Vanderbilt Institute of Chemical Biology

 

 

Discovery at the VICB

 

 

 

 

 

 

An Iron–Sulfate Metabolic Link

 

By: Carol A. Rouzer, VICB Communications
Published:  March 26, 2018

 

 

Genetic deficiency of an enzyme involved in the sulfate activation pathway leads to iron deficiency anemia.  

 

Sulfation is an important biochemical reaction that plays a role in detoxification of some xenobiotic compounds and the biosynthesis of molecules as diverse as condroitin sulfate and bile acids. The sulfotransferase enzymes that catalyze these reactions require activated sulfate in the form of 3´-phosphoadenosine 5´-phosphosulfate (PAPS, Figure 1). In mammalian cells, the enzyme responsible for PAPS biosynthesis is phosphoadenosine phosphosulfate synthetase (Papss2), which first adds inorganic sulfate to ATP to form adenosine 5´-phosphosulfate (APS) and then phosphorylates APS at the 3´-hydroxyl group to yield PAPS. Utilization of the sulfate group of PAPS in sulfotransferase reactions produces 3´-phosphoadenosine 5´-phosphate (PAP), which is then hydrolyzed to AMP. In the cytosol, the enzyme bisphosphate 3'-nucleotidase (Bpnt1) hydrolyzes PAP. Mice genetically deficient in Bpnt1 (Bpnt1 KO) exhibit generalized edema, liver failure, and impaired ribosomal biogenesis. Now, Vanderbilt Institute of Chemical Biology member John York and his lab show that these mice also suffer from iron deficiency anemia, revealing a previously unknown link between sulfate and iron metabolism and a potential genetic basis for this form of anemia [Hudson, et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published online March 5, DOI:10.1073/pnas.1715302115].

 

 

FIGURE 1. Sulfate activation pathway in mammalian cells. Papss2 catalyzes the reaction of ATP with inorganic sulfate to form APS and then the reaction of APS with ATP to form PAPS. PAPS serves as the sulfate donor for a wide range of sulfotransferase enzymes that add sulfate to substrates such as xenobiotic compounds, steroids, and carbohydrates. This converts PAPS to PAP, which is then hydrolyzed by Bnpt1 in the cytosol.

 

 

In Bpnt1 KO mice, failure to hydrolyze  PAP could lead to its accumulation (Figure 2). The investigators hypothesized that PAP toxicity might contribute to the development of anemia in the mice. To test this hypothesis, they engineered a Bpnt1 KO mouse that also carried a hypomorphic mutation in the gene encoding Papss2. In these mice, failure to synthesize PAPS prevents PAP accumulation even in the absence of Bpnt1, and consistent with their hypothesis, anemia did not develop.

 


FIGURE 2. Impact of enzyme deficiencies on levels of PAP in cells. (Top) In wild-type cells, hydrolysis of PAP by Bpnt1 results in physiological levels of the compound. (Center) In Bpnt1-/- cells, failure to hydrolyze PAP leads to its accumulation, reaching high levels. (Bottom) Double knockout (DKO) of Bpnt1 and Papss2 reduces the amount of PAPS to be converted to PAP, resulting in levels similar to those in wild-type cells. Figure reproduced under the Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND) from B. H. Hudson, et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published online March 5, DOI:10.1073/pnas.1715302115.

 

 

Prior work had demonstrated that Bpnt1 deficiency results in severe liver malfunction. The liver produces the hormone hepcidin that blocks the iron transporter ferroportin in the intestine. Under conditions of iron deficiency, hepcidin synthesis should be reduced to enable increased ferroportin activity. This response occurred normally in Bpnt1 KO mice, indicating an appropriate hepatic response to iron deficiency. This, coupled with the finding of pale, dilated, and abnormal nucleoli in the intestines of the mice suggested that the site of PAP toxicity leading to iron deficiency anemia is likely the intestinal epithelium.

 

To determine the effects of Bpnt1 deficiency in the intestine, the researchers developed a mouse model in which the gene encoding the enzyme was knocked out only in intestinal tissue (Bpnt1 KOint). These mice developed hair loss and anemia (Figure 3), but not the other abnormalities observed in Bpnt1 KO mice. Placing Bpnt1 KOint mice on an iron-deficient diet substantially exacerbated their anemia, leading to a much greater drop in hemoglobin and red blood cell counts than were observed in wild-type mice placed on the same diet. The researchers hypothesized that a failure to increase the expression of the intestinal epithelial apical iron transporter Dmt1 in response to dietary iron deficiency could be responsible for the severe anemia observed in Bpnt1 KOint mice. The finding that both mRNA and protein expression of Dmt1 were much lower in the intestines of Bpnt1 KOint mice than wild-type mice upon exposure to the iron-deficient diet provided support for this hypothesis.

 

 

FIGURE 3. Photomicrograph of red blood cells from wild-type mice (-/fl) and mice deficient in intestinal Bpnt1 (-/int). In the Bpnt1-deficient mice, the cells are smaller and pale because they contain less hemoglobin. Figure reproduced under the Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND) from B. H. Hudson, et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published online March 5, DOI:10.1073/pnas.1715302115.

 

 

The transcription factor hypoxia inducible factor 2α (HIF-2α) is primarily responsible for regulating the response to iron deficiency. Under iron deficient conditions, the rate of HIF-2α degradation decreases, so more of the protein is available to promote the transcription of genes bearing an HIF response element. The researchers performed a Gene Set Enrichment Analysis on intestinal epithelial cells from Bpnt1 KOint and wild-type mice that had been fed either a standard or iron-deficient diet. They noted differences in the expression of genes involved in sulfate and iron metabolism between the mice of the two different genotypes regardless of diet. Differences in the expression of HIF-2α target genes were also evident between the mice, but only if they were exposed to the iron deficient diet. Genes associated with iron metabolism that were differentially expressed included those encoding HIF-2α, the transferrin receptor, ferroportin, iron reductase, and Dmt1. The researchers confirmed that the reduced gene expression led to a corresponding reduction in expression of each of these proteins in Bpnt1 KOint as compared to wild-type mice. The low levels of HIF-2α expression in intestinal cells from Bpnt1 KOint mice (Figure 4) led the researchers to compare the patterns of gene expression in these mice with mice genetically deficient in HIF-2α, and they found similarities in their genetic signatures.

 

 

FIGURE 4. Expression of HIF-2α in intestinal epithelium from wild-type mice (-/fl), mice deficient in intestinal Bpnt1 (-/int), and HIF-2α knockout mice (HIF-2α  KO). HIF-2α is stained in green, and nuclei are stained blue. Figure reproduced under the Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND) from B. H. Hudson, et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published online March 5, DOI:10.1073/pnas.1715302115.

 

 

Together, the data suggest that Bpnt1 deficiency in the intestine leads to a high level of PAP that in turn disrupts HIF-2α-dependent signaling. The result is failure to respond appropriately to iron deficiency, leading to anemia. Although the exact mechanism by which PAP toxicity produces HIF-2α dysfunction is not fully understood, the findings demonstrate a previously unknown link between sulfate and iron metabolism. They also suggest a possible genetic origin for iron deficiency anemia that can be investigated clinically. If found, patients who develop anemia due to Bpnt1 deficiency might benefit from therapy targeting Papss2.

 

 

 

View Proc Natl Acad Sci U S A. article: Modulation of intestinal sulfur assimilation metabolism regulates iron homeostasis.

 

 

 

TwitterYouTube

 

 

The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center