Characterization of the Myoglobin and Its Coding Gene of the Mollusc Biomphalaria glabrata*

Sylvia DewildeDagger , Birgitta Winnepenninckx§, Marcio H. L. Arndt, Danielle G. Nascimento, Marcelo M. Santoro, Matty Knightparallel , Andre N. Millerparallel , Anthony R. Kerlavage**, Neil Geoghagen**, Eric Van MarckDagger Dagger , Leo X. Liu§§, Roy E. Weber¶¶, and Luc MoensDagger ||

From the Departments of Dagger  Biochemistry and Dagger Dagger  Medicine, University of Antwerp, B-2610 Antwerp, Belgium, § Koninklijk Belgisch Instituut voor Natuurwetenschappen, B-1000 Brussels, Belgium,  Departamento de Bioquimica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil, parallel  Biomedical Research Institute, Rockville, Maryland 20852, the ** Institute for Genome Research, Rockville, Maryland 20850, the §§ Harvard-Thorndike Laboratory, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston Massachusetts 02215, and the ¶¶ Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus C, Denmark

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature.

The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508).

The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Nonvertebrate Hbs occur episodically in most but not all nonvertebrate phyla and exhibit a much greater variability in their primary and quaternary structures and in their functional properties than their vertebrate counterparts (1-3). Within this structural/functional variability, two major groups can be distinguished: (i) intracellular (myoglobin-like) hemoproteins that occur in tissues and function as oxygen storage molecules and (ii) intra- or extracellular (hemoglobin-like) hemoproteins that transport oxygen between tissues. Intracellular globins mainly have low Mr values, whereas the extracellular Hb of nonvertebrates show a wide variety of Mr values. Their extracellular location makes a high Mr advantageous in minimizing loss by excretion. A high Mr has been achieved by different routes, exemplified in Annelida by the aggregation of many low Mr chains into a functional Hb or, as proposed for some Mollusca and Arthropoda, by duplication of the low Mr chains into polymeric globins. Despite this variability, there is compelling evidence that all globins are derived from a common ancestor that consists of a polypeptide chain of ~150 amino acids displaying the "globin fold" (4-6).

Within the phylum Mollusca Hbs may occur (i) intracellularly in circulating erythrocytes as monomers, dimers, or tetramers composed of single domain or two-domain globin chains (Bivalvia); (ii) intracellularly in the cytoplasm of specific tissues (Mbs)1 as monomers or dimers of single domain globin chains (Gastropoda, symbiont-containing bivalves, and Polyplacophora); or (iii) extracellularly dissolved in the hemolymph as high Mr aggregates of multidomain globin chains (Gastropoda) (for a review see Ref. 1 and references therein). The physico-chemical characteristics of these molecules are summarized in Table I.

                              
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Table I
Summary of molluscan Mb characteristics as described by Kapp et al. (5)

The gene encoding the ancestral globin chain is assumed to be interrupted by three introns inserted in the B (B12.2), E (E15.0), and G (G7.0) helix, as in plant legHbs. However, the conservation of this intron pattern and the exact insertion positions of the introns during evolution is a matter of ongoing debate. These features may reflect the ancestral configuration (that may be masked by subsequent intron removal and/or displacement exemplifying the introns early hypothesis), new insertion events (introns late hypothesis), or a combination of all these (7-11). Intron location and sequence may also shed light on the origin of polymeric globin genes occurring in molluscs and arthropods (12).

Globin and SSU rRNA sequences are commonly used to trace molecular evolution and phylogeny (13-15). The origin of molluscs as well as the phylogenetic relationships between the molluscan classes remain controversial. Recent molecular studies, using SSU rRNA as a parameter, failed to provide unambiguous results and suggest that Mollusca may not be a monophyletic group (e.g. Refs. 16 and 17).

The gastropod mollusc, Biomphalaria glabrata, an intermediate host of Schistosoma mansoni, contains an extracellular Hb in its hemolymph. This Hb has a Mr 1.75 106 and pI 4.6 (18) and is composed of multidomain globin chains of Mr ~180,000 with a minimum Mr of 17,700 per iron atom (19). It is a glycoprotein containing 2 mol of hexoses (mannose, galactose, and fucose) and 1 mol of glucosamine per Mr 17,700 (20). Mbs are well characterized in several molluscs (1). However, the presence of a Mb-like molecule in B. glabrata tissue has not been reported.

