The complete amino acid sequence of growth hormone of an elasmobranch, the blue shark (Prionace glauca)

GENERAL

AND

COMPAKATIVE

ENDOCRINOLOGY

73,252-259 (1989)

The Complete Amino Acid Sequence of Growth Hormone Elasmobranch, the Blue Shark (Prionace glauca)

of an

KAZUOYAMAGUCHI,AKTKAZUYASUDA,* URBAN J. LEWIS,‘FYOSHIHARU YOKOO, AND HIROSHI KAWAUCHI*" Tokyo Laboratory, Kyowa Hakko Kogyo Company LTD, Machida, Tokyo 194, Japan, fThe Whittier Institute for Diabetes and Endocrinology, La Jolla, California 92037, and *Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan Accepted August 5, 1988 The complete amino acid sequence of growth hormone (GH) from a phylogenetically ancient fish, the blue shark (Prionace glauca), was determined. The shark GH isolated from pituitary glands by U. J. Lewis, R. N. P. Singh, B. K. Seavey, R. Lasker, and G. E. Pickford (1972, Fish. Bull. 70, 933-939) was purified by reversed-phase high-performance liquid chromatography. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting peptide fragments were separated by rpHPLC and submitted to sequence analysis by automated and manual Edman methods. The shark GH consists of 183 amino acid residues with a calculated molecular weight of 21,081. Sequence comparisons revealed that the elasmobranch GH is considerably more similar to tetrapod GHs (e.g., 68% identity with sea turtle GH, 63% with chicken GH, and 58% with ovine GH) than teleostean GHs (e.g., 38% identities with salmon GH and 42% with bonito GH) except for eel GH (61% identity), and substantiates the earlier finding derived from the immunochemical and biological studies (Hayashida and Lewis, 1978) that the primitive fish are less diverged from the main line of vertebrate evolution leading to the tetrapod than are the modern bony fish. 6 1989 Academic Press, Inc.

Growth

hormone

(GH) and prolactin of pituitary hormones with considerable structural similarity and, thus, are thought to be derived from a common ancestral gene by gene duplication followed by evolutionary divergence (Bewley and Li, 1971, Niall ef at., 1971, Miller and Eberhardt, 1983). GH is involved in the regulation of somatic growth in most vertebrates, while PRL is known to have diverse functions among vertebrates (Bern, 1983). Accordingly, comparative structural information on these hormones from many vertebrate classes, especially lower vertebrates, is important in order to obtain an insight into their molecular evolution, the (PRL) are a family

’ To whom all correspondence should be addressed.

diversity of functions, and structurefunction relationships. Recently, chemical knowledge of these hormones from teleost species is rapidly expanding: salmon GH (Sekine et al., 1985, Kawauchi et al., 1986, Nicoll et al., 1987), salmon PRL (Yasuda et al., 1986; Kuwana et al., 1986; Song et al., 1988), rainbow trout GH (Agellon and Chen, 1986), carp PRL (Yasuda et al., 1987), eel GH (Saito et al., 1985, Yamaguchi et al., 1987), tuna GH (Sat0 et al., 1988), yellowtail GH (Watabiki et al., 1988; Kawazoe et al., 1988), bonito GH (Noso et al., 1988$, and it was found that there is the high evolutionary diversity in teleosts . Indeed, one should assume that teleost and mammalian GHs have evolved with different evolutionary 252

0016-6480/89 $1 SO Copyright All ri&ts

8 1989 by Academic Press. Inc. of repmductim~ in any form reserved.

STRUCTURE

rates, otherwise eel GH belongs to the tetrapods in the phylagenetic tree of GH (Kawauchi and Yasuda, 1988). The complete amino acid sequence of GH from a phylogenetically more ancient species than teleost, such as an elasmobranch is requisite for a reasonable phylogenetic relationship. GHs isolated from the pituitary glands of phylogenetically ancient fishes, blue shark (Lewis et al., 1972) and sturgeon (Farmer et al ., 198 1)) showed significant growthpromoting activity in the rat tibia test, while the teleostean GH showed very low activities. Moreover, immunochemical studies demonstrated that the shark and sturgeon GHs are more closely related to tetrapod GHs than to teleostean (tilapia) GHs (Hayashida and Iagios, 1969; Hayashida et al., 1975; Hayashida and Lewis, 1978). Based on these results, it has been suggested that there must be some significant differences in the structure of shark GH compared to the structure of GH from teleost fish. In the present report, we determined the complete primary structure of the shark GH; compared it with those of mammalian, avian, and teleostean GHs; and proposed one region specific to GH. MATERIALS

