dihydroxyphenylpropionic

acid (DHPPA), 3-methyl-2-benzothiazolinone hydrazone

(MBTH), kojic acid, arbutin, phenylthiourea, tropolone,

mushroom tyrosinase and keyhole limpet hemocyanin

(KLH)were supplied from Sigma (St Louis, MO, USA)

N-Tosyl-L-phenylalanine chloromethyl ketone

(TPCK)-treated bovine pancreatic trypsin was purchased from

Funakoshi Co Ltd (Tokyo, Japan) Phenyl-Sepharose

CL-4B, polyacrylamide gel plates for electrophoresis were

from Amersham Biosciences (Uppsala, Sweden) The other

chemicals were supplied by Wako Pure Chemical Industries,

Ltd (Osaka, Japan)

Analytical methods

PAGE under native conditions (native-PAGE)and

denaturing conditions (SDS/PAGE), and two-dimensional

PAGE (2D-PAGE)were performed using a PhastSystem

(Amersham Biosciences) For denaturing with a reducing

reagent, the protein was treated in the sample buffer

containing 2.5% (m/v)SDS, 5% (v)2-mercaptoethanol,

5% (v)glycerol and 62.5 mM Tris/HCl (pH 6.8)for

5 min at 95C For SDS/PAGE under nonreducing

conditions, the protein was treated using the sample

buffer from which 2-mercaptoethanol was omitted After

the run, the gel was stained for visualizing proteins with

Coomassie Brilliant Blue (CBB), or stained for detecting

of tyrosinase activity with 5 mM L-DOPA or 0.5 mM

L-tyrosine in 0.1 M sodium phosphate buffer (pH 6.8)at

room temperature

MALDI-TOF mass spectrometry experiments were per-formed on a Voyager-DE STR (Applied Biosystems, Foster City, CA, USA)according to the manufacturer’s instruc-tions Synapinic acid dissolved in a 2 : 1 mixture of 0.1% (by volume)aqueous trifluoroacetic acid and 0.1% (by volume) trifluoroacetic acid containing acetonitrile was used as a matrix for the analyses Spectrometry was performed in positive linear mode Bovine serum albumin (BSA)and horse heart myoglobin were used as mass number standards Amino acid sequences were analyzed by the gas-phase Edman degradation method using a protein sequencer PPSQ-10 (Shimadzu Corp., Kyoto, Japan), according to the manufacturer’s instructions

Protein content was determined by the method of Lowry

et al [36] or by measuring absorbance at 205 nm [37] using BSA as a standard Uronic acid, hexose and methylpentose were determined by the carbazole-sulfuric acid method [38], the phenol–sulfuric acid method [39] and the cysteine– sulfuric acid method [40], respectively

Copper analysis was performed using an atomic absorp-tion analyzer Z5010 with a graphite atomizer (Hitachi, Tokyo, Japan) Protein was dissolved in 1 mM sodium phosphate (pH 7.4), and analyzed by the standard addition method using commercially available copper standard solution The copper content of the buffer used was checked

in advance and found to be 0.1 ngÆmL)1 close to the detection limit

Purification of tyrosinase ST94 Ink sacs of I argentinus were homogenized with four volumes of acetone () 30 C)using a Waring blender, then filtered through a glass filter and the residue was dried

in vacuo The defatted powder (100 g)was extracted with

1 L of 10 mMsodium phosphate buffer (pH 7.4)at 4C for

12 h with stirring and then centrifuged (10 000 g, 30 min) to obtain the crude tyrosinase extract Ammonium sulfate concentration of the extract was brought to 30% saturation with solid ammonium sulfate and allowed to stand at 4C for 12 h The turbid solution was centrifuged (10 000 g,

30 min)and the supernatant was brought to 60% saturation concentration by addition of solid ammonium sulfate at

4C, and allowed to stand for 12 h The resulting precipi-tate was collected by centrifugation (10 000 g, 30 min) , dissolved in a small volume of 10 mM sodium phosphate (pH 7.4), and dialyzed against the same buffer (fraction AS60, 300 mL)

The fraction AS60 (100 mL)was added to 20 mL of 3M ammonium sulfate containing 10 mM sodium phosphate (pH 7.4)and applied to a column (2.6· 28 cm)of Phenyl-Sepharose CL-4B equilibrated with 0.5M ammonium sulfate in 10 mMsodium phosphate (pH 7.4) After washing with the same buffer, the column was eluted with a linear gradient of 0.5–0Mammonium sulfate in 10 mMsodium phosphate (pH 7.4)in a total volume of 1.5 L, and then further eluted with 10 mMsodium phosphate (pH 7.4)at a flow rate of 1 mLÆmin)1 Fractions of 10 mL were collected and analyzed for uronic acid using the carbazole–sulfuric acid method and for protein and pigment using the absorbance at 280 nm Tyrosinase activity was monitored