We here describe the structure of B. glabrata Mb and its coding gene, use it to study intron evolution as well as the origin and phylogeny of the Mollusca, and report its oxygen binding characteristics.

    EXPERIMENTAL PROCEDURES
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Procedures
Results & Discussion
References

Biological Material-- The snails, B. glabrata, were kindly donated and identified by Dr. Cecília Pereira de Souza from Instituto René Rachou (Fundação Oswaldo Cruz) and were maintained in glass aquariums.

cDNA Sequencing-- A B. glabrata neural tissue cDNA library in lambda ZAP, as well as a partial cDNA sequence of a putative myoglobin clone (RBGDA16T981) was provided by BRI and TIGR, respectively. A specific 5' forward 20-mer BIO1 (5'-TACTGTCACACAACCAGCCC-3') was designed upstream of the start codon based on that sequence information. The library was screened for a full-length myoglobin cDNA clone by PCR using the BIO1 primer and either the M13/pUC universal left or universal right primer. One µl of the cDNA library was added to a 50-µl PCR reaction mix (75 mM Tris-HCl, pH 9, 20 mM (NH4)2SO4, 0.01% Tween 20, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 unit of Taq polymerase) containing 100 ng of each primer. The PCR was carried out for 30 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min. The amplified product was purified and subcloned in pBluescript KS. Recombinants were confirmed by PCR and sequenced by the dideoxy chain termination method. A 3' reverse 20-mer BIO2 (5'-GAGTTGGACAGGATCCGTGG-3') was designed downstream of the stop codon based on the obtained cDNA sequence.

Purification of Myoglobin and Protein Sequencing-- After identification and sequencing the B. glabrata Mb at the cDNA level, the expressed protein was localized in the radular tissue and extracted. Freshly collected radulas from B. glabrata were homogenized in 0.1 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl. The protease inhibitors phenylmethanesulfonyl fluoride, tosylphenylalanyl chloromethyl ketone, iodoacetamide, EDTA, and pepstatin A were used to the following final concentrations: 1 mM, 100 µM, 50 µM, 5 mM, and 1 µM, respectively. Sodium nitrite was added to 0.8% final concentration in order to convert the Mb to metmyoglobin, and the mixture was centrifuged at 10,000 rpm for 10 min to remove cell debris. After centrifugation, this sample was applied to a Superose 12 HR gel filtration column (0.5-cm diameter; 25-cm length) and eluted in the same buffer used for homogenization. Fractions (1 ml) were collected and monitored at 415 and 280 nm. Sample homogeneity was tested by 15% SDS-polyacrylamide gel electrophoresis according to Laemmli (21).

The purified protein was digested with trypsin, and the resulting peptides were separated by reverse phase high performance liquid chromatography using a Vydac C4 column developed with 0.1% trifluoroacetic acid/CH3CN. Some of the peptides were sequenced in an ABI 471-B sequencer operated as recommended by the manufacturer.

Circular Dichroism-- The circular dichroism was conducted in a JASCO J20 spectropolarimeter with a constant flux of ultrapure N2 (White Martins). The myoglobin sample at 0.117 mg/ml in phosphate buffer 100 mM pH 7.0 was used in a 0.01-cm quartz cuvette. The spectropolarimeter was calibrated with respect to wavelength and signal amplitude using D-10 camphorsulfonic acid at 290.5 and 192.5 nm and D-pantolactone at 219 nm (22, 23). The data were converted to a residual molecular ellipticity and analyzed using the software Dicroprot version 2.3d using the Varselec procedure to evaluate the secondary structure content of this myoglobin (24, 25). All spectra from the data bank of the program were used as reference for the calculations.

The myoglobin concentration was estimated by absorbance at 278 nm using the extinction coefficient of Aplysia kurodai myoglobin (33.6 cm-1·mM-1) and by the Lowry method, giving similar results (26, 27).

Oxygen Equilibrium Measurements-- The Mb used for oxygen equilibrium measurements was reduced by dialysis at 5 °C against O2-free, N2- and CO-equilibrated 0.01 M HEPES buffer, pH 7.68, containing 0.5 mM EDTA and 0.1% sodium dithionite and further dialyzed against the same buffer in the absence of sodium dithionite. It was then concentrated by centrifugation in Ultrafree-MC Millipore tubes with 10,000 NMWL filters (Millipore Corp., Bedford, MA).

