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Bromide Ion is Essential for Life

By: Carol A. Rouzer, VICB Communications
Published: June 24, 2014

Bromide ion is required for basement membrane biosynthesis in multicellular animals with organized tissues containing epithelium.

The basement membrane is a thin fibrous layer of extracellular protein that underlies the epithelial cells of many tissues in multicellular animals. The primary component of basement membrane is a latticework of fibers formed by the assembly of triple-helical protomers of collagen IV. This lattice is stabilized by cross-links that join the NC1 domains of two associated protomers. The cross-links unite Met-93 of one NC1 domain to hydroxylysine- (Hyl-)211 of the neighboring NC1 domain through a sulfilimine (N=S) bond (Figure 1). The heme peroxidase peroxidasin catalyzes sulfilimine bond formation. The enzyme uses H2O2 to oxidize a halide ion to the corresponding hypohalous acid, which then reacts with the sulfur atom of Met-93 in the first step of the reaction (Figure 2). Peroxidasin can oxidize both Cl- and Br- in the presence of H2O2, and it seemed likely that the much more abundant Cl- ion would be utilized for the reaction in vivo. But now, Vanderbilt Institute of Chemical Biology member Billy Hudson and his laboratory report that Br- is absolutely required for sulfilimine bond formation and correct basement membrane assembly in living organisms [A. S. McCall, C. F. Cummings, G. Bhave, et al., (2012) Cell, 1380].

Figure 1. Cartoon representation of the molecular structure of the NC1 domains of two collagen trimers joined end-to-end to form a hexamer. Sulfilimine bonds between Met-93 of one monomer and Hyl-211 of the opposing monomer stabilize the structure. Image reproduced through the courtesy of Wikimedia Commons under the GNU Free Documentation License.

Figure 2. Reactions involved in sulfilimine bond formation. Peroxidasin catalyzes the reaction of H2O2 and a halide ion to form a hypohalous acid (top), which then reacts with the sulfur of Met-93 prior to formation of the cross-link. Dotted lines indicate bonds linking Met-93 and Hyl-211 to the remainder of the protein chain.

PFHR-9 embryonal carcinoma cells produce basement membrane in culture. The Hudson lab researchers showed that addition of SCN- or I- suppressed, while Br- enhanced, cross-link formation by these cells. The effects of Cl- and F- could not be determined in cell culture due to the abundance of Cl- in the medium and the toxicity of F-. So, the investigators grew the cells in the presence of I- to suppress cross-link generation and used the immature basement membrane they produced to explore sulfilimine bond formation in vitro. They discovered that addition of H2O2 plus F- generated no cross-links, while substitution of Br- for F- resulted in robust sulfilimine bond formation. Some cross-link production also occurred in the presence of H2O2 plus Cl-, but only at very high concentrations of the halide. This led them to hypothesize that the effect of Cl- was due to trace contamination by Br-. Indeed, carefully prepared Br--free Cl- was unable to promote sulfilimine bond formation in the presence of H2O2 in vitro. Furthermore, PFHR-9 cells grown in Br--free medium produced no collagen IV cross-links.

Collagenase digestion of basement membrane produced by cultured PFHR-9 cells yields free NC1 hexamers, which are formed when the ends of two collagen protomers associate to form the fibrous network (Figure 1). Sodium dodecyl sulfate gel electrophoresis separates the NC1 hexamers into monomers, and two cross-linked dimers, designated D1 and D2. Mass spectrometric analysis indicated that the D1 dimer contains a single sulfilimine cross-link, while the D2 dimer contains two (Figure 3). The researchers found that peroxidasin was more effective than myeloperoxidase or eosinophil peroxidase at catalyzing the formation of sulfilimine bonds, particularly with respect to the cross-links present in the D2 dimers. Peroxidasin used Br- preferentially to Cl-, and the researchers determined that the EC50 for Br- in the peroxidasin reaction is 4.5 μM, well within the physiological serum concentration of Br- (10 to 100 μM).


