Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins

Biocldmica et B?ophysica Acra. 1074(1Oql) 371 377

371

© 1991 ElsevierScience PublishersB.V. 031}4-4165/91/5113.511 ADONIS 031]441b591fJG292E BBAGEN 23553

Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins M i c h a e l E, U n g e r L2, T h o m a s T. C h e n .~.4 C o n s t a n c e M . M u r p h y 5 M a r t h a M . V e s t l i n g 5, C a t h e r i n e F e n s c l a u 5 a n d G . R o e s i j a d i ~,~,3 i Unit'erdty of Maryland. Chesaveake Oiologieal Laboratoq, Svlomons. MD (U.S A.). "~Umlersdty of Marylattd Prod,ram in T~cicolo,~,'. BafHmore, MD (U.S.A.). ~ Unirersay of Ma~'lm:d. ~k'tttt'rof )larme Biot¢chnviolzy. ~ultimore. MD (U.S.A.L z Unhersiry of Maryland-Baltimore Courtty, Department of Biological Sciences. Bal#mare. MD (U.S,A3 and 5 Unwersi~,o~ Mu~'land.Ballimore County Department of Chemist~, and Bioct]emisl6. and StmOnral Biochemisto' Center.Ballimore. MD (U.S,4.)

(Received 27 December Iqqlll (Revisedmaauscrip!received27 March ~q01)

Key words: Metanathionein:eDNA; Pnlymeras¢chain reaction: PCR: Tandemmassspcctrometff;Cadmium;N-Acet~lation: I.Motluse)~(G~'s'ie~")

The primary structure of metallothloneins (MT) of a mollusc, the oyster C ~ virginica, was determined by molecular cloning and mass spectrometry of purified proteins, The cloning strotesy included PCR amplification of the responsible ¢DNAs from total eDNA using completel,v degenerate oligonucketides (derived from the N.terminal amino acid sequence) and oligo(dT)zo as primers. Primer extension off mRNA was used as an independent determinaUon of t~e nudcotide sequence represented b) the degenerate PCR primers. The deduced amino acid sequence w ~ consistent with characteristics of" class I MT. Twenty.one cysteine residues, were arranged in nine Cys-X.Cys motifs, five as Cys-Lys.Cys. A single Cys-X-X-Cys motif was a l ~ observed. Two NITs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of ~ k peptides of purified Mrs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid seqtwnoe, Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with these of other species indicated a higher similarity with vertebrate ['wiTsthan with those ef other invertebrates.

Introduction Metallothioneins (MT) are inducible, low molecular mass, c~steine-rich, metal-binding polypeptides whose functions are believed to include the detoxification and intracetlular regulation of metals [1-3]. They are ubiquitously distributed among animals, plants, fungi, and prokaryotes. Associated diversity in biochemical struc. tore has ted to the proposal of a classification scheme

Abbreviations: MT, metallothione[n; PCR, polymerasechain reaction: bp, base pail,s); CvNAcMTand C,/MT, acetylated and nonacc~lated formsof me/al1015ioneinof Crassostrea rirginica. Correspondence: G. Rnesijadk Univcrsi~ tff Maryland, Center fur Environmentaland Estuarine Studies, Chesapeake BiolusicalLaboratory, P.O. Box 38, Solomons.MD 20688,U.S.A. The sequence data in thi~ pal~:r have bccn submitted to the EMBL/Geabank Dam libraries under the accessionnumberX59862+

that separates MTs into three classes based on primary structure and mode of synthesis [4]. Class I MTs are proteins whose p6mary structure is similar to that of equine renal MT [5], particularly in the locations of cysteine residues, which are conserved in several characteristic patterns (Cys-X-Cys, Cys-X-X.Cys and CysCyst. They include all vertebrate MTs, some invertebrate MTs (o.g., arthropods [6]) and MTs of some fungi 17]. Class II MTs are proteins with oysteines in positions only distantly related to those of equine renal MT and, thus far, include MTs of sea urchins [8], nematodes [9,10], yeast [11], and cyanobacteria [12]. Class III MTs are nontranslationally-synthcsized polypepfides and have only been reported in plants and some fungi [13]. The MTs of invertebrates are relatively diverse in structure and are included in both class ! and Il. However, the species studied to date represent a relatively small number of the invertebrate phyla and provide only a narrow picture of the evolutionary diversity that may actually exist.