Oxygen equilibria were measured using a modified diffusion chamber, where absorption of ultrathin layers of the Mb solution are recorded continuously during stepwise increases in the oxygen tension of equilibration gases supplied by cascaded Woesthoff pumps for mixing pure (>99.998%) nitrogen, oxygen, and air (3, 28). This procedure showed high reproducibility (P50 = 4.73 ± 0.04, n = 6, for stripped human Hb at 25 °C and pH 7.4) (29). Values of P50 and n50 were interpolated from Hill plots.

Genomic DNA Sequencing-- Genomic DNA was isolated from muscle tissue of B. glabrata by the N-cetyl-N,N,N-trimethylammoniumbromide method (30) and used as template in an asymmetric PCR reaction using Taq extender (Stratagene) with the primers BIO1 and BIO2. The first 10 cycles were carried out at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 5 min in the presence of the BIO2 primer only, and then BIO1 primer was added and another 25 cycles were carried out. Positively amplified products were purified and cloned in the pCRII vector (Invitrogen) and sequenced.

Southern Blotting-- Genomic DNA was digested separately with PstI and HindIII. Restriction fragments were separated by agarose gel electrophoresis and denatured DNA was transferred to a Hybond N membrane (31). After immobilizing by ultraviolet irradiation and prehybridization (in 40% formamide, 50 mM phosphate buffer, pH 7.4, 5 mM EDTA, 0.1% SDS, 5× Denhardt's, 0.9 M NaCl) at 42 °C for 3-4 h, the filter was hybridized overnight at 42 °C in the same prehybridization mixture with the denatured probe added. A genomic PCR fragment from the Mb of B. glabrata 32P-labeled by nick translation was used as a probe. The filter was washed subsequently at 65 °C, 1 × 15 min in 2× SSC, 0.1% SDS; 1 × 15 min in 1× SSC, 0.1% SDS; 2 × 15 min in 0.1× SSC, 0.1% SDS and exposed to autoradiography for 4 h at -70 °C.

Tree Construction-- SSU rRNA sequences of species related to those for which globin sequences are available were taken from the Van de Peer et al. (32) alignment. Globin sequences were aligned according to the nonvertebrate globin template (6). On the basis of globin and SSU rRNA sequences, neighbor-joining (33) and maximum parsimony trees were constructed, using the computer programs TREECON (34) and PAUP (35), respectively. For SSU rRNA data, neighbor-joining trees were derived on the basis of distance matrices calculated using the formula of Jukes and Cantor (36). Gaps were not taken into account. For globin amino acid sequences, neighbor-joining trees were derived on the basis of distance matrices calculated using the formula of Poisson as implemented in TREECON without taking gaps into account. Maximum parsimony trees of both globin amino acid and SSU rRNA sequences were constructed using the heuristic search option, with the tree-bisection-reconnection branch swapping option invoked. Gaps and phylogenetically uninformative sites were excluded from the maximum parsimony analyses. The confidence of neighbor-joining and maximum parsimony trees was tested by bootstrap analyses, running 1000 replicates. According to Hillis and Bull (37), nodes are considered to be reliable if they have a bootstrap value of at least 70%.

    RESULTS AND DISCUSSION
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Introduction
Procedures
Results & Discussion
References

The cDNA and Derived Amino Acid Sequence of the Biomphalaria Glabrata Myoglobin-- A B. glabrata cDNA library was used as template in a PCR with primer BIO1 and vector-derived primers. A full-length cDNA fragment of ~1500 base pairs was amplified, subcloned, and sequenced as described under "Experimental Procedures" (Fig. 1).


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Fig. 1.   cDNA and derived amino acid sequence of B. glabrata myoglobin. Primers are boxed and named.

The initiation codon is preceded by an incomplete untranslated region of 45 base pairs. Nevertheless, the absence of a leader sequence is obvious. This classifies the encoded protein as intracellular. The open reading frame extends for 148 codons and is followed by an exceptionally long (970-base pair) 3'-untranslated region. A normal polyadenylation signal is present.