Figure 3. Collagenase treatment of the basement membrane cleaves the NC1 domain of collagen IV from the remainder of the molecule. Because the NC1 domains of each of the three protein chains of collagen IV in the protomer have associated with the NC1 domain of a chain in the opposing protomer during basement membrane assembly, collagenase treatment releases the domains as a hexamer. SDS gel electrophoresis of these hexamers dissociates the individual subunits, revealing three bands, designated D1, D2, and M. The D1 and D2 bands are dimers containing one and two sulfilimine bonds, respectively, while the M band is the NC1 monomer.

Kinetics studies suggested that the D1 dimer is formed before the D2 dimer, leading to the hypothesis that D1 is formed when Met-93 from one monomer is cross-linked to Hyl-211 of the associated monomer. Then, Met-93 of that second monomer cross-links to Hyl-211 of the first monomer to form D2. A combination of mechanistic studies and molecular modeling provided an explanation for the preference of Br- over Cl- in sulfilimine bond formation (Figure 4). The investigators proposed that reaction of a hypohalous acid with the sulfur of Met-93 forms a halosulfonium ion intermediate. This unstable species can then react with water to produce methionine sulfoxide or with the amine of Hyl-211 to form the sulfilimine bond. Modeling of reaction energetics revealed that reaction of the chlorosulfonium ion with water is favored over its reaction with an amine. In contrast, reaction of the bromosulfonium ion with an amine is favored over its reaction with water. Thus, in the presence of Cl-, it is much more likely that methionine sulfoxide formation will occur, an outcome that actually blocks cross-link formation, while Br- promotes generation of the sulfilimine bond. Furthermore, once two monomers are linked by one sulfilimine bond as in the D1 structure, formation of the second bond yielding D2 is highly favored.

Figure 4.  Mechanism for Br--dependent sulfilimine bond formation. Both HOCl and HOBr can form a halosulfonium ion with Met-93. Reaction of the halosulfonium ion with water leads to sulfoxide formation, which prevents cross-linking. This reaction is favored for the chlorosulfonium ion. Reaction with the amino group of Hyl-211 leads to sulfilimine bond formation. This reaction is favored for the bromosulfonium ion.

Their in vitro studies strongly supported the hypothesis that Br- is required for efficient sulfilimine bond formation. To determine whether this conclusion also holds true in vivo, the investigators explored basement membrane synthesis in the fruit fly Drosophila melanogaster raised on a Br--free medium. In the first generation, flies retained some Br-, so the full effects of Br- deficiency were not observed. However, larvae of these flies exhibited developmental delay. By the second generation, a significant reduction in survival of the larvae was noted. These findings were reversed by adding Br- back to the medium. Eggs obtained from Br--free flies exhibited a marked reduction in hatching efficiency, and most larvae from the eggs that did hatch died prematurely. The basement membranes in these larvae were irregular, thickened, and diffuse, and marked by gross disruptions and splitting of the scaffold. Similar findings were observed in larvae from flies expressing mutant peroxidasin with low enzymatic activity.

During early development in D. melanogaster, a layer of collagen IV is deposited around the circumference of the egg. The collagen serves as a “corset” to prevent expansion of the center of the egg.  As a result, the egg elongates as it grows (Figure 5). The Hudson Lab investigators discovered that there is a dose-dependent relationship between Br- concentration and the aspect ratio (ratio of length to diameter) of the egg. As Br- concentrations increased, the aspect ratio also increased. These findings suggested that strong sulfulimine bond formation “tightens” the collagen IV corset about the egg, leading to a longer, narrower shape. Consistently, the researchers found that inhibition of peroxidasin, either by the small molecule inhibitor phloroglucinol or by siRNA knockdown, resulted in a decrease in egg aspect ratio.


Figure 5.  During egg development in D. melanogaster, a layer of collagen IV is deposited on the outside of the egg. This layer constricts the egg as it grows, causing it to elongate. Increasing Br- concentration favors cross-linking of the collagen IV layer, strengthening it and leading to more elongation. Inhibition of peroxidasin blocks cross-linking, weakening the collagen layer and preventing egg elongation.

Together the data support a role for Br- in sulfilimine bond formation that is required for basement membrane synthesis. As the sulfilimine bond is found in basement membrane over the broad range of multicellular animals with complex tissue organization, a requirement for Br- is supported in all of these organisms. This is the first demonstration that Br- is an essential nutrient in any species, and the first defined role for the ion in normal metabolism.







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