An Oxygen Molecular Sensor, the HIF Prolyl 4-Hydroxylase, in the Marine Protist Perkinsus olseni

Adaptations to changes in oxygen availability are crucial for survival and have been shown to be implicated in several disease states. Such adaptations are known to rely upon gene expression and regulation of hypoxia-inducible factors (HIF) and are mainly promoted by HIF Prolyl Hydroxylases (HPHs). These enzymes are involved in intracellular signaling and function as oxygen sensors. In the presence of molecular oxygen, HIF1α becomes hydroxylated at two specific prolyl residues and is targeted for destruction, whereas in the absence of oxygen (hypoxia conditions), HIF is not hydroxylated and thus remains free to activate HIF target genes. We have cloned the cDNAs corresponding to four different HPH transcripts from the bivalve parasite Perkinsus olseni, a unicellular eukaryotic organism, and determined the corresponding gene structure. Furthermore, we have analyzed the relationship between expression of this gene and parasite virulence. HPH-like genes were previously identified in other microorganisms, but only through comparative genomic analysis, and exclusively in pathogenic bacteria, thus suggesting that they may be involved in pathogen activity. To investigate a possible correlation between Perkinsus pathogenicity and HPH gene expression, pure cultures of this parasite were exposed to hemolymph from different bivalve species, either susceptible or resistant to Perkinsus, and HPH gene expression analyzed by real-time PCR. Our data show a massive increase in HPH expression in the presence of the susceptible host's hemolymph, indicative of the importance of HPH in infection mechanisms. In addition, the dependence of HIF-like enzymes on other factors or substrates known to be associated with their biological function, such as iron, 2-oxoglutarate (2OG), glycolytic enzymes and oxygen, was also investigated through the use of iron chelators, 2OG antagonists, glycolytic inhibitors and hypoxia conditions. A clear correlation was observed between the production of reactive oxygen species and HPH activity, in agreement with studies that indicate a stabilization of HIF by inhibition of HPH. Interestingly, it was also possible to conclude, through phylogenetic analysis, that Perkinsus HPH is closer to the actual HPH2 from mammals, thus providing additional evidence supporting previous data pointing to HPH2 as the most ancestral of the three HPH isoforms known in metazoans.

Introduction

Oxygen plays a vital role in all forms of life, especially in aerobic organisms, where O₂ acts as a final receptor of electrons in oxidative phosphorylation (Bruick 2003). Below normal levels of oxygen, organisms are in a state of hypoxia, which can lead ultimately to death. In adapting to this stress, cells have developed the ability to rapidly adapt the levels of oxygen through mechanisms involving transcription and/or post-transcriptional modifications (Giaccia et al. 2004). The main regulator of oxygen homeostasis is hypoxia-inducible factor (HIF), a transcriptional complex that, under hypoxic conditions, regulates target genes through the hypoxia-responsive element (HRE), thus promoting changes in energy metabolism, growth, angiogenesis, cell proliferation and survival (Dalgard et al. 2004; Safran and Kaelin 2003). HIF is an obligate heterodimer, composed of one α and one β (also known as aryl hydrocarbon receptor nuclear translocator – ARNT) subunit, both belonging to the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) protein family. HIFβ or ARNT is a partner of the aryl hydrocarbon receptor (AhR), as well as of HIFα, and is totally insensitive to O₂ concentrations, thus maintaining its levels constant under normoxic conditions. In contrast, HIFα is highly induced in response to low oxygen concentrations and is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system (Choi et al. 2003; Huang et al. 1999; Kallio et al. 1999).

Under normoxic conditions, O₂-dependent hydroxylation of HIF1α is directly involved in the stability of HIF. One of these hydroxylations is carried out by factor inhibiting HIF (FIH) through hydroxylation of an asparagine residue, leading to the inactivation of HIF by blocking the interaction between the transcriptional factor and its co-activator p300. On the other hand, proline residues within the HIF1α oxygen-dependent degradation domain (ODDD) are hydroxylated in the presence of HIF Prolyl Hydroxylases (HPH) (also known as Prolyl Hydroxylase domain-containing proteins or PHDs, or egg-laying-defective nine (EGLN)), thus inducing an interaction between von Hippel–Lindau protein (pVHL) and HIFα and subsequent proteasomal degradation.

In mammalian cells, three HPH isoforms have been identified: HPH1 (also known as PHD3 or EGLN3), HPH2 (PHD2 or EGLN1) and HPH3 (PHD1 or EGLN2) (Dalgard et al. 2004; Huang et al. 2002; Metzen et al. 2005). Although all three HPHs have been shown to hydroxylate key proline residues of HIFα in vitro, there is evidence that HPH2 has the primary role in vivo (Appelhoff et al. 2004; Berra et al. 2003; Epstein et al. 2001; Hirsila et al. 2003; Huang et al. 2002) and it was considered to be the ancestral form of this gene family in metazoans, based on conservation of amino acid residues and domain organization (Taylor 2001). Furthermore, available data suggest that each HPH has its own specific substrate, with HPH2 being the major form responsible for hydroxylating HIF-1α, and therefore the critical oxygen sensor for maintaining a low steady-state level of HIF-1α under normoxic conditions (Choi et al. 2003; Freeman et al. 2003; Huang et al. 2002; Masson et al. 2004; Masson and Ratcliffe 2003).

In this work, the HPH gene and cDNAs corresponding to its various isoforms were cloned from the eukaryotic parasitic protist Perkinsus olseni, and found to share higher similarities with the mammalian HPH2 form, thus supporting the hypothesis of its proximity to the ancestral form. The presence of this gene in Perkinsus olseni led us to investigate the main cellular processes involved in the regulation of HPH expression. Environmental factors, such as increased temperature and salinity and decreased levels of water-dissolved O₂, known to result in high clam mortality (Leite et al. 2004), also induce environmental hypoxia. Our data indicate that they also favor proliferation of Perkinsus, suggesting an association between factors inducing environmental hypoxia and increased host mortality. Accordingly, the effect of drugs with hypoxia-like effects, such as iron chelators, was also investigated on HPH gene expression in Perkinsus. Furthermore, to elucidate the role of this gene in pathogenicity, its expression was studied in Perkinsus cells exposed to hemolymph of permissive and non-permissive hosts, uncovering a direct association between expression of this gene and host permissive state.