372 In the invertebrates, complete amino acid sequences for MT have been deduced from nucleotide sequences in the nematode Caenorhabditis elegans [9,10], sea urchin Strongylocentratw purpuratu~ [8], and fruit fly Drosophila melanogaster [14,15], which are important as biomedical models. That of a crab, Scylla serrata, was determined by protein sequencing techniques [6]. Similar work has yet to be reported for other invertebrate species that are important in understanding responses of natural populations to metals. Recently, procedures were developed for the isolation and purification of MTs of an oyster, Crassostrea vir#Mca [16], a member of the relatively large molluscan phylum for which detailed information on MT structure was not previously available. This species possesses two MTs that differ only in that one is blocked at the N-terminus by a hydrophobic structure, which has been identified as an N-acetyl group [17]. N-terminal amino acid sequcnce analysis indicatod that MTs of this organism are more similar to vertebrate MTs than to MTs of other invertebrates [16]. We used this information to clone and sequence the MT eDNA in the present study. The deduced amino acid sequence was compared with the results of mass spectrometry of peptides generated from proteolytic digestion of purified MTs. The findings from molecular se..quencing and mass spectrometry were in accord with each other and the N-terminal amino acid sequence of the oyster MTs reported previously [16]. They supported classification of these proteins as members of class I MT.

Experimental procedures Collection of tissue Oysters used for molecular cloning of MT eDNA were obtained from the Chesapeake Bay Oyster Culture Company and maintained in tanks of flowing Chesapeake Bay water. Individuals were exposed to 50 ~g i -t Cd 2÷ as CdCl2 in bay water under static conditions for 14 days. Oysters were transferred to fresh exposure medium every second day, After the exposure, gill tissue was excised, frozen in liquid nitrogun and stored at -70°C. The tissue sample represented the pooled gills of several individuals. Oysters used for isolation of MTs to be analyzed by mass spectrometry were exposed to 200 ~.g 1-1 Cd 2÷ for 21 days as previously described [16].

Isolation of RNA Total RNA was extracted from the frozen tissue using a modification of methods based on extraction in guanidinium isothiocyanat¢ [18,19]. 10 g of tissue were ground to a powder in liquid nR:ogen with a mortar and pestle, homogenized[ with a Tekmar probe at room temperature in 230 ml GT buffer (4 M guanidinium isothiocyanate, 20 mM Tris-HC2 (pH 7.6) and 1 M

2-mercaptoethanol added immediately before homogenization), then adjusted to pH 5 with glacial acetic acid (100/zl). The RNA was precipitated with a half volume of ice-cold ethanol, incubated for 30 rain at -20°C and centrifuged at 39000 × 8 for 30 min at 4°C. The pellet was subjected to three rounds of resuspension in 40 ml GC buffer (8 M guanidinium hydrochloride, 20 mM sodium acetate and 20 mM sodium EDTA) and precipitation in ethanol. This was followed by an additional three rounds of suspension in 20 ml 20 mM sodium EDTA at 60°C, precipitation with 3 vol. of 4 M potassium acetate (pH 5.2), incubation at - 20°C for 30 rain, and centrifugation at 39 000 x g for 30 rain at 4°C. The pellet was then dissolved in 20 ml H20, heated to 60*(2 for 15 rain and centrifuged at 39000×g for I5 rain at 10*C. The supernatant was made up to 0.3 M potassium acetate and RNA was precipitated overnight by adding 2.5 eel. of ethanol. The final pellet was dissolved in H20, and the total RNA concentration was determined by measuring absorbance at 260 nm. PoIy(A) ÷ RNA was isolated by affinity chromatography on ollgo(dT) cellulose [20].

