Red blood with blue-blood ancestry: intriguing structure of a snail hemoglobin

Abstract

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin "plug" domain with three cysteines for subunit dimerization (total M(r) approximately 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical images (3 nm resolution); it suggests a 3 x 2-mer quaternary structure (M(r) approximately 1.43 MDa). Moreover, we identified a previously undescribed rosette-like hemolymph protein that has been mistaken for hemoglobin. We also detected expression of an incomplete hemocyanin as trace component. The combined data show that B. glabrata hemoglobin evolved from pulmonate myoglobin, possibly to replace a less-efficient hemocyanin, and reveals a surprisingly simple evolutionary mechanism to create a high molecular mass respiratory protein from 78 similar globin domains.

Fig. 1.

Identification of purified BgHb and BgRp. (a) Electron microscopy of HPLC-purified BgHb molecules. (a Inset) Three-dimensional reconstruction (see Fig. 5). (b) Electron microscopy of HPLC-enriched BgRp molecules. (b Inset) Class sum image from 10 top views aligned by IMAGIC. (Scale bars: 25 nm.) (c) UV-visible spectra of BgHb and BgRp. (d) Tandem-crossed immunoelectrophoresis of purified BgHb and BgRp against rabbit antibodies versus B. glabrata hemolymph proteins. Note the separate precipitation of the two protein peaks indicating nonidentity. (e) SDS/PAGE of BgHb (arrow) showing an apparent M_r of 180 kDa. A trace of the disulfide-bridged dimer of BgHb is also visible (arrowhead). Marker protein masses are indicated in kilodaltons. (f) SDS/PAGE of BgRP, showing two polypeptides (arrows) with 31 and 25 kDa, respectively. Traces of the hemocyanin subunit (arrowhead; see also Fig. 2) are also visible.

Fig. 2.

Identification of BgHc. (Left) Electron microscopy of BgHc molecules in the top view (arrow; note the lack of internal collar complex) and side view (arrowhead; the larger diameter is pretended by a flattening effect), together with contaminating BgRp molecules (double arrowhead; the views differ from that in Fig. 1b). (Scale bar: 50 nm.) (Left Inset) Two BgHc molecules from another preparation. (Right) The polypeptide encoded by the partial cDNA sequence of BgHc shares many identical residues (asterisks) with the functional unit HtH1-h (1) (and also with the other functional units of Haliotis tuberculata hemocyanin), but only five of the six copper-binding histidines are conserved (black arrows); one histidine is substituted for glutamine (gray arrow). (More BgHc sequences are available upon request.)

Fig. 3.

Multiple sequence alignment of the different globin domains. The aligned sequences are from BgHb1, BgHb2, and BgHb3 (Upper) and other molluscan heme-containing globins (Lower). The first two lines show the N-terminal “plug” domains BgHb1-p and BgHb2-p, each containing three conserved cystein residues (pink) and an N-terminal signal peptide (italic). Three potential N-glycosylation sites are marked in yellow. Note that all proximal histidines are replaced by glutamines (left double arrow), whereas the distal histidine (right double arrow) and two phenyalanines are strictly conserved (red). Other conserved residues are marked in blue (80%) or green (60%). A presumptive flexible linker region joining the 13 domains is marked by a bracket. The well known secondary structure of hemoglobin/myoblobin is added for better orientation. LsMb, L. stagnalis contig of CN810207/CN810223/CN810610/CN810699/CN810837/CN811024; BgMb, Biomphalaria U89283; NmMb, Nassarius (Nassa) mutabilis P31331 (Neogastropoda); BuMb, Buccinum undatum A44588 (Neogastropoda); SiHb, Scapharca inaequivalvis hemoglobin A chain S83524 (Bivalvia); BlHb, Barbatia lima α chain of the tetrameric (intracellular) hemoglobin D63933 (Bivalvia); AjMb, Aplysia juliana AB003277 (sea hare).

Fig. 4.

Molecular phylogeny of hemoglobin domains (Hb) and myoglobins (Mb) from mollusks. Note in the left unrooted tree the very large phylogenetic distance between pulmonate hemo/myoglobins on the one hand and other molluscan hemo/myoglobins on the other hand. (The branching order within the dashed field is not well supported.) Note that in the right tree, which has been rooted with the two Mb sequences, the topologically corresponding globin domains from the two polypeptides BgHb1 and BgHb2 are more similar to each other than any two domains within either BgHb1 or BgHb2, thereby indicating that the two polypeptides diversified by a single gene duplication event. The two as-yet-sequenced additional domains indicate a third polypeptide (BgHb3). The trees suggest that planorbid hemoglobin evolved by a series of gene duplications and fusions from an ancestral planorbid myoglobin. Posterior probabilities >0.95 are indicated at each node of the rooted tree. Nodes with lower supporting values are collapsed. The different BgHb domains are labeled according to the alignment of Fig. 3. Bg, B. glabrata; Ls, L. stagnalis; 1a–1m, domains BgHb1a to BgHb1m; 2a –2m, domains BgHb2a to BgHb2m; 3h and 3i, domains BgHb3h and BgHb3i

Fig. 5.

Three-dimensional reconstruction of the quaternary structure of BgHb. (a) 3D reconstruction of native BgHb (resolution, 3 nm). (b) Model of the BgHb polypeptide subunit with 13 globular masses for the heme domains and N-terminally a smaller mass for the plug domain (arrow). (c) The same view as in a, incorporating six polypeptide subunits as modeled in b, distinguished by different colors. By bringing together the N-terminal plug domains (arrows) of pairs of polypeptide chain subunits, this model is consistent with the occurrence of disulfide-bridged dimers of polypeptide chains, as was demonstrated biochemically. Additionally, subunits form triplets by joining C-terminal domains (white dot). Also see Fig. 6.