as follows A 5-lL sample was added to a microplate,

200 lL of 5 m -DOPA in 0.1 sodium phosphate buffer

(pH 6.8)was added to the plate, and the absorbance of the

mixture was measured using a Labsystems microplate

reader with a 492-nm filter after incubation at 25C for

10 min Fractions containing ST94 were detected by

native-PAGE (10–15% gradient gel), then concentrated and

desalted by ultrafiltration using YM10 membrane (Amicon,

Beverly, MA, USA) ST94 was purified further using

an anion-exchange column (4.6· 100 mm)of Poros

HQ/M (Applied Biosystems)with a BioCAD 700E HPLC

system (Applied Biosystems) For the elution (flow rate

10 mLÆ min)1), a linear gradient of NaCl, from 0 to 1.0M

over 16.6 min in 50 mM Tris/HCl (pH 7.0)was applied

(fractions of 2 mL) ST94 was detected by native-PAGE

(10–15% gradient gel) Fractions containing ST94 (eluting

at 0.27M NaCl)were concentrated and desalted by

ultrafiltration as described above, then recovered as a

solution of 1 mMsodium phosphate (pH 7.4)

Trypsin-treatment of ST94

ST94 dissolved in 10 mMTris/HCl buffer (pH 8.0)at a final

concentration of 250 lgÆmL)1 was treated with

TPCK-treated trypsin (2.5 lgÆmL)1) for 2 h at 25C The resulting

proteolyte of ST94, termed ST94t, was purified by gel

permeation HPLC Gel permeation HPLC was performed

with a L7100S HPLC system (Hitachi)using a column of

G3000SWXL(7.8 mm· 300 mm; TOSOH, Tokyo, Japan)

equilibrated with 0.2MNaCl in 0.1M sodium phosphate

(pH 7.0)(flow rate 0.5 mLÆmin)1) Fractions containing

ST94t were concentrated and desalted by ultrafiltration as

described above, then recovered as a solution of 1 mM

sodium phosphate (pH 7.4)

Assay of tyrosinase activity

Tyrosinase activity was assayed by the dopachrome method

[41] as follows The standard reaction mixture contained

5 mM L-DOPA, 0.1M sodium phosphate buffer (pH 6.8)

and the enzyme solution in a total volume of 3 mL The

reaction took place in a cuvette with a path length of 1 cm

and the absorbance at 475 nm was monitored continuously

using a spectrophotometer U-3210 (Hitachi)at 25C One

unit of tyrosinase was defined as the amount of enzyme

required to oxidize 1 lmol ofL-DOPA per min under the

above conditions, which was calculated using the molar

extinction coefficient of dopachrome (3600M )1Æcm)1)

The substrate specificity of tyrosinase was analyzed by the

MBTH method [42,43] The assay was carried out in 3 mL

of reaction mixture containing an appropriate

concentra-tion of substrate, 5 mM MBTH, 2% (by volume)

N,N-dimethylformamide and the enzyme solution in 50 mM

sodium phosphate buffer (pH 6.8) In the analyses for

monophenols, the corresponding o-diphenol at a final

concentration of 1 lMwas added to the reaction mixture

to shorten the lag period Formation of the MBTH-adduct

of o-quinone was followed at the isosbestic point wavelength

of each MBTH-adduct at 25C, and the steady-state rate

of oxidation of substrate was determined using the molar

extinction coefficient of MBTH-adduct at the isosbestic

point wavelength taken from the literature [43] The Kmand

Vmaxvalues for different substrates were obtained from the

Hanes–Woolf equation

Extraction of RNA and first strand cDNA preparation All DNA and RNA manipulations were carried out by standard techniques except where otherwise noted [44] PCR experiments were performed using a GeneAmp PCR System 9700 (Applied Biosystems) Poly(A)+ RNA was extracted from an ink sac (1.5 g) of I argentinus using a QuickPrep mRNA purification kit (Amersham Biosci-ences) A portion of the homogenate corresponding to

500 mg of ink sac was applied to an oligo(dT)-cellulose spun column Poly(A)+RNA eluted from a column was ethanol-precipitated, and redissolved in 50 lL of water First strand cDNAs for 5¢- and 3¢-RACE were prepared from the ink sac poly(A)+RNA using a SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions For each cDNA preparation, 0.46 lg of poly(A)+RNA was used After reverse-transcription, reaction mixtures (10 lL)were diluted by addition of 20 lL of 1 mMEDTA containing

10 mMTricine/KOH buffer (pH 8.5), heated at 72C for

7 min, then cooled on ice and used for further experiments Cloning and sequencing of tyrosinase ST94 cDNA Degenerate RT-PCR was carried out to detect ST94 cDNA using the first strand cDNA for 5¢-RACE as a template with sense and antisense primers corresponding to a portion

of the N-terminal amino acid sequence of ST94 (MVDVSQSD)and that of ST94t (MSPQEYIQ), respect-ively Sense (TYF1 and TYF2)and antisense primers (TYR1 and TYR2)were designed from these sequences to lower the degeneracy at the 3¢ end regions: TYF1, 5¢-ATGG TNGAYGTNWSNCARTCNGA-3¢; TYF2, 5¢-ATGGT NGAYGTNWSNCARAGYGA-3¢; TYR1, 5¢-TGDATR TAYTCYTGNGGNGACA-3¢; TYR2, 5¢-TGDATRTA YTCYTGNGGRCTCA-3¢ PCR was carried out using a TITANIUM Taq DNA polymerase (Clontech)in 25 lL of reaction mixture composed of the cDNA (0.5 lL), 25 pmol