AND METHODS

Mat&&. The starting material was shark GH isolated as described by Lewis et al. (1972). Enzyme used for fragmentation were lysyl endopeptidase (Wake Pure Chemicals, Lot No. CDL9675) and Stuphylococcus aureus protease (Sigma, Lot No. 84F-0357). ALI chemicals used for structural determination were sequential grade from Applied Biosystems and Wake Pure ChemicaIs . Reduction and S-carboxymethylation. Shark GH was reduced and carboxymethylated by a method derived from Hirs (1967) and Gurd (1967). One mg of the protein was dissolved in 1 ml of 1 M Tris-HCI @H 8.3) containing 6 M guanidine-HCl and 2 mM EDTA, reduced with dithiothreitol(O.3 mg) under nitrogen gas in the dark at 25” for 4 hr, and subsequently alkylated by adding 0.5 ml of 1.6 mM iodoacetic acid in 2 N NaOH for 15 min. The reaction was terminated by addition of glacial acetic acid. The rasulting precipitate was dissolved in 0.1% trifiuoroacetic acid (TFA). The protein

OF SHARK GH

253

solution was desalted by reversed-phase highperformance liquid chromatography (rpHFLC) on a TSK gel TMS-250 (TOSO Co., particle size 10 pm) coIumn (40.46 x 5 cm). After elution of the reagent and salts with 0.1% TFA, the adsorbed protein was eluted with 90% isopropanol containing 0.1% TFA, and Iyophilized. Chemical and enzymatic cleavage. S-carboxymethylated protein was cleaved with 100 mal excess of cyanogen bromide in 70% formic acid at room temperature in the dark for 18 hr (Gross, 1967). Digestion of an isolated cyanogen bromide fragment designated CBS was performed with Staphylococcus protease (E/S = l/60, by weight) in 0.05 M ammonium bicarbonate, pH 8.0, at 37” for 18 hr. The intact protein was also digested with lysyl endopeptidase (E/S = l/60 by weight} in 0.1 M ammonium bicarbonate, pH 8.2, at 37” for 4 hr. C&dative cleavage of trwtophanyl bond in the intact protein at the site ‘was performed with o-iodosobenzoic acid by a method of Mahoney antd Hermodson (1979). Five hundred micrograms of the protein and 500 pg of o-iodosobenzoic acid were dissolved in 100 ~1 of 80% acetic acid containiitg 4 M guanidine-HCI, and 5 yl ofp-cresol was added. The reaction was allowed to proceed at room temperature in the dark for 24 hr. Fractionation of peptide fragments. Fractionation of fragment peptides was performed exclusively by rpHPLC on a TSK gei ODS-12OT (TOSO Co., particle size 5 pm) column ($0.46 x 25 cm) using a linear gradient elution with 0.1% TFA and aceto&ile or isopropanol. Peptides were monitored by absorption at 210 or 220 nm. Amino acid analysis. Shark GH and its ,peptide fragments were hydrolyzed in constant boiling HCl containing 0.6% phenol (Muramoto et al., 1987) at 110” for 18-24 hr. Amino acid analysis was performed by a precolumn labeting method of Bidlinmeyer et al. (1984). Amino acids were derivatized wiih phenylisothiocyanate and the resulting phenylthiocarbamyl amino acids were identified by rpHPLC on a T$K gel ODS-8OT column @OSO,Co., particle size 5 pm) column ($0.46 x 25 cm) u&g gradient elutibu with 0.14 M sodium acetate containing 0.5% triethyIamine (pH 6.4) and acetotitrile. Half-cystine was deter&&d as cysteic acid after performic acid oxidation (Moore, 1963). Sequence analysis. N-terminal residues were determined by the Dansyl method (Gray, 1967). Autamatic microsequencing was performed with a gas-liquid sequencer (Applied Biosystems, Model 47OA) (Hewick et al., 1981). Manual sequencing was carried out by the method of Tarr (1986): Identification of phenylthiohydantoin amino acid was carried out by rpHPLC (Spectra Physics, SP8lOO system) WI a C8 column (Senshu Kagaku SlZIQ4, $0.46 x 30 cm, particle siie ‘7 pm) developed with a gradient of 40 niM sodium acetate (pH 4.9) and acetonitrile.