The cDNA translated amino acid sequence can be aligned unambiguously with globins with known tertiary structure, including those from three molluscan species (Scapharca inaequivalvis I, Aplysia limacina, and Lucina pectinata I), using the nonvertebrate globin template (6, 38-42) (Fig. 2). The alignment is confirmed by (i) the exclusion of polar residues from 33 invariant nonpolar sites listed in Lesh and Chothia (43), (ii) the alignment of Pro-C2, which determines the folding of the BC corner, (iii) the presence of the invariant His-F8 and Phe-CD1, (iv) the presence of a Gly-B6 essential for the near crossing of the B and E helices, and (v) the presence of a Trp-H8, which is indicative of invertebrate globins. This results in a low total penalty score for the sequence presented (Table II) and confirms the globin nature of the protein (6).


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Fig. 2.   Alignment of B. glabrata myoglobin with selected sequences with known tertiary structure. The helical segments of each globin three-dimensional structure are shown. Tryptic peptides from purified B. glabrata Mb (Fig. 4) that have been sequenced are indicated. Phys, P. catodon/Mb; Aplim, A. limacina/Mb; Lucina I, L. pectinata/HbI; ScaI, S. inaequivalvis/HbI; Bg, B. glabrata/Mb.

                              
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Table II
Penalty scores against the nonvertebrate globin template (4) of selected molluscan globin sequences

Since the molecule is intracellular, it must be interpreted as a tissue Hb or Mb type. As shown by the low penalty score for each motif, the presence of all the standard helical segments including a short D-helix can be accepted. Deviations of the standard pattern can be localized in the E-helix (penalty score: 1.7). This is mainly due to the presence of the hydrophobic Ile at external position E5 and of Val residues at positions E8 and E10, where normally a larger side chain is observed. The alignment of Fig. 2 was extended with 29 other molluscan globin sequences, and the penalty scores against the nonvertebrate template were calculated.2 The penalty scores of the other molluscan globin sequences are low, with the exception of the Hbs of the primitive deep sea clam Calyptogena soyoae (44), suggesting that they follow closely the standard globin fold (43). When B. glabrata Mb is compared with molluscan globin sequences, the highest similarity (30.2%) is observed with Cerithidea rhizophorarum Mb.

The hydrophobic lining of the heme pocket of B. glabrata Mb is normal but is less occupied by aromatic side chains than that of A. limacina and L. pectinata Mb. In B. glabrata, Mb-specific side chain substitutions occur at several positions. In the B-helix, Val-B10 and Trp-B12 are unprecedented. Position B10 is usually occupied by a large hydrophobic residue (nonvertebrate globins studied (3) have Leu (27.8%), Phe (27.8%), Tyr (27.8%), Met (11.0%) and Trp (5.6%)). Its side chain can be turned into the heme pocket and become involved in the control of O2 affinity through stabilization of ligand binding, as shown for nematode and trematode Hbs (45, 46). It is very unlikely that the small Val can fulfill such a ligand stabilization role in B. glabrata Mb. However, the nature of the B10 side chain is correlated to the nature of the distal (E7) residue. Indeed, when a Gln-E7 is present, 80% of the nonvertebrate globins display a Tyr-B10. The adjacent residue at position E11 is also important in determining oxygen affinity. However, no correlation could be found between residues at positions E7/E11 and B10/E11.

The Gln-E7 of B. glabrata is shared with C. soyoae and L. pectinata, whereas Aplysiidae (A. limacina, A. juliana, and A. kurodai) display Val-E7 and all other mollusc globins His-E7. The Val-E7 of the monomeric Mbs of the Aplysiidae, Bursatella leachii and Dolabella auricularia, does not contact the heme directly (39, 47). Solution 1H NMR indicates that the hydrogen bonding of the bound ligand by the standard His-E7 is taken over by Arg-E10. This hydrogen bonding is responsible for the relatively high ligand affinity and the slow dissociation rate (48, 49). In contrast, the sequence of the dimeric Mbs of Busycon canaliculatum, C. rhizophorarum, Nassa mutabilis, and Buccinum undatum shows the classic His at the distal position and consequently no Arg-E10. Neither the monomeric nor the dimeric Mbs of the gastropods have residues capable of hydrogen binding with the ligand at position B10 (45, 50). It is clear that the ligand stabilization system in B. glabrata Mb differs from the unique mechanism described in the Aplysiidae. A combination Val-B10, Gln-E7, and Ile-E11 is unprecedented.