of sense primer (TYF1 or TYF2), 25 pmol of antisense primer (TYR1 or TYR2), 0.2 mMdNTPs, 0.5 lL of the Taq DNA polymerase and 1· PCR buffer under the following conditions: after holding (94C, 1 min), 30 cycles of denaturing (94C, 10 s), annealing (56 C, 30 s)and elongation (72C, 30 s), followed by holding (72 C,

3 min) The amplified product (about 200 bp) that occurred

in the reaction mixture containing the primers TYF1 and TYR1 was subcloned into pT7 Blue T-vector (Novagen, Madison, WI, USA)following purification by 2% (m/v) agarose gel electrophoresis using a MiniElute purification kit (Qiagen, Tokyo, Japan) The clones were subjected to DNA sequencing on both strands by the dideoxy chain termin-ation method The sequencing reaction was performed using

a Thermo Sequenase Primer Cycle Sequencing kit (Amer-sham Biosciences)with Texas red-labeled M13 forward primer () 21)or M13 reverse primer () 29) The samples were analyzed with a DNA sequencer SQ-5500 (Hitachi) For 5¢-RACE of ST94 cDNA, the specific primer, TYR3 (5¢-CGTCTGCCGATTTCCAATTCTTCTG-3¢), was designed from the sequence of part of the RT-PCR product 5¢-RACE was carried out using a SMART RACE cDNA Amplification kit according to the manufacturer’s instructions with 0.25 lL of the first strand cDNA for

5¢-RACE as a template and 10 pmol of TYR3 in 50 lL of

the reaction mixture Touchdown PCR was performed as

follows: five cycles of denaturing (94C, 5 s)and annealing/

elongation (72C, 2 min), five cycles of denaturing (94 C,

5 s), annealing (70C, 10 s)and elongation (72 C, 2 min),

followed by 25 cycles of denaturing (94C, 5 s), annealing

(68C, 10 s)and elongation (72 C, 2 min) The amplified

products were subcloned into pT7 Blue following

purifica-tion by agarose gel electrophoresis and subjected to DNA

sequencing as described above

3¢-RACE was performed to amplify the whole ST94

cDNA A primer, TYFW (5¢-GATATGAGGATGAA

ACCACACTTGG-3¢), corresponding to nucleotide 4–28

in the cDNA sequences (Fig 6), was designed from the

sequence of the largest 5¢-RACE product PCR was carried

out using a KOD Plus DNA polymerase (Toyobo, Osaka,

Japan)to ensure high fidelity in 50 lL of reaction mixture

containing 2.5 lL of the first strand cDNA for 3¢-RACE,

15 pmol of TYFW, 7.5 lL of 10· universal primer A mix

(a component of a SMART RACE cDNA Amplification

kit), 0.2 mMdNTPs, 0.8 mMMgSO4and 1 unit of KOD

Plus DNA polymerase in the PCR buffer supplied PCR

was performed under the following conditions: holding

(94C, 1 min), 30 cycles of denaturing (94 C, 5 s)and

annealing/elongation (68C, 3 min) The PCR products

about 2.2 kbp in length were subcloned into pT7 Blue with a

Perfectly Blunt Cloning kit (Novagen)following

purifica-tion by agarose gel electrophoresis Clones of the amplified

fragments were subjected to DNA sequencing on both

strands by primer walking using a BigDye Terminator cycle

sequencing kit with a DNA sequencer, ABI PRISM 3100

(Applied Biosystems) The nucleotide sequence data are

available in the DDBJ/EMBL/GenBank databases under

the accession numbers AB107880 and AB107881 for the

squid tyrosinase ST94 cDNA-1 and cDNA-2, respectively

Sequence analysis

Sequences were analyzed using a softwareDNASIS(Hitachi

Software, Tokyo, Japan) A homology search was carried

out withNCBI-BLAST2.0 program available at DDBJ web

server (http://www.ddbj.nig.ac.jp) Phylogenetic tree was

deduced by a neighbor-joining analysis based on the

alignment of amino acid sequences constructed using the

CLUSTALWprogram available at DDBJ web server

Results and discussion

Purification of tyrosinase ST94

ST94 was isolated from the ink of I argentinus by

ammonium sulfate fractionation, and Phenyl-Sepharose

and anion-exchange chromatography The ammonium

sulfate fractionation recovered 95% of tyrosinase activity

in the ink as a precipitate (fraction AS60): the dialyzed

solution of AS60 (300 mL)obtained from 100 g of defatted

ink powder contained 2.9 g of protein, 0.29 g of hexose,

0.25 g of uronic acid, 0.12 g of methylpentose and 5400

units of tyrosinase activity The compositional analysis

revealed that the IPG [23–25] of the ink was also

concen-trated in fraction AS60 The elution profile of fraction AS60

on Phenyl-Sepharose CL-4B chromatography is shown in

Fig 1 Chromatography of squid tyrosinase on a Phenyl-Sepharose CL-4B column (A)Fraction AS60 (100 mL)was applied to a Phenyl-Sepharose CL-4B column (2.6 · 28 cm), and eluted with a linear gradient of 0.5–0 M ammonium sulfate in 10 m M sodium phosphate (pH 7.4)(flow rate 1 mLÆmin)1, fractions of 10 mL) d, tyrosinase activity, absorbance of L -DOPA oxidation reaction mixture at

