A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation

Abstract

Background: The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the alpha subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1alpha in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).

Results: Patients with IE were screened for changes in the HIF-1alpha coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.

Conclusion: The Pro582Ser change represents a common polymorphism of HIF-1alpha that does not impair HIF-1alpha prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1alpha it does not diminish the association of HIF-1alpha with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.

Figure 1

Detection of C1744T polymorphism. Sequencing of exon 12 of the HIF-1α detected a heterozygous base change of C to T at nucleotide 1744 in 8 patients (Figure 1B) when compared to the normal sequence (Figure 1A). One patient, D1, was homozygous for this base change (Figure 1C). The position of the mutation is indicated by an arrow. Bases are as follows: G = black; A = green; T = red; C = blue. The C1744T mutation destroys the restriction site for Tsp 45I enzyme and the normal exon 12 PCR product is restricted in the presence of this enzyme into 4 fragments of 213, 156, 139 and 92 bp; in the presence of the C1744T mutation the 156 and 139 bp fragments are replaced with one of 295 bp. (Figure 1D). M1: 100 bp DNA ladder; M2: 25 bp ladder; 1: C1744 control individual; 2: heterozygous individual for C1744T polymorphism; 3: individual D1 homozygous for C1744T polymorphism.

Figure 2

Effects of the Pro582Ser polymorphism on HIF-1α hydroxylation. (A) GST pulldown assay. 5 μg of recombinant (His)₆FlagPHD2 were incubated with 3 μg of either GST, GST-HIF-1α (531–575), or GST-HIF-1α (569–589) prebound to GSH agarose under hydroxylation conditions, as indicated. The resins were washed, and were subsequently incubated with ³⁵S-labeled, in vitro translated HA-VHL. Bound VHL was eluted and then subjected to SDS-PAGE and autoradiography. The position of HA-VHL is indicated. "In" designates 10% of the input HA-VHL. One of two representative results is shown. (B) VBC pulldown assay. ³⁵S labeled, in vitro translated wild type or Pro582Ser GAL4-HIF-1α (531–652) was incubated with 1 μg of (His)₆-PHD2 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with (His)₆-Flag-VBC, and then by immunoprecipitation with anti-Flag antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. "In" designates 10% of the substrate. One of three representative results is shown. (C) GST pulldown assay. 1.5 μg of recombinant (His)₆PHD2 was incubated with 5 μg of either GST, GST-HIF-1α (531–652), or GST-HIF-1α (531–652) Pro582Ser prebound to GSH agarose under hydroxylation conditions, as indicated. The resins were washed, and were subsequently incubated with ³⁵S-labeled, in vitro translated FlagVHL Arg200Trp. Bound VHL was eluted and then subjected to SDS-PAGE and autoradiography. The position of VHL Arg200Trp is indicated. "In" designates 10% of the input VHL Arg200Trp. One of two representative results is shown.