Aplysia myoglobins with an unusual amino acid sequence

J. rVol. Riol.

Aplysia

(1984)

180, 1179-1184

Myoglobins

with an Unusual Amino Acid Sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana. a species distributed world-wide, has been determined and compared with t,he sequence of the myoglobin of Aplysia Zinuzcina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one. the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference. Rossi-Fanelli & Antonini (1957) were the first to isolate a new type of myoglobin from dplysia lima&a, a common gastropod mollusc from the Mediterranean, and subsequently Tentori et aE. (1968,1973) confirmed that it lacked the usual distal histidine residue. We have succeeded in isolating native oxymyoglobin (MbOz) directly from the radular muscle of Aplysia, kurodai, a common species around the ,Japanese coast, and have examined its spectral and stability properties; the spectrum of Aplysia MbOz is very similar t’o those of mammalian oxymyoglobins. but it’s stability is quite different (Shikama & Katagiri, 1984). Here, we describe the complete amino acid sequence of the myoglobin isolated from Aplysia juliana, a common coastal species throughout the world, and compare it with those of A. limacina (Tentori et al., 1973) and A. kurodai (Suzuki et al.. 1981). These sequence data seem to be of great interest if compared with that of sperm whale myoglobin as a reference, and may provide a basis for elucidating the role of the distal histidine residue and the heme environments in myoglobin chemistry as well as in myoglobin evolution. Specimens of A.,juliana were collected around the Onagawa coast, Miyagi. Japan. The myoglobin was isolated from the radular muscle and purified by gel chromatography on Sephadex G-75 followed by chromatography on DEAEc+ellulose as described (Shikama & Katagiri, 1984). Heme was removed by extraction with 2-butanone at acidic pH (Yonetani, 1967). The lyophilized apomyoglobin (25 to 75 nmol) was dissolved in 180 ~1 of 0.1 M-NH,HCO, (pH S-5), and enzymatic digestion carried out at 37°C for four hours by adding 20 ~1 of TPCK-treated trypsin (1 mg/ml in 1 mM-HCl; Worthington) or of staphylococcal protease (1 mg/ml in distilled water; Miles). The reaction mixture was subjected directly to high-pressure liquid chromatography to separate the resulting peptides. Larger tryptic peptides were furt)her digested with chymotrypsin (Worthington), and thermolysin (Daiwakasei), for three to five hours in 0.1 M-NH,HCO, (pH 8.5) at 37”C, or prolinespecaific enzyme (Seikagaku Kogyo) for 24 hours, or with staphylococcal protease 117’) 0022-%836/X4/361

179-06

$03.00,‘0

0

1984

Academir

Press

Inc.

(London)

Ltd.

1180

T.

TAKAGI

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(Miles) for 24 hours in O-1 M-phosphate buffer (pH 78) to cleave both aspartyl and glutamyl bonds. The apoprotein was also treated with heptafluorobutyric acid/formic acid in the presence of CNBr to cleave tryptophanyl bonds (0~01s et al., 1977). Purification of peptides was carried out by high-pressure liquid chromatography with a Hitachi model 636-50 instrument. The column (4 mm x 250 mm) was packed with Lichrosorb RP-8 (Merck) and equilibrated with 0.05 M-ammonium acetate (pH 6.8). Elution was carried out with acetonitrile in a linear concentration gradient, and monitored by measuring absorbance at 215 nm. Some of the peptides were further purified on a short column (4 mm x 150 mm), which had been equilibrated with O*1o/o (v/v) trifluoroacetic acid, and developed with acetonitrile in a linear concentration gradient. Peptides (1 to 5 nmol) were hydrolysed in 6 M-HCI containing 0.2% (w/v) phenol at 110°C for 16 hours in evacuated sealed tubes. Amino acid analysis was performed with a Hitachi 835-50 amino acid analyser. The amino acid sequence of peptides was determined by the manual Edman method, according to the procedure of Tarr (1977) but with some modifications (Takagi et al., 1983). Phenylthiohydantoin (PTH)-amino acids were identified by high-pressure liquid chromatography according to the method of Zimmerman et al. (1977). The

TABLE

1

Amino acid compositions of Aplysia myoglobins Amino acid residue Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

14.4 1.9 16.0 11.4 5.2 12.0 29.4 6.8 2.8 3.5 12.7 0.0 14.6 5.0 1.0 4.1 N.D.

Total t The values determined. $ Taken from 4 Taken from

Number of residues A. kurodaij

A. julianaf (15) (2) (18) (11) (5) (11) (28) (7) (3) (4) (13) (0) (15) (5) (1) (4) (2)

19 1 15 7 6 9 26 9 3 4 12 2 13 11 1 4 2

144 in parentheses Suzuki Tentori

are the

et al. (1981). et al. (1973).