492 nm; s, uronic acid, absorbance of assay reaction mixture at

530 nm; ——, absorbance at 280 nm Fractions containing ST94, indicated with horizontal bar, were pooled (B)Native-PAGE of the tyrosinase-active fractions Samples were run on 10–15% gradient gels and stained with CBB (left), with L -DOPA (center)and with L -tyrosine (right) Lane M, marker proteins, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and BSA (66.4 kDa); Lane 1, fraction no 170; lane 2, fraction no 180; lane

3, fraction no 190; lane 4, fraction no 198; lane 5, fraction no 204 The arrow indicates the band of ST94 (lanes 3 and 4) (C) IEF-native 2D-PAGE of the pooled fraction containing ST94 The first dimen-sion, IEF (pH 3-9); the second dimendimen-sion, native-PAGE (10–15% gradient gel) The gels were stained with CBB (left) and with L -DOPA (right) Lane M, mass marker proteins Positions of pI marker proteins were indicated at the top; amyloglucosidase (pI 3.50), soybean trypsin inhibitor (pI 4.55), b-lactoglobulin A (pI 5.20), bovine carbonic anhydrase B (pI 5.85), horse myoglobin (pI 7.35)and lentil lectin (pI 8.65) The arrow indicates the spot of ST94.

Fig 1A Most of the IPG was eluted in the breakthrough

fraction and separated from tyrosinase; these two

compo-nents could not be separated by anion-exchange

chroma-tography and gel permeation chromachroma-tography [26] In

native-PAGE of the tyrosinase-active fractions (Fig 1B),

the protein bands were not observed in the position

corresponding to the strong enzyme activities, which yielded

wide, and smeared bands As ST94 showed weak activity

compared with other tyrosinase-active components, ST94

was detected by PAGE analyses during purification, as

shown in Fig 1B On isoelectric focusing (IEF)-native

2D-PAGE, ST94 was separated from other tyrosinase-active

components and was observed as a clear spot corresponding

to the position of a protein with pI 4.1 and a molecular mass

of 94 kDa The tyrosinase activity of ST94 was also

observed by activity staining (Fig 1C) ST94 was further

purified by anion-exchange HPLC and obtained as a

homogeneous preparation (seen in Fig 2)with a yield of

0.73 mg protein from 100 g of defatted ink powder and a

specific tyrosinase activity of 23.5 U per mg protein

Activation of ST94 by treatment with trypsin

As described above, most of the tyrosinase activity in the ink

of I argentinus originated from tyrosinase-active molecules

showing a broad smeared band on native-PAGE followed

by activity staining Despite their high tyrosinase activities,

these molecules showed indistinct bands when stained with

CBB, which suggests that their specific activity was high

compared with that of ST94 From these observations,

ST94 was presumed to be a partially activated molecule that

was capable of becoming a more active molecule As

arthropod prophenoloxidases [10,11,14–16] and a bivalve

tyrosinase [34] have been shown to be activated by

proteolysis, we examined the effect of trypsin treat-ment on ST94 During the treattreat-ment, the tyrosinase activity was increased to about four times the maximum after incubation for 2 h under the conditions described in Materials and methods As shown in Fig 2A, ST94 generated a proteolyte, termed ST94t, showing slightly higher mobility than ST94 on native-PAGE ST94t in the proteolysate of ST94 was purified as the enzyme prepar-ation with a yield of 0.23 mg protein from 0.30 mg of ST94 and a specific activity of 103 U per mg protein by gel permeation HPLC The results indicated that ST94t was an activated tyrosinase molecule bearing the stable catalytic domain of ST94

Molecular mass of the squid tyrosinase ST94 electrophoresed to a position corresponding to that of

a protein with a molecular mass of about 70 kDa on SDS/ PAGE under reducing conditions, whereas it migrated

as a  140 kDa protein under nonreducing conditions (Fig 2B,C) In MALDI-TOF mass spectrometry, a parent ion signal of ST94 was observed at m/z 140.2 kDa These results indicated that ST94 is a 140.2-kDa protein composed

of two 70.1-kDa subunits that are linked, probably by a disulfide bond These estimates of the molecular mass of ST94 were supported by the result of gel permeation HPLC (Fig 3) There was a minor band between 140 and 232 kDa

in the native-PAGE of Fig 2A (lane 1), implying that ST94 enables to form an oligomeric protein On SDS/PAGE, ST94t electrophoresed to a position corresponding to that

of a protein of about 65 kDa under reducing conditions, while migrated as an 130 kDa protein under nonreducing conditions (Fig 2B,C) In MALDI-TOF mass spectro-metry, the trypsin-treated reaction mixture of ST94 showed