PCR amphfication of MT ~ecifw eDNA PeR from eDNA was based on the protocol of Berchtold [21]. The template (10 n8) was total first strand eDNA [22]. Primers (t00 ng each) were a mixture of all possible codon combinations of 20-mer oligonucleotides ( 5 ' - G A ( T / C ) C C ( T / C / A / G ) TG(T/C)AA(T/C)TG(T/C)AT(T/C/A)GA-Y), derived from the N.terminal amino acid sequence of the oyster MT [16], and an oligo(dT)z0. A Sail restriction site and six additional nudeotides at the 5' end of both sets of primers were included to facilitate subsequent manipulation of the DNA. The Cctus GeneAmp Reagent Kit was used according to manufacturer's instructions with a single modification to optimize reaction conditions: the MgCI 2 concentration was increased from 1.5 to 2.5 mM. MT-encoding eDNA was amplified in three rounds of PCR, 25 cycles per round. Reaction products were screened by Southern hybridization using a 39-met oligonuelcotide probe (5'GCATGCGCAAGTGCCAGTITCAATGCAGTTACAAGGATC-3 °) derived from the N-terminal amino acid sequence of the oyster MT [16]. In the first round, the denaturation, annealing and extension temperatures were 94, 37 and 72~, respectively. The reaction product was separated by 1% agarosc gel clectrophoresis, and the band containing the putative MT eDNA was excised and purified with GencClcan (Biol01). This eDNA was subjected to two additional rounds of amplification by PCR and purification by electrophoresis. The annealing temperature was increased to 51PC in the latter reactions.

373

Cloning and sequencing of PCR amplified eDNA The PCR amplified eDNA and the plasmid pGEM3Z (Promega) were digested with SaII and ligated with T 4 DNA ligase. Eschenchia coli (strain JM109) cells were transformed with the ligation mix [23]. Colonies were screened by colony hybridization [24] using the 39-met probe. Plasmid DNA from positive clones was isolated [24], digested with SalI and screened by Southern hybridization using the 39-mer probe. Both strands of c]ones containing appropriate inserts were sequenced directly in pOEM-aZ [25] or subcloned into the single strand sequencing vectors M13mpl8 and M13mpl9 and sequenced [25]. It is known that the use of completely degenerate primers for PCR may lead to less than perfect matches between template and primer, resulting in amplified products with sequences in the the primer region that may not accurately represent the sequence of the original template [26]. Therefore, the sequences in the regions corresponding to the N-terminal PCR primer and the 5' noncoding region were obtained by direct sequencing off the poly(A)+ RNA by primer extension [27] with reverse transcriptase and a primer derived from the nucteotide sequence determined above for the clones+ The complete eDNA sequences were constructed as a composite of sequences obtained from the clones and from the primer extension reaction.

crete band of approx, 400 base pairs (bp) (Fig. 1A, fane I), which corresponded to the expected size of 250-400 bp for the oyster MT eDNA. The eDNA in this region was isolated from the gel and used as the template in two additional rounds of PCR. Hybridization of the 3q-mer probe in Southern blots was enhanced with the additional PCR amplifications (Fig. IA, lanes I| and Ill). The final PCR product (Fig. 1A, lane III) was cloned into pGEM-3Z. Four E. coli colonies hy. bridized to the 39-mer probe during screening of clones, two of which were shown by Southern hybridization to have positive inserts. Both of the latter were 400 bp in size (Fig. IB), which is consistent with that of the PCR products. The PCR-amplified-cDNA clones differed from each other at positions of three nuclcotides, two at the degenerate third position of cations and one in the 3' untranslated region. They included identical sequences in the region corresponding to the PCR primers and, therefore, were the products of polymerase chain reactions initiated by the same primer sequence in the oligonucleotide mixture (sequence of the responsible PCR primer not shown). The putative polyadenylation sequence, AATAAA. was identified 16 nucleotides bee. p C ~ l - ~ elm~es

&IsCFIPrcgtvo~) i

II

II

I

111

Mass spectrometry of MT tryptic peptides Two oyster MTs, C'vMT and CvNAcMT [17], were purified, earboxyamidomerhylated and digested with trypsin as described previously [t6]. Resultant peptides were dissolved in 0.1% trifluoroacetic acid and analyzed in thioglyceroi or 3-nitrobcnzyl alcohol with a JEOL H X I I 0 / H X l l 0 tandem mass spectrometer (EBEB) (Tokyo, Japan). The sample was desorbcd by xenon fast atom bombardment. The accelerating voltage and the resolution were 10 Kv and 100~, respectively. The observed masses of MH + ions were reported as the most intense peak in each isotopic cluster [2g]. Observed masses of the tryptic peptides were aligned with portions of the amino acid sequence deduced from the nueleotide sequences, Results

PCR amplification of MT specific eDNA PCR was used to amplify MT eDNA sequences since initial studies indicated an apparent low abundance of the responsible mRNA (Unger, M.E., Chert, T.T. and Roesijadi, G., unpublished data). After the first round of PCR, ethidium.bromide stained electrophoretic gels showed a smear of cDNAs of different sizes along the length of the gel Southern blot analysis with thc 39omer probe resulted in binding to high mok+alar weight components in the smear and a dis-

•..-~,.: -2

-1 -~a 2

-4

-4

-5

-S -S

'

.