It can be assumed that in B. glabrata Mb Ala-B5, Leu-B9, and Met-B13 and Asn-B8, Trp-B12, and Asn-B16 form two ridges between which a single ridge of the G-helix (Phe-G15, Asn-G11, and Pro-G7) is packed to form the B/G-helical contact (43). As such, a strong hydrophobic interaction can be expected among Leu-B9, Phe-G15, and Trp-B12 to stabilize the helical contact.

The observation that Cys (EF8) (which is considered to be responsible for dimerization and is conserved in all dimeric Mbs) is absent in the B. glabrata sequence confirms its monomeric nature.

Since the B. glabrata Mb was identified starting from a cDNA clone, its expression in the animal itself was verified. As described under "Experimental Procedures," a heme-containing protein with an apparent Mr ~17,000 could be isolated from the radular muscle (Fig. 3). Sequencing of the intact protein clearly proves that the amino terminus is inaccessible for Edman degradation, suggesting that the mature protein is acetylated as in all other mollusc Mbs observed so far (Table I). The amino acid sequences of several tryptic peptides clearly indicate the sequence identity of the isolated protein with the cDNA-derived amino acid sequence of the B. glabrata Mb (Fig. 2).


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Fig. 3.   Purification of B. glabrata myoglobin. A, gel filtration of radula extract of B. glabrata on a Superose 12 HR column equilibrated with Tris-HCl 0.1 M buffer, pH 7.4, containing 0.2 M NaCl. B, 15% SDS-polyacrylamide gel electrophoresis of gel filtration fractions. Lane 1, molecular weight markers; lane 2, crude radular extract; lanes 3-5, respectively, fractions 15, 16, and 17 from panel A.

The circular dichroism analysis of the B. glabrata Mb shows 46% of alpha -helices, 17% of beta -sheets, 14% of turns, and 23% of coil (Fig. 4). This alpha -helix content is low if compared with the vertebrate globins but is quite consistent with the circular dichroism data obtained for A. kurodai Mb (26). However, the crystallographic structure of A. limacina Mb (39) reveals 76.6% of alpha -helices, indicating the high structural similarity of these mollusc myoglobins with its vertebrate relatives. Therefore, the circular dichroism data obtained for B. glabrata Mb were interpreted as confirmative of the standard globin folding for the B. glabrata Mb.


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Fig. 4.   Circular dichroism of the myoglobin of B. glabrata. Mb at 0.117 mg/ml in 100 mM phosphate buffer, pH 7.0. Path length was 0.01 cm.

We thus conclude that the B. glabrata Mb is effectively expressed in the radular muscle and occurs as a monomeric molecule with a calculated Mr of 16,049.

Oxygen Binding by B. glabrata Mb-- B. glabrata Mb exhibits P50 values of 0.09, 0.21, 0.38, and 0.66 mm Hg at 5, 15, 20, and 25 °C, respectively (Fig. 5). The n50 values observed (0.85-0.91) show the absence of cooperativity, which is in agreement with the monomeric structure of the pigment.


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Fig. 5.   Oxygen binding of B. glabrata Mb. A, oxygen equilibria measured at 5 °C (triangles), 15 °C (squares), and 24 °C (circles) in the presence of 0.01 M HEPES buffer, pH 7.53 (at 15 °C), containing 0.5 mM EDTA. B, Hill plots (log (S/1 - S) versus log PO2, (where S represents fractional oxygen saturation of the Mb and the slopes of the linear regressions give the Hill cooperativity coefficients). C, van't Hoff plot, showing relation between P50 and absolute temperature (T). [Hb] = 0.049 mM.

The O2 affinities are high compared with those reported for Mbs of other invertebrates (Table III). Although the O2 affinity of B. glabrata Mb is similar to that of Arenicola marina Mb I (0.38 mm Hg), it is higher than that of A. marina Mb II (0.72 mm Hg),3 whose lower affinity compared with Mb I correlates with an E6 Ser/Pro exchange that may change the surroundings of His-E7 (91).

