By David N. Leff

If you've never heard of aceruloplasminemia, you're not alone. The inherited disease was first described only a couple of years ago, and to date there's one documented family in Ireland and two in Japan.

"People with aceruloplasminemia," said molecular and cellular biologist Paul Fox, "had severe problems with overloading of iron in their tissues. They had a defect in their ceruloplasmin gene, and were very sick."

Fox's research laboratory at the Cleveland Clinic focuses on the functions of ceruloplasmin in the body.

"Affected members of those aceruloplasminemia families," he told BioWorld Today, "acquire a common disease called hemochromatosis, which means iron loaded in many of their tissues. But the version of it they get is different, and much worse, because metallic iron crystals get deposited in their eyes and brain. In the latter, that causes severe neurological problems, such as loss of memory, speech and balance."

Ceruloplasmin (Cp) gets its poetic name because of its cerulean sky-blue color. And it gets that color from seven atoms of copper in every molecule of Cp. Its gene sits on the long arm of human chromosome 3.

The protein that gene expresses can, among other unknown functions, Fox observed, "cause the oxidation of ferrous Fe² to ferric Fe³. That's why the old name for ceruloplasmin was ferroxidase.

"And that old idea," he went on, "said that this ferroxidase can drive iron into transferrin, which transports iron around the body, or ferritin, an iron storage protein. Those two proteins, the story went, lacked ferric iron, so Cp could get it into them.

"The new idea that we now have," Fox said, "is that this can also drive iron into a form recognized by a ferric transporter on the cell surface."

Meanwhile, he went on, "two other labs started cloning new genes involved in iron metabolism in baker's yeast [Saccharomyces cerevisiae]. One of the genes they came up with was homologous to Cp. That was what motivated us to look at Cp and iron in cultured human cells."

This week's issue of Science, dated Jan. 30, 1998, tells what he and his co-authors discovered. Their paper's title: "Role of ceruloplasmin in cellular iron uptake."

They came up with a surprise.

"The old idea," Fox explained, was that Cp was important for getting iron out of cells. One easy way to think about that," he suggested, "is these patients with aceruloplasminemia have iron loaded in all their tissues. So you would say: 'Okay, no Cp; iron accumulates. You need Cp to get it out.'"

He found just the opposite to be true. Instead of Cp taking iron out of tissues, it puts the iron in. That's exactly the same effect those other investigators reported in their yeast.

One salient implication of this reversal of understanding, Fox surmised, "is that it may help explain not just aceruloplasminemia, in which things are more complicated than first thought. We think it may also help to explain both iron-deficiency anemia, which is common in infants and toddlers, and the anemia of chronic inflammation, which is frequent in adults."

He pointed out that "30 percent of the world's population is anemic."

A potential therapeutic payoff from his current finding and continuing research, Fox observed, "might be developing inhibitors that block Cp's interactions with the cell, and could potentially prevent, say, the anemia of inflammation.

"We're looking now," he added, "at specific sites on the Cp molecule responsible for its activities, for example, its ability to oxidize iron. At the molecular level of those sites, using mutagenesis, we could develop site-specific inhibitors to block individual Cp activities."

In the nearer term, he pointed out: "Cp is a copper protein. All very young children get iron supplements in their feeding formulas. While looking very carefully at how much iron we should give them, we should also be looking carefully at how much copper they ought to be getting. Because you need copper to make ceruloplasmin."

In his ongoing investigation, Fox is pursuing two questions: "First, we want to understand the molecular mechanism by which Cp transports iron into cells. We've shown that it happens. Now we want to know how the molecule does it."

Yeast Genes Blaze Human Trail

"Second, if a cell's iron-deficient, it makes Cp to get iron back into it. We've shown that that occurs by DNA transcription. Now we want to know how that transcription is regulated at the molecular level." He added, "And we have some very nice clues, because we're following the yeast."

As described in his Science paper, Fox and his co-authors measured iron taken up by normal liver cells in a simple radioactive assay. "Then we showed that Cp increased the uptake." Following this finding, the team determined that "iron deficiency actually induced Cp synthesis."

Wilson's disease (WD), a life-threatening inherited disruption of copper metabolism, used to be blamed on lack of ceruloplasmin — but no more. As Fox tells the story, "It was once thought that by a guy named David Gitlin. And then, 30 or 40 years later, his son, Jonathan Gitlin, showed that it's not the case of a mutant Cp, that WD is due to another gene."

Although Cp is off the hook as a bad actor in WD, Fox pointed out, "There is a relationship. And that is, our studies show, that you only get defects in cellular iron metabolism if you drop the Cp levels below 10 percent of normal. Now exactly the same thing," he went on, "was found in WD. Those people have very low Cp, and they're fine with respect to iron, because their Cp, though low, is not terribly low.

"In some rare WD patients," he concluded, "their Cp levels drop lower than that minimum 10 percent of normal. And those people do have iron problems. But there's no genetic relationship between our findings and Wilson's disease." *