!i!!_

.':: .

• --7

"7

Fig. I. ,%uthem hybridization of P(::R-anzplified oyslcr ~ eDNA and isotated pGEM-3Z clones. L~) PCR-amplificd eDNA was elcc-

ttophore~d on a 1% asamse gel, transferred to n.y~n membrane and hybridized to 5' end-]ahe[ed39-mer probe
dem:rihed ia A. The molecular weight markerswere lambda phage -DNA digestedwilh Hind II1. The sizes for the markers are l, 23.1 kb;,2, q,42 kb; 3, &68 kb; 4, 4.36kb; 5, 2.32kb; 6, 2.03 kb; 7. 0.56 kb (kb is kilobasc pairs).

374

Cys-X-X-C,ys motif (i.e., Cys-Lys-Cys-Ala-Oly-Cys), No Cys-Cys arrangements existed in the sequence. In the deduced amino acid sequence, the initiator methionine is adjacent to the serine previously reported as the N-terminal amino acid in the mature protein [161. Removal of this methionine would result in a mature protein with 74 amino acids and a mass of 7187 Da. The protein is rich in glyeine (12 residues; I6.2%) as well as cysteine (28.4%) and, characteristic of MT, lacked the aromatic amino acids and histidine.

fore the start of the poly A sequence, indicating that the complete Y region of the eDNA was isolated. A single sequence corresponding to the 5' end of the eDNA was detected by primer extension and assigned to both clones. This sequence contained the coding region corresponding to the PeR primers, the start codon ATG and the 5' noncoding region. It differed from the sequences of the two PCR-amplified clones at three positions in the region of the PeR primers. For reasons discussed in the Experimental procedures section on the possibility of less than perfect matches between PeR primers and template, the sequence obtained by primer extension was regarded as a more accurate representation of the PeR primer region. Thus, the entire cDNA sequences for the oyster MTs (Fig. 2) were constructed as composites of the sequences obtained from the two pGEM-3Z clones of PCR-amplified eDNA and the single primer extension sequence. These nucleotide sequences encoded identical proteins of 75 amino acid residues and a molecular mass of 7318 Da (Fig. 2). Characteristic of MT, the amino acid sequences contained 21 ~steine residues arranged in nine Cys-X-Cys motifs (five of these are Cys-Lys-Cys). Two cysteines not included in Cys-X-Cys motifs were adjacent to lysines. One of the cysteines (residue 40) belonging to a Cys-Lys-Cys was also involved in a

Mass spectrometry of MT tryptic peptides Tandem mass spectrometry showed that this oyster MT exists in acetylated and nonacetylated forms [17]. Tryptic digestion of these MTs resulted in peptides whose masses corresponded with those predicted from tryptic cleavage of the deduced amino acid sequence if N-terminal modifications are considered (Table I). Peptides identified as T1 are the N-terminal peptides which differ in mass due to the N-acetyl group on CvNAcMT [17]. TI of both proteins corresponded to residues 2 to 25 of the deduced amino acid sequence if the N-acetyl group of C-X,NAcMT was not included in the comparison. The masses of the remaining peptides corresponded to those of predicted peptides that would result from ttyptic cleavage of the deduced amino acid sequence. A dipeptide VaI-Lys at residues 43 and 44

ptk~r rl~lion for Pt;R primer ¢'.~11~2

%w2CACAGACA CAAA~%ACT TeACCTTTAT ATAA~J~ A ~ G T C T GAC ~ ..............................................................

5'

~GT

AAC

TGT

ATT

GAG

63

Met Set Asp P~O CMJ Ann Cym Ila G ~

primer ~k,n 64 ACT GGC ACC %X3T 6CG TG? qCO CAT TCC ~

fo~ p,'imere~lonsion

CCA ~2G ACe GGA TGT ~AA ~

GCa% ~

(~CaK 123

............................................................

Thr GIy Thr CyB Ala emil S~r AS~ Set e¥~

Pro A/a ~

~.ly e'2a byS C ~

81y PrO Gly

..........................................................

eye LyS eMIl O~[y Asp ASp eMn 184 GAA

~

. . . . .