                              
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Table III
Oxygen affinity of some nonvertebrate globins

B. glabrata Mb has an Gln-E7 in combination with Val-B10. Zhao et al. (51) found that an E7 His/Gln mutation lowers O2 affinity in sperm whale Mb but that this effect is reversed by B10 Leu/Phe substitution, whereas human Hb E7 His/Gln shows a strong increase in affinity (52). Ascaris suum Hb has a very high oxygen affinity (on the order of 300 times higher than that of the mammalian counterparts), which has been assigned to the combination of Tyr-B10, Gln-E7, and Ile-E11. Ascaris Mb has the same side chains at B10, E7, and E11, yet its oxygen affinity is 60-fold lower. These discrepancies may be explained by assuming that other residues are essential to correctly position the B- and E-helices to enable hydrogen binding between Tyr-B10 and the bound oxygen (53). Trematode globins have tyrosine residues at both B10 and E7 and, like Ascaris Hb, exhibit very high oxygen affinities (Ref. 46; Table III). The Val-B10 residue in B. glabrata Mb projects into the heme cavity but cannot make a hydrogen bond with the bound oxygen as Tyr does in Ascaris and the trematodes globins (46, 50). B. glabrata, however, does have a high oxygen affinity, but it is unclear how this is obtained in the given configuration.

These observations therefore suggest that (i) O2 affinity cannot be correlated uniquely to single substitutions in the heme pocket, since there are so many other exchanges that may have an influence, (ii) the same substitutions may have different effects in different globins (as indicated by opposite effects of E7 His/Gln substitution in globin chains), and (iii) there may be a different molecular mechanism determining high oxygen affinity in B. glabrata Mb.

The Structure of the Biomphalaria glabrata Mb Gene and Comparison with Other Nonvertebrate Globin Genes-- Primers flanking the coding region were made based on the cDNA sequence and used in a PCR on genomic DNA to determine the presence, site, and size of introns in the Mb gene. A fragment of 3.29 kilobase pairs was amplified, suggesting a total intron sequence length of 2.7 kilobase pairs. The gene fragment was subsequently cloned and sequenced as described under "Experimental Procedures" (Fig. 6).


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Fig. 6.   Complete genomic DNA sequence of B. glabrata myoglobin. The coding sequence is translated in boldface type.

The coding sequence is interrupted by three introns that are 1116, 1008, and 582 base pairs long. All exon/intron boundaries have the expected acceptor/donor splice sites. Possible branch point sequences are found in 2 out of 3 introns: CTAACT and CCAAC, beginning 37 and 67 base pairs upstream from the 3' splice junctions of intron II and intron III, respectively. No known branch point sequence is detected in intron I. Immediately upstream from the 3' splice site, no large polypyrimidine tract is found (54).

Using the alignment of Fig. 2, the intron insertion positions can be assigned as A3.2, B12.2, and G7.0.

A three-intron/four-exon pattern with an intron inserted in the B-, E-, and G-helix, is considered to be ancient. According to Hardison (8) and Lewin (4), all other gene structures evolved out of the ancestor by intron loss. Most nematodes still have this ancient gene structure. However, the globin genes of nematodes (55) as well as the midge larvae (56) display central or E-helix introns at positions different from the plant central intron (Fig. 7), suggesting that the possibility of independent intron insertion events cannot be excluded.


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Fig. 7.   Comparison of the intron insertion positions of relevant globin genes. The helical position and phase of intron insertion are indicated. , leader sequence; square , intron; square , precoding intron; black-square, exon.

No central intron is observed in the beta - and beta '-globin genes of Anadara trapezia (57, 58), and this also applies to the A and B globin genes of S. inaequivalvis (59) and the c-gene of the annelid Lumbricus terrestris (60). Therefore, if there was a central intron in the ancestral globin gene of annelids and molluscs, it might have subsequently been lost before the divergence of these phyla.

There are many eukaryotic genes encoding for secretory proteins that have an additional intron (precoding intron) near the junction of the DNA encoding the signal sequence with that of the mature protein (61). This intron is very likely of recent origin and has been captured with upstream sequences, being the leader sequence. The extracellular two-domain globins of the nematodes Pseudoterranova and Ascaris display an intron at position A4.1, which can be considered as precoding (62, 63). This is not the case for the A3.2 intron in B. glabrata Mb, since there is no leader sequence. It must therefore be considered as a newly inserted intron. It is striking that precoding or A-helix introns are only observed in genes encoding for didomain Hbs (with or without leader sequences), the B. glabrata Mb gene being an exception. A similar precoding intron is localized two bases before the start codon of the two-domain globin of the mollusc Barbatia reeveana Hb (Fig. 7). The derived bridge intron that separates the DNA sequences encoding the two domains can be considered as "precoding" for the second domain (64).

