Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD)

Abstract

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O(2)- and Fe(2+)-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis.

Fig. 1. Multiple sequence alignment of partial amino acid sequence of HIF-α from eastern oyster with HIF sequences from Pacific oyster, shrimps, nematodes (C. elegans and C. briggsae) and human

Helix-loop-helix (HLH) DNA binding domain and PAS fold domains are shown with stripped and white boxes, respectively, as per PFAM prediction and Li and Brouwer (2007). Identical residues are shown with dots (.), and gaps are indicated as dashes (-). The mRNA sequence for HIF-α transcript from C. virginica is deposited in GenBank (NCBI accession number HM441076).

Fig. 2. Minimum evolution tree of representative metazoan HIF-α sequences based on the Dayhoff distance

Support of internal branches was evaluated using the bootstrap method, and only values higher than 50% are shown. HIF-α sequences from nematodes were used as outgroup. Each sequence is identified by the name of the species and its Ensembl or GenBank accession number. Detailed list of sequences used is given in Suppl. Table 1.

Fig. 3. Multiple sequence alignment of partial amino acid sequence of PHD from eastern oyster with EGLN-1,-2 and -3 sequences from human and EGL-9 homolog from Caenorhabditis elegans

Shared 2-oxoglutarate- and Fe²+-dependent oxygenase domain (2OG-FeII_Oxy) is shown with white and shaded boxes, as per PFAM prediction and Aravind and Koonin ( 2001), respectively. Asterisks (*) and underscores (_) mark predicted catalytic residues and residues that share physicochemical properties among the entire 2OG-Fe(II) dioxygenase superfamily, respectively, per Aravind and Koonin (2001). The mRNA sequence for PHD homolog from C. virginica is deposited in GenBank (NCBI accession number HM441077).

Fig. 4. Minimum evolution tree of representative metazoan PHD sequences based on the Dayhoff distance

Support of internal branches was evaluated using the bootstrap method, and only values higher than 50% are shown. EGL-9 sequences from nematodes were used as outgroup. Each sequence is identified by the name of the species and its Ensembl or GenBank accession number. Detailed list of sequences used is given in Suppl. Table 2.

Fig. 5. Tissue-specific mRNA expression of HIF-α (A) and PHD (B) in C. virginica under different experimental conditions

mRNA expression was determined using qRT-PCR in tissues of control oysters (normoxia), oysters maintained for 2 weeks in 5% oxygen (hypoxia), exposed for 6 days in air with no gapping (anoxia) or for 30 days to 50 μg L⁻¹ Cd in normoxia (Cd). HP – hepatopancreas. A post-hoc LSD test was used to carry out the following comparisons: 1) within each tissue type, all experimental treatment groups were compared to each other and to the respective controls; 2) within each treatment group, mRNA levels for the target genes were compared between different tissues. For the first type of comparisons, asterisks in panel B indicate values significantly different from the respective hypoxic values in the same tissue type (P<0.05); all other experimental conditions were not significantly different from each other or from the respective controls (P>0.05). For the second type of comparisons, different letters denote values that are significantly different between the tissues within each experimental condition (P<0.05). No letters mean that there were no significant differences in gene expression between different tissues within the given experimental condition (e.g. in normoxic, hypoxic and Cd-exposed groups) (P>0.05). N=4–10.