254

YAMAGUCHI

RESULTS

ET AL. TABLE 1 AMINOACIDCOMPOSITIONOFBLUESHARKGH

Shark GH isolated from pituitary glands Amino acid Residues of the blue shark by Lewis et al. (1972) was '/2Cys0 4.0b (4)C further purified by rpHPLC (Fig. 1). The 18.2 (16) Asp protein eluted as a single peak with a retenGlu 29.7 (23) tion time of 20 min and gave a single band Ser 16.2 (16) with an apparent molecular weight of His 7.2 (6) 22,000 estimated by SDS-PAGE and a sinGUY 9.1 (7) 13.8 (10) gle N-terminal residue of valine. The amino Arg Thr 8.3 (8) acid composition of the protein is shown in Ala 13.1 (14) Table 1. PI-0 7.7 (8) The complete amino acid sequence of the 7.5 (7) Tyr shark GH was determined by analyses of Val 5.3 (5) Met 3.7 (4) the S-carboxymethylated protein and its Be 4.9 (5) peptide fragments prepared by cleavages Leu 17.6 (24) with cyanogen bromide, o-iodosobenzoic Phe 6.6 (IO) acid, lysyl endopeptidase, and S. aureus 2.0 (2) Trpd protease. Amino acid sequence analyses of 12.8 (14) LYS the S-carboxymethylated protein allowed Total 187.3 (183) the assignment of N-terminal 44 residues. a Determined as cystic acid. Fractionation of the S-carboxymethylated b Values indicate the number of residues/molecule. protein by rpHPLC after cyanogen bromide ’ Numbers in parentheses represent the number of cleavage resulted in the separation of five residues determined by sequence analysis. peptide fragments (Fig. 2). Amino acid d Determined by a method of Muramoto et al. compositions and N-terminal residues are (1987). summarized in Table 2. CB5 was localized to the N-terminal of the protein, since only the peptide gave the same N-terminal resi- digested with S. aureus protease and the due as the intact protein. CB5 was further resulting peptide fragments were separated by rpHPLC (data not shown). Sequence analyses of CBS-SP12 reconfirmed the sequence of residues 1 to 38th of the intact / bluesharkGH ,’ protein. CB5-SP9 provided additional 13 F ,,’ residues in succession to SP12. Thus, the ,/’ ,’ /’ residues identified totaled up to 68 in the /,’ N-terminal of the protein. /’ ,’ ,’ The total amino acid sequences of the ,’ four fragments, CBl, CB2, CB3, and CB4, were determined without further fragmentation. They could be aligned at the Cterminal region of the protein with the arrangement of CB4-CB3-CB2-CB 1, exhibiting the extensive homology to the sequences of mammalian and teleostean Time (min) GHs. The residues identified totaled up to FIG. 1. High-performance liquid chromatography of 67 in the C-terminal of the protein. shark GH (400 pg) on a TSK gel ODS120T column In order to determine amino acid se(40.46 x 2.5 cm, particle size 5 Fm). Broken line represents a gradient of acetonitrile in 0.1% TFA. quence of the middle portion of the protein

STRUCTURE

25%

OF SHARK GH

TABLE 2 A~INOACIDCOMPOSITIONOFCBFRAGMENTSOFBLUESHARKGROWTHHORMONE Amino acid

CBl

v2cys ASP Gill Ser His GUY Arg Thr Ala Pro TV Val Met Be Leu Phe Trp LYS N-terminal residue Sequence in shark GH

2.0 2.4 1.3 1.2

1.2 1.1 1.0

0.1 -

CB2

CB3

CB4

CBS

2.4

3.2 5.3 3.7

1.0

4.2

0.9

1.1 1.1 1.8

9.8 19.4 12.5 5.1 4.1 9.8 4.6

0.9

1.1

+

+

1.5 0.7

3.5 3.0

1.0

1.0

1.3

1.0

1.0 1.9 + 0.9

0.7

11.1 7.0 4.2 2.7 -I4.0 14.5 4.8

1.0 1.0

2.0

2.6

2.4

Asn

His

LYS

LYS

TF

172-183

162-171

149-161

117-148

l-116

and to obtain overlapping peptides to connect CB fragments, the intact protein was digested with lysyl endopeptidase. The resulting peptide fragments (LE) were sepa-

Y

5.3

rated by rpHPLC (Fig. 3). Analyses of LE33 provided the sequence that overlapped with that of CB5-SP9 and thus extended the additional 27 residues up to

I 110

0 Time

(min)

FIG. 2. High-performance liquid chromatography of cyanogen bromide fragments of S-carboxymethylated shark GH on an ODS column by a linear gradient elution with isopropanol in 0.1% TFA.