Fig 2 PAGE analyses of ST94 and ST94t Purified ST94 and the trypsin treatment reaction mixture of ST94 were subjected to PAGE analyses Lane M, molecular mass markers; lane 1, ST94; lane 2, the trypsin treatment reaction mixture of ST94 The closed arrow and the open arrow indicate the band of ST94 and that of ST94t, respectively (A)Native-PAGE Samples were run on 10–15% gradient gel with the same marker proteins as in Fig 1B, and stained with CBB (left)and with L -DOPA (right) (B) SDS/PAGE under reducing conditions Samples were run on 12.5% gel and stained with CBB Marker proteins used were, phosphorylase b (97.2 kDa), BSA (66.4 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (29.0 kDa), soybean trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa) (C) SDS/PAGE under nonreducing conditions Samples were run on 4–15% gradient gel and stained with CBB Marker proteins used were, myosin (212 kDa), a 2 -macroglobulin (170 kDa), b-galac-tosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa).

a parent ion signal of ST94t at m/z 127.6 kDa These results

indicated that ST94 was digested by trypsin to generate

ST94t as a 127.6-kDa protein composed of two 63.8-kDa

subunits that remained linked after treatment with trypsin

The molecular masses of the subunits of ST94 and ST94t

were similar to those of the proenzymes ( 70–80 kDa)and

the activated enzymes by proteolysis ( 60–70 kDa)of

a bivalve tyrosinase [34] and arthropod phenoloxidases

[10–13,16]

ST94 was found to contain copper atoms at a content of 0.18 ± 0.01% (by mass): the copper concentration of ST94 solution of 28 lg proteinÆmL)1 was determined to be 50.2 ± 1.5 ngÆmL)1 The result indicates that ST94 has two copper atoms per a subunit of 70.1 kDa For control experiment, the copper content of KLH was also analyzed

by the same procedure and determined to be 0.24% (by mass)in good agreement with the value calculated from the literature [7] (16 copper atoms per subunit): 17.5 ± 0.3 ng copperÆmL)1 was detected in KLH solution of 7.3 lg proteinÆmL)1 The ST94 solution was also subjected to the analysis of manganese, but no manganese was detected The N-terminal amino acid sequences of ST94 and ST94t were shown to be NH2-MVDVSQSDGLQSXLDRFADD (X represents an amino acid undetermined)and NH2 -ISTLATMSPQEYIQ, respectively, which indicated that the N-terminal region of ST94 was truncated with trypsin to generate the N-terminal of ST94t Each analysis for ST94 and for ST94t showed a single N-terminal sequence, allowing us to speculate that ST94 is a homodimeric protein Enzymatic properties of the squid tyrosinase

Effects of pH and temperature on stabilityand acti-vity As ST94 and ST94t showed the identical results on pH- and temperature-effects, we describe the data of ST94t in this section ST94t retained more than 90% of its activity after incubation at 4C for 24 h within a pH range of 6.5–11 (Fig 4A) The optimum pH for o-diphenolase activity of ST94t was determined to be pH 8.0 (Fig 4B)with correction

by subtraction of the increasing baseline caused by sponta-neous oxidation of L-DOPA; we routinely assayed the tyrosinase activity at pH 6.8 to avoid spontaneous oxidation

of o-diphenols and their oxidized products (o-quinones) observed particularly under alkaline conditions

ST94t was shown to be stable up to 30C and complete inactivation was observed at 70C when ST94t was

Fig 3 Molecular mass estimation of ST94 by gel permeation HPLC.

ST94 (2 lg)and molecular mass standard proteins (each 2.5 lg)were

chromatographed on a column of G3000SW XL (7.8 · 300 mm)

equilibrated with 0.2 M NaCl in 0.1 M sodium phosphate (pH 7.0)

(flow rate 0.5 mLÆmin)1) The eluate was monitored at 205 nm.

Molecular mass standard proteins used were: 1, catalase (232 kDa); 2,

lactate dehydrogenase (140 kDa); 3, BSA (66.4 kDa); 4, ovalbumin

(45.0 kDa); 5, chymotrypsinogen (25.0 kDa) The arrow indicates the

elution volume of ST94.

