Emergence of the acute-phase protein hemopexin in jawed vertebrates

Abstract

When released from damaged erythrocytes free heme not only provides a source of iron for invading bacteria but also highly toxic due to its ability to catalyze free radical formation. Hemopexin (Hx) binds free heme with very high-affinity and thus protects against heme toxicity, sequesters heme from pathogens, and helps conserve valuable iron. Hx is also an acute-phase serum protein (APP), whose expression is induced by inflammation. To date Hx has been identified as far back in phylogeny as bony fish where it is called warm-temperature acclimation-related 65 kDa protein (WAP65), as serum protein levels are increased at elevated environmental temperatures as well as by infection. During analysis of nurse shark (Ginglymostoma cirratum) plasma we isolated a Ni(2+)-binding serum glycoprotein and characterized it as the APP Hx. We subsequently cloned Hx from nurse shark and another cartilaginous fish species, the little skate Leucoraja erinacea. Functional analysis showed shark Hx, like that of mammals, binds heme but is found at unusually high levels in normal shark serum. As an Hx orthologue could not be found in the genomes of jawless vertebrates or lower deuterostomes it appears to have arisen just prior to the emergence of jawed vertebrates, coincident with the second round of genome-wide duplication and the appearance of tetrameric hemoglobin (Hb).

Fig. 1. Characterization of the unknown 75 kDa shark serum protein

(a) whole nurse shark plasma run on 6% non-reducing SDS-PAGE and (b) IMAC-purified plasma run on 12% reducing SDS-PAGE. The lane marked m is molecular weight marker, white arrows indicate pentameric IgM (pM) and monomeric IgM/IgNAR (mM/N), black arrows unknown protein p75 (characterization of the unlabelled bands in Fig 1b will be detailed elsewhere. Dooley et al., manuscript in preparation).

Fig. 2. Amino acid alignment of nurse shark (Gc) and little skate (Le) Hx with that of human Hx (Hs) shows conservation of residues important for function in these species. These residues are also conserved in fugu (Tr) WAP65-2 but not WAP65-1

The two hx domains are shaded, the conserved histidine residues which co-ordinate Hb are highlighted by white text on black, conserved aromatic and basic residues in the Hb binding region are shown with darker shading and bolded. Potential N-linked glycosylation sites are underlined.

Fig. 3. Northern blot results indicate that shark Hx is expressed only from liver

Full length Hx cDNA was used to probe total RNA from multiple nurse shark tissues.

Fig. 4. SDS-PAGE of Hb- and Ni²⁺-immunoprecipitated proteins shows shark Hx binds heme

Immunoprecipitated nurse shark plasma, run under reducing conditions. Proteins eluted from Ni²⁺-sepharose incubated without (lane 1) or with plasma (lane 2) and Hb-sepharose without (lane 3) or with plasma (lane 4).

Fig. 5. Neighbour-Joining tree showing the phylogenetic relationships between Hx and WAP65 from different species

Hx from the cartilaginous fish are shaded. Two letter abbreviations for genus and species are used as follows: Human (Homo sapiens), mouse (Mus musculus), opossum (Monodelphis domestica), cow (Bos taurus), chicken (Gallus gallus) Hx, clawed frog (Xenopus tropicalis), channel catfish (Ictalurus punctatus), Fugu pufferfish (Takifugu rubripes), european seabass (Dicentrarchus labrax), Japanese medaka (Oryzias latipes), nurse shark (Ginglymostoma cirratum) and little skate (Leucoraja erinacea). The tree was rooted with the human hx domain-containing protein vitronectin (VTN).

Fig. 6. Schematic illustrating the putative evolution of Hx

The second round of genome wide duplication in the jawed vertebrate ancestor (2R) and teleost specific genome wide-duplication (3R) are shown. The divergence times of the lineages (shown as millions of years ago) are based upon those of Blair and Hedges (2005).