C4~C T~T A

A~A

T~T

GGG

Ly6 eyn Ala Gly eyn I/~s val ~,s c~ll ~

~¥ii Thr 8or

GAG

~

AAG

TGC

Ace

GGT

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

elu Gly Gly eys Lys Cys r.ly Glu Lys eym ~ r 244 (~GC TGC AC,C TGC

AAG AAG TGAC-CGCTCG ............................

C

CCC

GCA

ACC

I~C

AAA

GGA

~

243

. . . . . . . . . . . . . . . . . . . . . . . .

GLy Pro Ala Tbz ey$ 5ys Cy| Gly Set

GCU.AATGAGA GGCGTTCTCC TGTACCGAGT A.............................

3CI

GIy C~,s Set cys ny~ Lys 3C2

TC~TGTATTG ATCTATTGI~ CTAAAAATAT TOT?AATATT ............................................................

C-C-GATCd~A'~L"1~

.......................................

ATTT~ATAAA

361

3'

Fig. I cDNA sequences for oyster MT obtained from PCR-amplified eDNA clones and primer extension off mRNA. PCR-amplified cDNA clones were sequenced in beth pGEM-3Z plasmid and M13mplS/M13mp19 bsctoriophage sequencing vectors by the dideoxy chain termination method [25]. The 5' resion of the eDNA was sequenced by primer extension off the mRNA [27], This region of DNA included tile N-terminal

PCR primersand eDNAupstreamof Iheseprimers.An oligonucleulidederivedfromthe pGEM-3Zcloneswas usedas the primerfor lhc primer extensionreaction.The entireeDNAsequencewasconstructedas a compositeof ~equcnccsobl ,,nedby the twomethods.

375 "FABLE I Obsen'ed and predtcted masses of tG~ptic z:e:~ide~rfor ¢'c~rbn.',3'amldomethytated (~ M7 and CcNAcMT and their afignmem ~'fth the sections of the rleduced amino acid seqoence that corre~y~ond m ma.s.~

Observed ma,~se~of try.pt[c peptidea wc~c determined by mass sI~eclr~metr':as described in lhe text. The predicted masxeswere calcttlated for peplides that would resull fromthe predicted try~rie cleavagesof lhe ~lminoacid sequencede~luced fromthe eDNA.The peptidea, numberedTI tO T8. were listed [a order of increasingdistance from ~he Nqerr~[nus(TI is the N terminal !0eplide~.Values fur (~bservedma~,~esare for the mosl i~teme peak in each isot(rplcch~sler[28]. I0 2() 311 ~11 50 60 70 MSDPCNCIUTGTCACSDSCPATGCKCGPGCKCGDDCKCAGCKVKCSCI%EGGCKCGEK(71"GPATCKCGSGCSCKK I-

I

T1

i

T2

I

T3

~

T4

t

I

"1~5

Peplide [Residues]~'

CvMT observed m / :

CvNAcMT observed m / :

"rt 12-251 a

Z71~9]~ 678.5 754.7 595.4 1145.9 4q3.4 894.8 q15.8

2751,5 fi783 754.3 595.3 1145.6 4q3.4 894,4 915.2

T2 [26-31] T3 [32-371 T4 [38-42] T5 [45-54] Tfi [55-58] "1"7159-66] T8 167-74]

1

T6

l

T7

II

T8 E~l~cted MH" -~71610' 2752,0 a 678.3 754.3 595.2 1145.4 493.2 894.4 t)15.3

e Peptides containingresidues43 through44 and 75 were not detected. Low ma.~sitmssuch as these are often not distir~goishablein the matrix haekgroun d , b The initiator methionine in peptide TI is deleted in the mature protein [t61 and was omitted in cornpari~onsbelweenob~rved and e~peetea ~,a Calculated without (e) and with Id) an acety] group on the N-terminus[17].

and the carbox'yt terminal lysine were not detected. Smalt peptides such as these are often not distinguishable in the matrix background. Overall, 96% of the primary structure of the sequence was confirmed by mass spectrometry,