Fig 4 Effects of pH on stability (A) and o-diphenolase activity (B) of ST94t (A)ST94t solution (6.8 lgÆmL)1of 20 m M buffer with various pH)was incubated for 24 h at 4 C, followed by measurement of residual activity by the dopachrome method (B)The o-diphenolase activity of ST94t was measured under various pH conditions at 25 C; the assay mixture (3 mL)contained 5 m M L -DOPA and ST94t (0.36 lg)in 0.1 M buffer (pH 3.6– 9.2) The reaction was monitored at 475 nm The data were corrected by subtraction of the increase caused by auto-oxidation of L -DOPA The following buffers were used: d, sodium acetate buffer (pH 3.6–5.6); s, sodium phosphate buffer (pH 5.7–8.0); j, Tris/HCl buffer (pH 8.0–9.0);

h, sodium carbonate buffer (pH 9.2–10.8); m, sodium phosphate buffer (pH 11.2–11.9).

incubated at pH 7.4 for 20 min (Fig 5A) Similar stability

toward temperature has been reported for the subunit of

hemocyanin from a gastropod, Rapana thomasiana grosse

[45] The conformational stability of hemocyanin was

influenced generally by the aggregation state; the association

of structural subunits to hemocyanin increased the stability

[45] Covalently linked dimeric form, the characteristic

structure of ST94 possibly contributes to the stability

The effects of temperature on the o-diphenolase activity

of ST94t were investigated in the range 4–50C using

L-DOPA as a substrate Tyrosinase from mushroom was

also examined for comparison with ST94t As shown in

Fig 5B, the o-diphenolase activities of both enzymes

correlated linearly with temperature according to the

Arrhenius equation, within the range from 4 to 40C

When the reactions were carried out above 45C, however,

the reaction rates could not be determined accurately due to

inactivation of the enzymes It was noteworthy that the

rate of decline for o-diphenolase activity of ST94t was

considerably smaller than that of mushroom tyrosinase

Although no other cephalopod tyrosinase for comparison

has been characterized so far, the high activity of I

argen-tinus tyrosinase at low temperature seems to be an

adaptation to the cold living environment of the squid

[46] For example, it has been reported that hemocyanin of

the Antarctic octopod, Megaleledone senoi, showed the

highest level of oxygen affinity among the cephalopods, and

the thermal dependence of the affinity was remarkably

smaller than those of temperate cephalopod hemocyanins

[47]

Effects of inhibitors As shown in Table 1, the L-DOPA

oxidizing activities of ST94 and ST94t were inhibited by

phenylthiourea, tropolone, kojic acid and arbutin, potent

inhibitors of tyrosinase [10,35,48,49] EDTA, which is

known to inhibit some tyrosinases [49], did not affect the

activities of ST94 and ST94t

Substrate specificity The substrate specificity of ST94t was investigated using five diphenols and three monophen-ols as substrates and compared with that of ST94 The rate parameters for oxidation reactions of these substrates by ST94 and ST94t are summarized in Table 2 Both enzymes were shown to be able to catalyze the oxidation of all monophenols and o-diphenols tested The oxidation of monophenol by these enzymes showed a characteristic lag period, as reported for other tyrosinases, which was shortened by addition of each mono-oxygenation product (diphenol)as a cofactor [2,28] From the comparison of reaction efficiency (k0/Km), dopamine appeared to be oxidized most effectively by ST94 as well as by ST94t, whereas DHPPA, which is known to be good substrate for several tyrosinases [42,43], was shown to be a poor substrate for these enzymes The K value forL-tyrosine was higher

Fig 5 Thermostability (A) and temperature-dependency of o-diphenolase activity (B) of ST94t (A)ST94t solution (6.8 lgÆmL)1of 20 m M sodium phosphate buffer, pH 7.4)was incubated at 20–75 C for 20 min and residual activity was measured by the dopachrome method Activity after incubation on ice is taken as 100% (B)The o-diphenolase activity of ST94t (d)was measured at 4–50 C using the dopachrome method (0.30 lg per assay) Mushroom tyrosinase (s)was also examined for comparison (3.8 lg per assay) The reaction rates, v (DA 475 Æmin)1per mg protein)were plotted according to the Arrhenius equation (T, absolute temperature).

Table 1 Effects of tyrosinase inhibitors on the o-diphenolase activities of ST94 and ST94t The o-diphenolase activities of ST94 and ST94t were measured by the dopachrome method in the presence of inhibitor at

25 C Conditions were, 5 m M L -DOPA, 0.1 M sodium phosphate buffer (pH 6.8), several concentrations of inhibitor and the enzyme (ST94, 0.35 lgÆmL)1; ST94t, 0.10 lgÆmL)1) The IC 50 value represents the concentration of inhibitor needed to inhibit the o-diphenolase activity by 50%, which was obtained from each graph of the reciprocal

of reaction rate vs inhibitor concentration.