Several mechanisms have been proposed for the duplication of the gene. In Pseudoterranova for example, it appears that the duplication has resulted in a direct head to tail arrangement with the original genomic copy, which may be due to an unequal cross-over involving the coding sequence of the last exon of the first repeat (62). It can also be that the duplication of the gene occurred by recombination events involving genomic DNA or that it occurred during a mispaired gene conversion event, which is capable of producing genes that are a fusion of two original genes. In all cases, it is not clear whether the precoding and bridge intron were already present before the duplication. The genes encoding for the two nine domain globin chains of Artemia do not contain precoding or bridge introns. The domains are ancestrally related and are presumed to be derived from copies of an original single domain gene (12).

However, in Barbatia it has been accepted that a cross-over has occurred between the precoding intron and the 3' noncoding region, as indicated by sequence similarity in these regions (64).

The B. glabrata Mb gene, that contains an unprecedented A-helix intron at position A3.2 encodes for a monodomain globin. However, B. glabrata not only expresses a Mb but also a polymeric Hb (18, 65).

A comparison of the sequence of the A-helix intron, that can be considered as precoding, with that of the 3'-noncoding region, shows places of similarity (data not shown). Thus, the multidomain Hb of B. glabrata may have evolved from the duplication of the ancestral Mb gene involving the A-helix intron. The determination of the structure of the gene encoding for the polymeric Hb of B. glabrata and comparison with the B. glabrata Mb gene may elucidate these polymerization events.

To test the similarity between B. glabrata Mb and Hb as well as to preliminarily classify the number of Mb-encoding genes, a genomic Southern blotting experiment was done as described under "Experimental Procedures." The hybridization was carried out at low and high stringency, and in both genomic DNA digestions (PstI and HindIII) only a single fragment ranging in length from ~ 8000 to 12,000 base pairs was detected (data not shown). This result indicates that the Mb must exist in the B. glabrata genome as a single gene and that it lacks marked similarity with the Hb gene(s).

Recently, it has been suggested that the DNA sequence coding for the central exon of S. inaequivalvis tetrameric Hb is part of an open reading frame displaying all features of a functional gene in the flanking intron sequences (66). The existence of this putative minigene is used as an argument for the "exon theory of genes" (67). If the central exon and its flanking introns are considered as a putative minigene, then it should be possible to trace it back in other nonvertebrate globins as well. Inspection of B. glabrata Mb as well as other available globin genes clearly demonstrates that several start and stop codons, in frame with the functional heme binding domain, could be found in the same gene. This suggests that they occur at random and therefore cannot be used as a proof for the existence of "minigenes." Moreover, there is little selective pressure to keep the intron sequences conserved, and it is therefore unlikely that the start and stop codons of putative ancestral minigenes would be conserved until now (68).

Molecular Phylogeny of the Molluscan Phyla Based on Globin Sequences-- A neighbor-joining tree constructed on the basis of 41 metazoan globin sequences is shown in Fig. 8. The platyhelminth Paramphistomum epiclitum is used as an outgroup. Considering for the interpretation of the tree only branching points with bootstrap values higher than 70%, seven molluscan groups can be distinguished: the Polyplacophora, Opisthobranchia, Pulmonata, Prosobranchia, Pteriomorphia, and two heterodont groups. Surprisingly, the monophyly of some very well established groups, such as the Heterodonta and Gastropoda, cannot be retrieved. Also, molluscan monophyly is not advocated by the tree topology, but there is no bootstrap support for refuting it. None of the deeper branching points in the tree shows high bootstrap values. The maximum parsimony topology (not shown) on the basis of the same globin data set confirms all of these findings.


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Fig. 8.   Neighbor-joining tree of 28 molluscan and other metazoan globin sequences. The platyhelminth P. epiclitum was used as an outgroup. Bootstrap values are indicated above the nodes.