Discussion Although molluscan MTs have been the subject of numerous studies that have dealt with the regulation and toxicology of metals [3], few detailed studies on the biochemistry of MTs in this widespread phylum have been reported. Recent success in purification of MTs from the o t t e r and information derived from N-terminal amino acid sequence analysis [16] enabled us to develop a strategy for determination of the complete primary structure based on two approaches: (i) development of probes and primers to be used in PCR of MT eDNA, followed by molecular cloning of amplified products and (ii) use of mass spectrometry for analysis of proteolytically-generated peptides of purified proteins. With these two approaches, we were able to deduce the entire primary structure from the eDNA sequence and confirm this sequence by direct comparison of masses of specific proteolylically-generated peptides with those of peptides predicted to occur from the enzymatic cleavage of the deduced sequence. The results were entirely consistent with an earlier determi-

nation of the N-terminal amino acid sequences of two nearly identical MTs [16]. In the earlier study, the N-terminal amino acid residue of the oyster MTs is serine, and one of the two proteins carries an hydrophobi¢ N-terminal block, the only feature that distinguished the two from each other. The N-terminal block was later identified as an acetyl group by tandem mass spectrometry [17]. By comparison of the N-terminal peptide sequence with the amino acid sequence deduced from the nucleotides, it is clear that the N-terminal serine is the penultimate ceded residue and that the ,,'nitiator methionine has been removed, most likely as a result of co-translational events [29,30]. Thus, the mature protcin differs from the coded amino acid sequence by the lack of this methionine residue. The presence of the N-acetyt group on one of the oyster MTs is expected if currently-accepted general rutes [29,30] regarding Nterminal acetylation of proteins applies in our ease. The presence of the unblocked MT is not predicted and may represent a precursor to the acetylated form, which accumulated in the cell as a result of cadmium exposure [17]. During the isolation of the oyster MT eDNA, two clones of nearly identical nucleotide sequences were observed. The two sequences differed in three base substitutions that did not alter the deduced amino acid sequence since they were at the third position of cations

376 or in the 3' noncoding region. Several explanations for this situation exist. First, genetic polymorphism among individual oysters may have resulted in the presence of two nearly identical nucleotide sequences in our preparation since poly(A) + RNA was isolated from the pooled gill tissue of several animals. Second, gene duplication may have resulted in the evolution of two very related sequences for the oyster NIT. Third, the lower fideli~ of Taq polymerase in comparison with other DNA polymerases [31] may have resulted in minor cloning artifacts introduced by the PCR. It should be noted here that a single unambiguous primer extension sequence was derived from the same total mRNA that was used as the template for PCR. Exhaustive screening and characterization of oyster eDNA and genomie DNA libraries for MT gene or genes of individual oysters can be used to test for the above possibilities. However, the origin of the two sequences is of minimal consequence to the structure of the gene product, since the two coded for the same amino acid sequence.

The single deduced oyster NIT amino acid sequence assigned to the two clones was compared with those of all other MTs in the NBRF, OenBank and EMBL databases with the FASTA algorithm [32] as implemented by the Genetics Computer Group's Wisconsin Package I33]. Both percent identity and percent corresponding residues ( = identical residues plus conservative replacements) were also determined for comparisons of the oyster MT to the trout MT-A [34], representative mammalian MTs [35-37], and invertebrate MTs [6,8-10,14,15,381 (Table !I). Overall, FASTA scores indicated .greater similarity of oyster NITs to vertebrate MTs than to those of invertebrates. They

could be divided into two groups with higher scores ranging from 135 to 159 fur comparisons with all vertebrate MTs and lower scores of 91 to 135 for comparisons with all invertebrate MTs. Additionally, it should be noted that the oyster MTs were more similar to the class ! MTs of vertebrates in comparison with the class I MTs of invertebrates. The percent similarity, which ranged from 31 to 41% identical residues and 64 to 83% corresponding residues,' did not exhibit discernable patterns of affinity of the oyster MTs to either group. However, the significance of the percentage values for the invertebrate MTs was decreased relative to those obtained for the vertebrates due to the shorter length of the overlap producing the former values. The FASTA scores, on the other hand, take into account the length of overlap as well as the percent identical and corresponding residues. On this basis, molluscan MTs are more similar to the vertebrate M r s than to other invertebrate MTs, a premise originally proposed as a result of analysis of the N-terminal amino acid sequence of oyster MTs [16]. In addition to the presence of a high number of eysteines and the numerous Cys-X-Cys motifs, the existence of the Asp-Pro doublet in the N-terminal region was particularly intriguing since it occurs in all vertebrate MTs and none of the invertebrates except the oyster. Furthermore, the similarity with the fish MTs in this region can be extended beyond the doublet to include Asp-Pro-Cys-X-Cys as a local region of high similarity. Mammalian MTs exhibit an insertion of asparagine immediately following the Asp-Fro doublet. The high glycine content appears characteristic of molluscan MTs, based on comparisons with the amino acid contents of other species [39]. The lack of the initiator

TABLE [l FASTA scores, % identity, % corresponding redlsdltes and length of t~vfdapping region for the comlmrtson of the deduced amino arid sequence of the oyster MT with representative vertebrate and invertebrate MT amino acid sequences

Comparisonsare ranked by highestFASTAoptimizedscore. Species

Clan a

OptimizedFASTAscore

Percentidentity

Percentcorrespondence Lengthof overtap

Re[.