Inhibitor

IC 50 (l M )

than that for theD-isomer The same tendency was observed

for isomers of DOPA This catalytic stereospecificity of

I argentinus tyrosinase was similar to that reported for

S officinalistyrosinase [28] From the comparison of ST94

and ST94t, it appeared that the trypsin treatment of ST94

caused a fall in the Km value and a rise in the k0 value,

resulting in approximately five to 70 times higher reaction

efficiency of ST94t for oxidation of each substrate These

results suggest that a limited proteolysis for activation is

involved in the natural regulation system of tyrosinase in

cephalopods, as in the case of arthropods [14–17]

Molecular cloning of ST94 cDNA

The cDNA cloning of tyrosinase ST94 was carried out

by degenerate RT-PCR and RACE using the first strand

cDNA of poly(A)+ RNA extracted from an ink sac of

I argentinusas a template First, in order to detect the target

cDNA, RT-PCR was carried out using degenerate primers

designed from the N-terminal amino acid sequences of ST94

and ST94t For lowering degeneracy at the 3¢ side of the

primers, each two primers for sense and for antisense, which

differed only in the triplet corresponding to the Ser codon at

the 3¢ side, were prepared and used for RT-PCR The

amplified product about 200 bp in length was observed only

in the reaction carried out using the pair of degenerate

primers TYF1 and TYR1 The DNA sequence analysis of

the RT-PCR products revealed that the two distinct DNA

fragments of 194 bp corresponding to nucleotide 176–369 in

the cDNAs (Fig 6)were amplified On the 5¢-RACE

performed using the specific primer TYR3 (nucleotide 290–

314 in the cDNAs), designed from the internal sequence of

the RT-PCR products, DNA fragments of about 310 bp,

which contained the sequences of nucleotide 62–314 in the

cDNAs with an additional universal primer sequence, were

mainly amplified Fragments about 370 bp in length, which

contained the sequences of nucleotide 1–314 in the cDNAs

(Fig 6)were obtained as the largest product in this study

Finally, using a primer TYFW corresponding to nucleotide

4–28 in the cDNAs, the full-length cDNAs of about 2.2 kbp

were amplified by 3¢-RACE The complete nucleotide

sequences were determined for 12 plasmid clones of the full-length cDNAs

Comparison of the cDNA sequences revealed that two distinct messages for the squid tyrosinase, represented as cDNA-1 and cDNA-2, were expressed in the ink sac of

I argentinusused for extraction of poly(A)+RNA in this study, as summarized in Fig 6; seven and five clones carried cDNA-1 and cDNA-2, respectively These two cDNA sequences were also confirmed by other PCR experiments performed using the first strand cDNA independently prepared from the same poly(A)+RNA preparation as a template Both of the cDNAs covered the complete open reading frames of 1878 bp (nucleotide 122–1999)encoding putative 625-amino acid proteins with 121 bp of the 5¢ untranslated regions and the 3¢ untranslated regions containing polyadenylation signals (AATAAA)at three positions and the poly(A)-tails The criteria for a consensus translation initiation site were observed around the putative initiator ATG codon (CCGAAATGG)of the largest open reading frames [50] The molecular mass numbers calculated for the encoded proteins in the open reading frames of cDNA-1 and cDNA-2 were 70 975 and 71 046, respectively Deduced amino acid sequences

The amino acid sequence deduced from the nucleotide sequence of cDNA-1 was shown to contain sequences that agreed with the N-terminal amino acid sequences of ST94 and ST94t determined by Edman degradation, whereas the nucleotide sequence of cDNA-2 was different from that of cDNA-1 at 15 positions, resulting in amino acid tions at four positions (Fig 6) In particular, the substitu-tion from Gly27 to Glu27 caused by the single base substitution, which did not agree with the result obtained from the N-terminal amino acid sequence analysis of ST94, was observed in cDNA-2 In the N-terminal sequence analysis, a portion of the ST94 preparation isolated from about 200 ink sacs was subjected to Edman degradation, but no similar amount of phenylthiohydantoin (PTH)-Glu

to PTH-Gly was detected at the corresponding cycle in this study despite the similar existence ratios of the two cDNAs

Table 2 Rate parameters for the oxidation of several o-diphenols and monophenols catalyzed by ST94 and ST94t The steady-state rate of the oxidation of substrate was measured by the MBTH method at 25 C Assay conditions were 50 m M sodium phosphate buffer (pH 6.8), 5 m M MBTH, 2% N,N-dimethylformamide, differing substrate concentrations and the enzyme (0.1–0.7 lgÆmL)1) In the analyses for monophenols, the corresponding o-diphenol at a final concentration of 1 l M was added to the reaction mixture The V max values are expressed as micromoles of substrate oxidized per min per mg of protein The k 0 values were calculated using molecular masses of 140.2 kDa and 127.6 kDa for ST94 and ST94t, respectively.

Substrate

K m

(m M )

V max

(lmolÆmin)1Æmg)1)

k 0

(s)1)

k 0 /K m

(m M )1 Æs)1)

K m

(m M )

V max

(lmolÆmin)1Æmg)1)

k 0

(s)1)

k 0 /K m

(m M )1 Æs)1)

Ratio of k 0/ K m

(ST94t/ST94)

Furthermore, the nucleotide sequences of cDNA-1 and

cDNA-2 were almost identical: the calculated homology

was 99.3% except for the poly(A)-tails Therefore, the

cDNA-2 was thought to be of an allelic variant message for

tyrosinase ST94, expressed in the individual of I argentinus

used for poly(A)+ RNA extraction in this study Both

amino acid sequences deduced from the two cDNAs were revealed to possess two putative copper-binding sites (critical regions for tyrosinase activity), as described below, and no amino acid substitution was observed in these two sites (Figs 6 and 7) Therefore, the variant of ST94 was thought to be able to function as tyrosinase as well as ST94

Fig 6 Nucleotide and deduced protein sequences of ST94 cDNA-1 The nucleotides (upper)are numbered from the first base; the amino acids (lower)are numbered from the initiating methionine Base substitutions at 15 positions and amino acid substitutions at four positions observed in cDNA-2 are shown in italics The N-terminal amino acid sequences of ST94 and ST94t obtained by Edman degradation are underlined The putative copper ligands are circled A potential N-linked glycosylation site is shown by an asterisk Cysteine residues are indicated by d The putative polyadenylation signals are indicated by double underlining.

