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Ironing out a mysterious protein

Strong lab sheds light on function of NGAL, which helps body rob key ingredient from disease-causing bacteria
A three-dimensional model shows the protein NGAL with the iron-enterochelin complex bound in the "cup" (lower right). The background is rat kidney tissue stained to highlight components of the developing kidney - the nephrogenic precursors (the "branches") and the ureteric buds (the "leaves"). In the tube is a preparation of NGAL, which is red when purified from E. coli. Crystals of NGAL are used for the X-ray crystallographic analysis to determine its three-dimensional structure. Illustration courtesy Dr. Roland Strong

When your doctor recommends adding iron to your diet, it's easy enough to reach for a box of Special K or a multivitamin.

But when infectious bacteria need that essential ingredient, acquiring it is a hard-won battle. Inside the human body, competition for iron among microbes can be fierce.

Free iron - that is, not bound to proteins like hemoglobin that ferry it throughout the blood and tissues - exists in minute quantities in the body. Without it, bacteria, like humans, can't survive.

So it's not surprising that the human immune system attempts to thwart bacterial growth by robbing every last trace of this precious mineral from disease-causing microbes.

New research

Exactly how the immune system accomplishes this task is now clearer, thanks to new research from Dr. Roland Strong's laboratory in the Basic Sciences Division. The lab's work sheds light on the function of a previously mysterious protein called NGAL, which the lab found to be central to this clever defense mechanism.

A second study performed in collaboration with researchers in New York revealed that the same protein also is critical for normal kidney formation in the earliest stages of mammalian development.

And there's more: NGAL may even play a role in the growth advantage that cancer-tumor cells display compared to their healthy neighbors.

The studies are published in two papers and featured on the cover of the November issue of Molecular Cell. An editorial about the discoveries appears in the Nov. 27 edition of Cell.

Long, winding journey

The experimental journey that illuminated roles for this do-it-all protein was a long and winding one, Strong said.

"Science is always a combination of hard work and unexpected events," he said, "but this project was serendipity magnified a few times."

The story began when Strong's graduate student David Goetz, now a postdoctoral fellow at the University of California in San Francisco, set out to determine the three-dimensional structure of NGAL. The lab's interest in the protein stemmed from its sequence similarity to a family of proteins known as lipocalins.

"Lipocalins are small proteins that cells send out to bind things and carry them back," Strong said. "They are found in many different cell types and organisms."

NGAL is found in neutrophils, white blood cells that are among the first defense cells on the scene at the site of an injury or infection. The cells are packed with granules that contain proteins, including NGAL, that display antibacterial action.

Goetz elucidated NGAL's structure, using a technique known as X-ray crystallography. Yet the protein did not resemble known lipocalins and its unusual structure - teacup-shaped - did not initially yield clues to what molecules it might bind.

The first breakthrough came when Goetz attempted to manufacture large quantities of NGAL for further structural studies.

Previously, the lab had obtained pure preparations of NGAL from colleagues who had engineered insect cells to overproduce it. When those supplies ran low, Goetz turned to a strain of the bacterium E. coli that had been manipulated to serve as an NGAL factory.

Surprisingly, after Goetz purified NGAL from these modified bacteria, the resulting protein preparation was bright red.

"The preparation made in insects was colorless," Strong said. "That suggested that NGAL was binding to a pigmented molecule in E. coli."

'Blob' of iron

Goetz repeated the crystallography analysis on the red protein, which revealed a "blob" lodged inside NGAL's teacup-like cavity.

Signals produced by the analysis indicated a metal was contained within this unknown bacterial molecule. That finding, combined with the red color, immediately suggested that the substance contained iron.

Goetz determined the identity of the molecule associated with NGAL to be a bacterial iron-scavenging molecule known as enterochelin. A few strains of E. coli make a second iron-scavenging molecule known as aerobactin, which causes those strains to be particularly virulent. Aerobactin has a distinct structure from enterochelin and does not associate with NGAL.

Strong said this information helped explain why aerobactin is what is known as a "virulence factor."

"NGAL binds up all the enterochelin during an infection, inhibiting bacterial growth by removing available iron," he said. "But NGAL doesn't recognize aerobactin, so a strain of bacteria that produces aerobactin would be resistant to NGAL's antibacterial action."

Potent agent

In the laboratory, NGAL is a potent antibacterial agent as well. Even trace amounts added to a culture of E. coli stops growth in its tracks. For this reason, Strong is investigating whether NGAL can bind to iron-scavenging molecules from other types of pathogenic bacteria with the hope of potentially exploiting NGAL for therapeutic use.

While this clarifies the antibacterial role of NGAL in neutrophils, Strong noted that NGAL has been implicated in the scientific literature in diverse biological processes, including kidney-cell development and cancer.

In collaboration with researchers at Columbia University and Memorial Sloan-Kettering Cancer Center in New York City, Strong's group found that during the early stages of mouse embryonic growth, the developing kidney uses NGAL to acquire iron necessary for further differentiation. This finding was the first discovery of an alternate iron-delivery pathway distinct from the major iron-delivery pathway in mammals, which is mediated by a molecule called transferrin.

NGAL's ability to deliver iron may explain why it is overproduced in certain tumors, where it might confer a growth advantage by better competing for iron than in healthy cells. Studies are under way to address NGAL's role in tumor formation.

Strong said the NGAL story represents a rare case in which a crystallography lab has the opportunity to determine the function of a protein.

"Typically, we contribute to the understanding of proteins whose functions are already established," he said. "But in the case of NGAL, determining its structure was the key to figuring out its biological role."

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