Verlchlarcs Trout MT-A Human MT-IB

] I

159 155

37.5 4L.0

7f.g 72.1

64 61

[34] [35]

RabbitMT-I Mouse MT.I

[ [

154 153

37,1 38.7

74,2 70.1

~2~ 6Z

[36] [37]

Sea UrchinMTa Crab MT-2 Crab MT-I Sea UrchinMTh

Ii I

3%0 33.3 31.0 33,3

74.1 71.7 72.4 68,3

54 61} 58 63

[8] [6]

11

135 133 127 127

Nematode MT-I

It

124

40.4

70,1

57

lgl

DrosophilaMTn DrosophilaMT0 NematodeMTQ

I 1 11

114 100

4L7 41.4 32.2

83,3 S2.8 64.4

36 29 59

[14] [15] []0]

Invertebrates

]

91

~6]

438]

" Classification according Io Fowler el al. [4], The oyster MTs reported in this study were classified as c]ass | NITs accordins to these criteria and were most similar to the vertebrate class t MTs.

377 methionine was similar to reported structures of most other invertebrate MTs [2]. Motifs comprised of X-Cys-X-Cys-X-X-X-Cys-X-Cys or Cys,X.Cys.X,X-X-Cys-X-Cys-X are representative of the 'central segment" of MT, which is proposed as a characteristic feature of the molecule [8]. W e detected several regions in the oyster NiT sequence that were consistent with the features of this central segment. Residues 65 to 74 align exactly with the first aloe of the 10 amino acids in the central segment of the sea

urchin MT [8] if this local region is taken out of the context of the overall alignment. The significance of this relationship is not clear, and analysis of MTs from other taxonomic groups may clarify the biological importance of the 'central segment'. In summary, we have deduced the entire primary structure of the oyster MT from the eDNA sequence and confirmed the structure by mass spectrometry. The mature protein is N-terminally-modified and lacks the initiator methionine. Thus far, it appears that these oyster MTs are more similar to MTs of vertebrates rather than other invertebrate species. Sequences of other molluscan kiTs have yet to be reported. Interestingly, the protein occurs as acetylated and nonaeety-

luted forms [17] whose significance has yet to be established. Acknowledgements We acknowledge Drs. C-M. Lin and D,B. Bonar for technical advice and assistance, and Drs, D.A. Powers and IL Colwell for initial encouragement, Dr. Powers provided use of his laboratory facilities during the initial stages of this study. The faculty of the Summer Workshop on Molecutar Biolo~ (held at Smith College) provided valuable insights on cloning strategies. This study was supported in part by Grants DI~.FG0586ER60469 (U.S. Dc1~artment of Energy; GR), 14-080001-G1900 (U.S. (~;,Aogical Sutwev; GR), BBS 8% 14238 (National Science Foundation; CF), 14-08-001G1496 (U,S.G.S.; TTC) and University Research Initiative from the Office of Naval Research (TIC). References 1 Hamer, D.H. (1986) Annu, Rev, Bioehem. 55, 913-951. 2 Kasi, J.H.R. and Kojlma, Y. (1987`1in Metallothionein il {Kagi, J.H.R. and Kojima,Y,, uds.), pp, 25-61, Birkhauser-Veflas, Basel. 3 Easel, D.W. and Btouwer, M. (1989) Adv. Cutup. Environ, Pllysiol. 5, 54-75. 4 Fowler, B.A., Hildebrand, C.E., Kojim~ Y. and Wcbb, M. (1987) in Metallothionein It (Kagi, J.H.R. and Kojiraa. Y., eds.), pp. 19-22~ Birkhauser-Verla$, Basel. 5 Kagl. J,H.R. and Vallee. B.L. (1960) I. Biol. Chem. 235, 34603465. 6 Lurch, K., Ammer. D, and Olafson, R.W. (1982) L Biol. Chem. 257. 2420-2426.

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