A. limaeina§ 17 2 13 7 6 11 28 10 3 4 10 0 15 12 1 4 2

144 number

of residues

determined

145 by

sequencing.

N.D.,

not

LETTERS

TO

THE

11x1

EDITOR

reagents used for sequence determination were of sequanal grade obtained from Wako Pure Chemicals. Table 1 shows the amino acid composition of A. julianu myoglobin compared with that of A. kurodai and A. liwmcina myoglobins. There are clear differences in the content of glutamic acid, tyrosine and lysine. The amino acid sequence was mainly determined for the tryptic peptides by overlapping their subpeptides obtained by digestion with chymotryptic. thermolytic, or proline-specific protease. The overlaps between the tryptic peptides were made using the staphylococcal protease peptides and the tryptophanyl bond-cleaved peptides of the apoprotein. The N terminus of the whole protein was blocked, as in the case of other Aplysia myoglobins, but thermolytic digestion of the N-terminal chymotryptic peptide released acetyl-

A. k~urodai A. juliana A. limacina

AC-SW AC-AlaAC-Srr

Leu-Ser-

Glu Ala- Ala- Asp-AlaGlu

Lys Asp Ser- Asp- Ala- Asn- GlyLys Asn

Asp GlyAsp

10 Val Gly Leu-Leu-AlaAla Gly

20 Lys Tyr Gin- Ser- Trp- Ala- Pro- Val- Phe- Ala- AsnLys

30 40 Asn Leu Ser Glu Lys Asn Asn Ala- Ser- Phe- Leu- Val- Ala- Leu- Phe- Thr- Gln- Phe- Pro- Glu- Ser- AlaGlu Lys Asp ASP 50

Ala TY~ Asn- Phe- Phe- Asn- Asp- Phe- Lys- GlyAla

Ile Lys- Ser- Leu- Ala- Asp- IleVal

60 LYS Gln- Ala- Ser- Pro- Lys- LeuLYS

70 Arg- Asp-Val-

Ser- Ser- Arg- Ile-

Thr Phe-AlaThr

Arg-Leu-Asn-Glu-

Phe-Val-

80 Asn Ser- Asn- Ala- Ala- AspAsn Asp Asn

90 Ala- Gly-

Gly-

Ser Lys- Met-GlySer

Ala Ser Ser- Met- Leu-GlnAla Ser

Glu Ser. Ala- Gin- Phe-Gln-

Asn-Val-

100

SfX Val Gln- Phe- Ala- Thr- Glu- His- Ala- Gly- Phe-GlyLys Val

\‘a]-

110

I 20

Arg-Ser-

Met-Phe-Pro-

Ala Gly-

Phe-Val-

Ala- Ser. Leu-Ser. Val Ala

130 $]a-

Pro * Asp Pro* - Ala- Ala- Asp-AlaPro Gly Ala Asp

Lys Ala Gln- Ser- Ala- GlyLys Ala FIG. and A. centre Suzuki

Lys

LYS Ala- Trp- Asn-SerLeu- Phe-GlyThr

Leu- Ile-

Val Ile-

120 Ala Ser- Ala- LeuAsp

(OH)

1. Amino acid sequence of the myoglobin from A.juliana compared with those of A. kurodai liwzacina. Residues that differ in A. kurodui and in A. limacina are shown above and below the sequence, respectively, and a deletion is indicated by an asterisk. Sequence data are taken from et al. (1981) for A. kurodui, and from Tentori et al. (1973) for A. Zimacina.

1182

T.

2 m .c 2 f Et P T I

TAKAGI

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AL.

2.0

0

-2.0

-4.0

-4.0

I 1

I

t

1 0

1 50

I 100 Sequence

FIG. 2. Hydropathy profiles of Aplysia myoglobins of 7 residues. See the text. (a) A. juliana myoglobin residues); (c) A. Zimacina myoglobin (145 residues).