Time

(min)

FIG. 3. High-performance liquid chromatography of lysyl endopeptidase digest of shark GH on an ODS column with a gradient of isopropanol in 0.1% TFA.

256

YAMAGUCHI

ET AL.

95th. LE27 overlapped with the N-terminal of CB4. Three peptides, LE21, LE3, and LE12, clarified the connections between CB4 and CB3, CB3 and CB2, and CB2 and CB 1, respectively. Oxidative cleavage of the tryptophanyl bond in the native protein with o-iodosobenzoic acid was carried out to determine the unidentified 9 residues corresponding to the C-terminal region of LE33. The resulting fragments were separated by rpHPLC (data not shown). The peptide designated IB was subjected to sequence analysis. The results provided the sequence of 19 residues, which overlapped with that of LE33. Although peptides to connect between Glu55 and Thr-56, and Lys-104 and Leu-105 blue

shark

could not be found, there are extensive sequence identities with other vertebrate GHs. We, thus, propose the complete amino acid sequence of 183 residues for the blue shark GH as shown in Fig. 4. DISCUSSION This investigation provides, for the fist time, the complete primary structure from the most primitive species studied so far. Shark GH has four half-cystine residues that are characteristic of all vertebrate GHs. Amino acid sequence of shark GH is compared with those of other species of GHs by placing the conserved half-cystine residues in homologous positions (Fig. 5).

GH 10

20

Pro Leu Ser Asp Leu Pbe Ala Lys Ala 1*tad--------------------------------------------------------------------

Val

His

Arg

Ala

Gin

His

Leu

Lys

Tyr

Ile

Pro

Glu

Glu

Gin

30

His

Leu

Val

Ala

AITJ His

Ser

His

Lys

Glu

40

Ala Glu !Ihr Thr Lys Asp P&e Glu Aq Intact---------------------------------------------------------------------

(~*---------------50

Lys

SEC Ser

Pro

Ser

Ala

60

Phe Cys Gin

Ser

Glu

sp,2------------------------------------)

AspAIa

'I%r

Ile

Pro

Ala

Pro

Thr Gly

(spg------------------------------

GInGlnArg~?+spArgGlu

J&u I&

La

TyrSerLeuI~%Ieu

IleGlnSer

LF33-----------------------------------------------------------------------spg------------90

100

Trp~uAsnProIleGln~n~uSerRlaPheArg?hrSerAspArgVal~AspLys m33----------------------------------(IB============-===-==============-====--110

120

LeuArgAspLeuGIuGluGlyIIePheALaLeuMetLys~~uGlu~GlyGZySer

(-------------------------

(LE2,===-================-==================~ 130

Ser Gin Gly Phe Ala Trp Leu Lys ~-------------------------------------------------------------------

140

Phe

Ser

(E2,

'&T

Glu

Arg Pbe Asp Gly

3x=========z==

150

Glu Ala LeuMet Lys AmTyr Gly Leu Ieu Ala c3&----------) (~--------------------------------~---~-~~~-~~) LEg ==============)

FIG. 4. Proposed automatic sequence

Lys Val

I.eu Ser

Glu

160

Q's

Phe Lys

Lys Asp&t

Met Asn Cys Lys Krg phe Ala (~,-----------------------------------------~ (IJg 2=============)

HisLysVal (c@-------

(L,E3===G====)

170

Glu ti 'QT Leu a2----------------------I

?sn

========= zsz=======x

780

Glu

Ser

amino acid sequence of shark GH. (---) Represenfs analysis; (= = =) sequences defermined by manual

183

A.m Cys WC Val

CH

sequences determined sequence analysis.

by

STRUCTURE 20

30 +

+

huntan bovine chicken sea turtle blue shark

257

OF SHARK GH 40 *

50

60 +

+

70 +

80 +

90

10

+

+

AFm~m~Lm-1 TF~~~~~~~T= A~~~~NQI :* : ** **

*: **

:**

:t::*

:**

**it

*: :

*

c **:t*

*:

:*:*:

:::