To assess the value of globin sequences for phylogeny inference, the results of Fig. 8 were compared with a neighbor-joining tree constructed with 19 complete metazoan SSU rRNA sequences (Fig. 9), selected as representatives of the same higher groups (phyla, classes, or subclasses) as those included in the globin-based tree (Fig. 8). The SSU rRNA tree supports the monophyletic origin of the Pteriomorphia, but consistent with the globin-based tree, it indicates molluscan polyphyly. However, as the deeper branches have only low bootstrap values, the possibility of Mollusca being monophyletic cannot be refuted either. In contrast to Fig. 8, the SSU rRNA-based tree, however, does support gastropod and heterodont monophyly and confirms the existence of a eutrochozoan clade, including all molluscan representatives, in accordance with previous morphological and molecular findings (16, 69, 70). A monophyletic gastropod and heterodont cluster are also present in the globin-based tree but have only very low bootstrap values. The results of Fig. 9 are confirmed by the maximum parsimony tree (not shown) found on the basis of the same SSU rRNA data set.


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Fig. 9.   Neighbor-joining tree on the basis of 19 complete metazoan SSU rRNA sequences. The platyhelminth Opisthorchis viverrini was used as an outgroup. Bootstrap values are indicated above the nodes.

Both the SSU rRNA and the globin data set fail to confidently resolve deeper branching points. This may reflect the rapid way in which the metazoan higher groups radiated at the Precambrian-Cambrian boundary (71-74). Information on the branching pattern of taxa, which radiated in an explosive way, will only be recorded by very variable, fast evolving sites. Yet, if the explosive radiation happened a long time ago (in the case of Metazoa, 700 million years or more), these informative sites will have undergone many subsequent changes, obscuring the original information. This phenomenon was already previously described for SSU rRNA-based trees (16, 75-77). Globin sequences seem to be even more subject to this phenomenon. Indeed, some well established phylogenetic relationships, which can be retrieved by SSU rRNA sequences, cannot be confidentially retrieved by globin data. Apparently, globin sequences are, as previously concluded for SSU rRNA sequences (16), better suited to trace more recent metazoan divergences, such as the molluscan intraclass or even intrasubclass relationships. Indeed, many branching points within the molluscan clades (e.g. the pteriomorph and prosobranch clade) are robustly supported by bootstrap analysis.

A problem with the use of globin sequences in phylogenetic analyses is the fact that they constitute a multigene family, in which case it may be very difficult to recognize orthologous genes necessary for species phylogeny inference. This problem holds especially for nonvertebrate metazoan globins, since they show a broad array of structures. Globin-based trees are in fact a mixture of gene and species trees and include information on the evolution of the species as well as the gene. A comparison of Fig. 8 with the corresponding SSU rRNA tree (Fig. 9) shows that the information on the monophyly of some molluscan groups is apparently superimposed on the gene phylogeny. Yet, apparently one has to be more cautious when considering relationships at more restricted taxonomic levels. Indeed, for example within the pteriomorph clade, species phylogeny is overshadowed by gene phylogeny, resulting in a clustering of gene types rather than of closely related congeneric species. It is not clear whether the extent to which orthologous and paralogous genes are combined in our analysis can be responsible for the discrepancies between the globin and the SSU rRNA tree.

In conclusion, at this time it seems that globin sequences, like SSU rRNA sequences, contain insufficient information to resolve the metazoan radiation pattern or to recover molluscan monophyly but that they seem to yield confident results at more restricted taxonomic levels. Apparently, in resolving deeper branching points, globin sequences perform worse than SSU rRNA sequences. Moreover, in contrast to SSU rRNA sequences, the use of globin sequences in unraveling species phylogeny suffers from the facts that it is nearly impossible to identify which globin genes are orthologous and that gene and species trees are always superimposed on each other. All of these conclusions are only preliminary, since they are based on a globin data set in which several metazoan taxa are absent and others are still poorly sampled. However, the globin tree presented in Fig. 8 does raise serious doubts on the monophyly of the Mollusca.

    ACKNOWLEDGEMENT

We thank Prof. Roney Elias da Silva (GIDE-UFMG-Brasil) for instruction on the dissection of the radula. The anonymous referees are acknowledged for valuable suggestions.

    FOOTNOTES

* This work was supported by Belgian National Science Foundation Grant 9002394 (to L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U89283.

|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Tel.: 32 3 8202323; Fax: 32 3 8202248; E-mail: lmoens{at}uia.ua.ac.be.

1 The abbreviations used are: Mb, myoglobin; PCR, polymerase chain reaction; SSC, disodium citrate; SSU rRNA, small subunit rRNA; P50, half-saturation oxygen tension; n50, Hill's cooperativity coefficient at half-saturation.

2 The alignment and penalty scores are available upon request.

3 R. E. Weber, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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