These findings suggested the occurrence of other ST94

variants in the gene pool of I argentinus Sequence

poly-morphism of the tyrosinase gene has also been observed in

Neurospora crassa[51]

The N-terminal sequence of ST94 was found to start at

the 19th amino acid residue in the open reading frame

encoded in cDNA-1, which was preceded by 18 amino acid

residues, indicating that the subunit of ST94 was expressed

as a premature 625-amino acid protein, followed by excision

of the preceding 18 amino acid residues considered to be a

signal sequence to form the mature subunit polypeptide

Thus, the subunit of ST94 was thought to consist of 607

amino acid residues from Met19 to Lys625 with a molecular

mass of 68 993 Da, on the basis of the amino acid sequence

deduced from cDNA-1 The N-terminal sequence of ST94t

was shown to start at the 70th amino acid residue, indicating

that the N-terminal 51 amino acid residues of ST94 were

digested with trypsin to generate the N-terminal of ST94t,

resulting in a reduction in molecular mass of 5807 Da per

subunit However, the molecular mass of ST94 was reduced

by 6.3 kDa per subunit by trypsin treatment, in the mass

analyses Therefore, ST94 seemed to be digested by trypsin

not only at the N-terminal but also (a few amino acids)at

the C-terminal Although there is no evidence, one

candi-date for trypsin cleavage site is at Arg620-Asn621 in the

C-terminal region to release a peptide of five residues

(Asn621 to Lys625) The molecular mass of the N- and C-terminal-truncated subunit, a 551-amino acid polypeptide composed of Ile70 to Arg620, was calculated to be

62 644 Da, which gives a value for the reduction in molecular mass (6349 Da per subunit)concordant with that observed in the mass analyses

Both molecular mass numbers deduced for the subunits

of ST94 (68 993 Da)and ST94t (62 644 Da)from cDNA-1 were approximately 1.1 kDa less than those obtained from the mass spectrometry (70.1 kDa for ST94 and 63.8 kDa for ST94t) The cause of the difference of about 1 kDa except for mass of two copper atoms (127 Da)remains unclear; however, the difference was presumed to be due to post-translational modifications, for example, glycosylation

at the unique potential glycosylation site for N-linked carbohydrate found at Asn333

It was demonstrated that mature ST94 subunit could undergo the digestion of N-terminal 51 amino acid residues

by trypsin In prophenoloxidases in two insects, Bombyx moriand Manduca sexta, the N-terminal region consisting

of 51 amino acid residues were also cleaved by the prophenoloxidase-activating enzyme (PPAE)to generate active phenoloxidases [11,13] This similarity suggests that the conformational change of ST94 caused by the elimination of the N-terminal region is required principally for its activation Furthermore, the cleavage site of ST94

Fig 7 Comparison of the amino acid sequences at two putative copper-binding sites, Cu(A) and Cu(B), in ST94 and other type 3 copper proteins Numbers indicate positions of the amino acid residues in each sequence Gaps (–)have been introduced to optimize the alignment The putative copper ligands of histidine residues conserved in all proteins are labeled with d, those conserved in molluscan proteins and tyrosinases with an s, and those conserved in arthropod proteins with an h The identical residues are shaded IaY, I argentinus tyrosinase ST94; OdHc and OdHe, Octopus dofleini hemocyanin functional unit c and e, respectively (SWISS-PROT, accession No O61363); SoHh, S officinalis hemocyanin unit h (SWISS-PROT, P56826); HpHg, Helix pomatia b c -hemocyanin unit g (SWISS-PROT, P56823); McH2c, M crenulata hemocyanin unit 2-c (SWISS-PROT, P81732); MmY, Mus musculus tyrosinase (SWISS-PROT, P11344); GgY, Gallus gallus tyrosinase (DDBJ, D88349); CeY,

C elegans hypothetical protein K08E3.1 (DDBJ, Z81568); AoY, Aspergillus oryzae tyrosinase (DDBJ, D37929); SgY, Streptomyces glaucescens tyrosinase (SWISS-PROT, P06845); BmP, B mori prophenoloxidase subunit 1 (DDBJ, D49370); PlP, Pacifastacus leniusculus prophenoloxidase (DDBJ, X83494); PiH, Panulirus interruptus hemocyanin subunit a PROT, P04254); LpH, Limulus polyphemus hemocyanin II (SWISS-PROT, P04253).