In mammals the primary role of the APP Hx is to bind free heme and prevent oxidative damage whilst sequestering iron away from bacteria. Hx is expressed by the liver and is present at 0.5–1.25 mg/ml levels in serum; however its expression is greatly upregulated (50-fold within 24 h) by the inflammation that accompanies injury or infection (Wang et al., 2001
; Poli and Cortese, 1989
). During the present study we identified one of the major serum proteins in nurse sharks as the cartilaginous fish orthologue of Hx. Cartilaginous fish diverged from a common ancestor with other vertebrates ~500 MYA and are members of the most ancient extant gnathostome lineage. However mammalian and elasmobranch Hx both show the ability to bind their respective Hbs and comparison of their sequences shows the residues important for function are highly conserved in these two lineages. The situation in the teleost fish is more complex with a number of species now being shown to express two forms of WAP65, the teleost Hx homologue. The two WAP65 genes are divergent in both their sequence and pattern of tissue expression; WAP65-1 shows mutation of many of the ‘conserved’ Hx residues and shows constitutive, multi-tissue expression whilst WAP65-2 is more conserved in both sequence and expression characteristics (Sha et al., 2008
; Sarropoulou et al., 2009
; Kikuchi et al., 1995
). Phylogenetic trees () show the teleost WAP65s cluster separately from Hx of all other species and the WAP65-1 and -2 genes group separately within the cluster. As the ancestor of the majority of ray-finned fish is thought to have experienced an additional, lineage-specific genome-wide duplication (so-called 3R) ~320 MYA (Vandepoele et al., 2004
) the separation of the two WAP65 genes strongly suggests they are paralogues resulting from this additional round of duplication. Initial studies also suggest the two forms have different physiological functions (Sha et al., 2008
; Hirayama et al., 2004
; Sarropoulou et al., 2009
) indicating duplication has been followed by diversification and neofuctionalization.
Searches for Hx in the genomes of more ancient species (lamprey, hagfish, sea urchin and amphioxus) have been conducted by us and others (Wicher and Fries, 2010
) however no orthologue has yet been found. This strongly suggests that Hx evolved after the emergence of the cyclostomes (jawless fishes) but prior to that of the jawed vertebrates, coincident with the 2nd round of genome wide duplication (2R; ). Additional searches with individual hx domains in these species returned only matrix metalloproteases (MMPs) or MMP-like molecules, proteins which also use hx domains as mediators of protein:protein interaction. Thus, we have not identified an unambiguous candidate related to the Hx ancestor.
Schematic illustrating the putative evolution of Hx
Despite the crystal structure of Hx clearly showing that the heme-binding site lies between the two hx domains, bounded by the interdomain linker, when subject to proteolysis the isolated N-terminal hx domain is also able to bind heme (Morgan and Smith, 1984
). This finding, combined with the high sequence identity between the two hx domains, has lead to the suggestion that Hx evolved from another protein containing a single Hx domain (Baker et al., 2003
). We propose that this molecule was an MMP-like protein, similar to those we found in the genomes of the jawless vertebrates and invertebrates, which arose at approximately the same time as the second round of genome wide duplication (2R) in the ancestor of the jawed vertebrates. Freed from selective pressure the duplicate MMP-like gene could lose its pro-peptide and catalytic domains and duplicate its hx domain, thus generating the proto-Hx gene. The concomitant appearance of tetrameric (α2-β2) Hb in jawed vertebrates enabled more sophisticated regulation of oxygen binding (as compared to the monomeric/dimeric Hbs of the jawless fish) (Coates, 1975
) but also proved much more harmful when released from erythrocytes; as the interdomain binding site gives much greater control of heme binding and release (Morgan and Smith, 1984
) this improved mode of binding by Hx, once evolved, would be under very strong positive selection.
One feature that remains difficult to explain is the very high (>10 mg/ml) level of Hx in the serum of outwardly healthy sharks (10–20X the level of serum Hx in mammals). Studies in mammals have shown that released heme is removed by Hx, however when Hb is released from erythrocytes it is cleared by another acute-phase serum protein, haptoglobin (Nielsen et al., 2010
and references therein). Whilst we have found high levels of haptoglobin in nurse shark plasma, in our hands it does not bind Hb-coupled sepharose (Dooley et al.
, manuscript in preparation). Thus it seems that nurse shark Hx could bind either heme or Hb, or maybe even binds both, perhaps explaining why such high levels of serum Hx are required. This would be even more likely if there is a need to conserve valuable iron in these species and/or sequester it away from whatever bacteria routinely infect these animals? Additionally, recent work in mice has shown that Hx can suppress the production of TNF and IL6 by macrophages in response to LPS (Liang et al., 2009
). As bacteria can be routinely cultured from the organs and blood of healthy sharks (Mylniczenko et al., 2007
) perhaps high levels of Hx act to ‘dampen’ the immune response to their commensal microflora? Although this important question remains unresolved, with the cloning of shark Hx we have finally identified the 5S ‘pink protein’ which was first described during chromatography of shark serum over 45 years ago (Marchalonis and Edelman, 1965