I 150

number

along the amino (144 residues);

acid sequence (b) A. kurodui

at a span setting myoglobin (144

alanine, which was determined as a form of dansyl-acetylhydrazine and dansylalanine from its hydrazinolysis and subsequent, dansylation according to our previous method (Suzuki et al., 1981). A. juliana myoglobin was composed of 144 amino acid residues, was acetylated at the amino terminus, and contained a single histidine residue at position 95,

LETTERS

TO

THE

11x3

EDITOR

which most likely corresponds to the heme-binding proximal one, characteristic for all Aplysia myoglobins examined so far. Its complete amino acid sequence is shown in Figure 1, where it is compared with those of A. kurodai and A. limacina myoglobins. The overall degrees of similarity in the sequence are very high, as shown below: A. kurodai

A. juliana

A. limacina

Figure 2 shows the hydrophilic and hydrophobic profiles of Aplysia myoglobins along their polypeptide chains. In these computations, made by the method of Kyt.e & Doolittle (1982), a hydropathy scale was adopted in which each amino acid is assigned a value ranging from + 4.5 for isoleucine to -4.5 for arginine based on the transfer free energy (kcal mol -I. . 1 cal = 4.184 J) of each side-chain from condensed water vapour to water. We then determined the average hydropathy within a segment of seven residues as it advances through the sequence by starting at the amino-terminal residue and moving on by one residue. The resulting score is plotted above the middle residue of each segment. The first value therefore corresponds to the average hydropathy of residues 1 to 7 and is plotted at location 4, the second value corresponds to the average value for residues 2 t’o 8 and is plotted at location 5, and so on. At the same time, a midpoint line of -0.4 was adopted to correspond to the ground average of the hydropathy of the amino acid compositions found in most of the sequenced soluble proteins (Kyte & Doolittle, 1982). It is clear from Figure 2 that the hydropathic profiles of Aplysia myoglobins obtained from three speciesin different distributions are very similar. This may be a strong indication that Aplysia myoglobins follow a very similar geometry in

I 50

1 100 Sequence

FIG. 3. Hydropathy profile of sperm whale at a span setting of 7 residues. See the text.

myoglobin

I 150

number

(153 residues)

along

the amino

acid sequence

11x4

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AL.

their globin folding. It was therefore of great interest to compare the hydropathic profiles of Aplysia myoglobins with that of sperm whale myoglobin as a reference, which was then computed (Fig. 3). It is evident that Aplysia myoglobins are quite different from sperm whale myoglobin in their hydrophilic and hydrophobic characters along the amino acid sequence. Whereas sperm whale myoglobin shows a large hydrophobic lobe with its maximum centered at, position 70, corresponding to the region containing the distal histidine residue (E7) at position 64, Aplysia myoglobins do not show such a hydrophobic character on the distal side of the heme iron. Instead, two lobes of strong hydrophobicity centered at positions 117 and 136 in the carboxyl-terminal end are characteristic for all Aplysia myoglobins. Our previous observation also indicated that the magnitude of the circular dichroism of myoglobin at 222 nm is markedly different from the value of -24,000 (+500) deg. cm2 dmol-’ for sperm whale, being - 18,500 (+ 1000) deg. cm2 dmol- ’ for A. kurodai (Shikama et al., 1982). In conclusion, all of these structural data seem to indicate that the folding of the globin of Aplysia myoglobin is quite different from that of mammalian myoglobins. We are indebted to T. Suzuki, T. Nemoto and T. Katagiri stage of this work. Biological Institute, Sendai 980, Japan

Tohoku

University

for their assistance at an early

TAKASHI TAKAGI SHIGEO IIDA ARIKI MATSUOKA KEIJI SHIKAMA~

Received 23 July 1984

REFERENCES Kyte, ,J. & Doolittle,

R. F. (1982). J. MoZ. Biol. 157, 105-132. Ozols, J., Gerard. C. & Stachelek, C. (1977).J. Biol. Chem. 252, 5986-5989. Rossi-Fanelli, A. & Antonini, E. (1957). Biochimie, 22, 336-344. Shikama, K. & Katagiri, T. (1984). J. Mol. Biol. 174, 697-704. T., Takagi, T. & Hatano, M. (1982). Shikama. K., Suzuki, T., Sugawara, Y., Katagiri, Biochim. Biophys. Acta, 701, 138-141. Suzuki, T., Takagi. T. & Shikama, K. (1981). Biochim. Biophys. Acta, 669, 79-83. Takagi, T.. Tobita, M. & Shikama, K. (1983). Biochim. Biophys. Acta. 745, 32-36. Tarr. G. E. (1977). Methods Enzymol. 47, 335-357. Tentori, L.. Vivaldi. CT., Carta, S.. Antonini, E. & Brunori, M. (1968). Nature (London), 219, 487. Tentori, L.. Vivaldi, G.. Carta, S., Marinucci, M., Massa, A.. Antonini. E. & Brunori. M. (1973). Jnt. .J. Pept. Protein Res. 5, 1877200. Yonetani, T. (1967). J. Biol. Chem. 242, 5008-5013. Zimmerman, C. I,., Appella, E. t Pisano, J. J. (1977). Anal. Riochem. 77, 569-573. Edited

by G. A. Gilbert

t Author to whom correspondence should be sent.