***

:**:***+a*

*:I

*

YPLLPLSDLFAKAVHRAQHLHLVAAETTKDFERKYIPEEQRHS=HKSSPSAFCQSETIPAPTGKEDAQQRSDRELLLYSLLLIQSWLNPIQNLS=-=-=

eel chum salmon tmi to yellow hi1

110 +

120

130 +

140 i

150 +

160 +

170

180

~~IV~=S~~F

hwm bovine chicken sea turtle

SLvut;~~~~~PR~IF=~==T~~~D~

blue shark

==AFRTSD=RVYDKlRDLEEGIFALM~LEDGGSSQGFA~LK==FSYERFDGNL=SEEALk~~Y~LACFKKD~ETYL~~~FA~S~~~V

eel churn salmon bonita

SLMFC~GIF~~~G~IYI=EDV=R=NL

yellow

=GGSALRN=QISPIi@ED(~L~ITAN~MFSDVSALQLA~@YQS%&t&!#&@i% _____._____ _---_-_--cg -- ----- --

,, NL~~~&~E~PRGPQLL=R==P~~~~~~~~ SL~~~~~LRCFQVL=R==P~~I~~~ :: :

tail

190

**

I:: ::

*:r*r*r* :* :*

** *I

*:

***

: :: : **:

:;:**:*****t

:* :*I *:

*:

: :t*

r+:**:

* **:*:::

****t********iiP*

*

*:

:* :'

*:

:* **i

:

SL~~~=QIS~~~ITGS~L~DDNDSQQLP ~==GAQRN-QISE~L~[~~~MF~SS~QLA -- _.__

Dg ^--------------_-_--

FIG. 5. Comparisons of the ammo acid sequences of shark GM with salmon GH (Sekme et al., 1983, yellowtail GH (Watabiki et al., 1988; Kawazoe ef al., 1988) bonito GH (Noso et ai., 1988), eel GH (Yamaguchi et al., 1987), sea turtle GH (Yasuda et al., 1988), chicken GH (Souza et al., 1984), bovine GH (Graf and Li, 1974), and human GH (Martial et al., 1979). (*) Represents alignment position with residues identical to sea turtle GH; (:) alignment position with conserved residues; (=) indicates deletion. The residues identical to that of sea turtle GH are also shaded in each alignment position.

Deletions are introduced in several positions maximizing homology. The comparison reveals that shark GH is more similar to higher vertebrate GHs than to teleost GHs: it shows 58% identity with bovine GH, 63% with chicken,GH, 68% with sea turtle GH, and 61% with,eelGH, but-only 3840% with teleosts such as salmon, bonito, tuna, and yellowtail GHs. On the basis of such sequence comparisons, a molecular phylogenetic tree of GH can be constructed. Assuming that teleost and tetrapod GHs had evolved with a sim-

ilar rate of change, the shark GH branched between eel and tetrapod GHs in the phylogenetic tree. Therefore, Kawauchi and Yasuda (198’S) hypothesized that the phylogenetic tree qonsists of at least two trunks which have their own rates of evolutionary change and the rate of change of the’teleost trunk is larger than that of the tetrapod trunk. Thus, the shark GH is a key to construct the phybgenetic tree of GET. The above observation is in good accordance with the results of rat tibial activities and immunochemical relatedness of GN from

258

YAMAGUCHI

various vertebrate species. Hayashida and Lewis (1978) suggested that the blue shark GH appears to be less diverged from the main line leading to tetrapods GHs than are GHs from teleost, and the structure resembles those of tetrapods more closely than those of modern bony fish. The results obtained in the present study clarified their conclusions. Structure-function relationships of GH and PRL have been investigated on chemical modification of amino acid residues or chemical or enzymatic fragmentation of mammalian hormones. In contrast, comparison of hormone structures at many phylogenetic levels would provide useful information to predict possible relationships to biological activity. Nicoll et al. (1986) have discussed the structure-activity relationships of GH and PRL molecules on the basis of amino acid sequences of the hormones from homeothermic species and proposed the GH specific region, the segment 1-134. We have proposed that there are four highly conserved regions on the primary structure of GHs, located in alignment position 5-35 (A), 50-90 (B), 93-134 (C), and 158-184 (D) and the C region may be responsible for the activity specific to GH (Yamaguchi et al., 1987, Kawauchi and Yasuda, 1988). Although shark GH also shares these regions, there are a number of deletions between B and C domains in the alignment positions 95-108. Therefore, the third-conserved region can be shortened by 110-131 in the alignment. In conclusion, the complete amino acid sequence of blue shark GH was determined in the present study. This is the first report on the structural information from a more ancient species than teleost in the phylogeny. The sequence comparison revealed that the elasmobranch GH is considerably similar to tetrapod GHs than to teleost GHs, being consistent with the earlier observation from immunochemical and biological similarity to tetrapod GHs reported by Hayashida and Lewis (1978).

ET AL.

ACKNOWLEDGMENT This study is supported in part by a grant-in-aid from the Ministry of Education to H.K.

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STRUCTURE

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