Biological tissue’s ‘glue’ identified
Researchers at Vanderbilt University Medical Center have discovered a novel chemical bond in biological tissue, a fundamental discovery that helps explain evolutionary adaptation and may shed light on human disease.
The novel sulfilimine bond, between a sulfur and a nitrogen atom, acts like a “fastener” to reinforce a collagen IV network found in connective tissue throughout the body. It is “an apparent adaptation crucial for … evolution,” the researchers reported in this week's Science magazine.
Bill Hudson, Ph.D.
“Every tissue in your body has got this bond,” said Billy Hudson, Ph.D., senior author of the paper and director of the Vanderbilt Center for Matrix Biology. “It is the 'glue' … that helps hold together the extracellular matrix.”
The extracellular matrix provides structural support in all tissues, and molecular cues for influencing cell behavior.
Roberto Vanacore, Ph.D.
The finding “is just scratching the top of the iceberg,” added the paper's lead author, Roberto Vanacore, Ph.D., research assistant professor of Medicine. The Vanderbilt researchers are now searching for the enzyme that makes the bond, and for diseases that may be caused by a defective bond.
Hudson, the Elliot V. Newman professor of Medicine, Biochemistry and Pathology, has been studying the glomerular basement membrane of the kidney for more than 40 years.
The basement membrane, which supports cells in the extracellular matrix, consists largely of type IV collagen. The collagen, in turn, is constructed from a family of proteins called alpha chains that twist around each other to form triple helical “protomers,” like cables supporting a bridge.
While at the University of Kansas Medical School in Kansas City, Hudson and his colleagues discovered two previously unknown alpha chains, which are defective in an inherited kidney disorder called Alport syndrome.
One of the chains, they found, is also involved in a rare, auto-immune kidney disease called Goodpasture syndrome.
There is some evidence that the misguided antibody attack on the kidney's basement membrane may be triggered by exposure of a previously hidden part of collagen caused by the absence or breakage of the sulfilimine bond, Vanacore said.
A native of Chile, Vanacore was a first-year graduate student in Hudson's lab at Kansas in 2000 when he decided to test a well-accepted hypothesis that the chains of collagen IV molecules were held together by disulfide bonds — two sulfur atoms.
If they were actually disulfide bonds, chemicals called reducing agents should break them apart. But Vanacore couldn't break them. After he moved with Hudson and his lab to Vanderbilt in 2002, Vanacore doggedly tried to determine the bond's true structure.
With Amy-Joan Ham, Ph.D., research associate professor of Biochemistry, he broke the collagen chains into pieces and used mass spectrometry at the Vanderbilt Proteomics Laboratory to analyze the fragments.
They found the first evidence for a covalent bond between methionine and hydroxylysine amino-acid residues that connects the collagen chains.
To determine the chemical structure of the bond, they sent the fragments to Thomas Conrads, Ph.D., and Timothy Veenstra, Ph.D., at the National Cancer Institute lab in Frederick, Md. Using high-resolution mass spectrometry they found the first evidence to support Vanacore's suspicion that the bond was sulfilimine — not disulfide.
Back at Vanderbilt, Chuck Sanders, Ph.D., professor of Biochemistry, and Markus Voehler, Ph.D., research assistant professor of Chemistry, got the same answer when they applied another technique, nuclear magnetic resonance (NMR) spectroscopy.
By now, “we were scratching our heads,” Vanacore recalled. “Is it possible?”
He and Hudson asked Barry Sharpless, Ph.D., a Nobel laureate and expert in sulfur chemistry at the Scripps Research Institute in San Diego, and his colleague, Philip Dawson, Ph.D., to collaborate on additional experiments to determine the chemical nature of the bond.
The collaborations led to the discovery of the sulfilimine bond between the sulfur atom of methionine and a nitrogen atom of hydroxylysine.
By comparing gene sequences for collagen from different species, the Vanderbilt researchers found evidence that the bond may be conserved across nearly all of evolutionary scale, from hydra-like creatures to humans.
“Maybe at some point it was necessary in order to survive to make this network (of collagen fibers) stronger,” Vanacore speculated.
Vanacore, who earned his doctorate in Biochemistry in 2005, credited Hudson with encouraging him to pursue the search for the bond. “You have to reach as high as you can,” he said. “That's part of being a scientist.”
The research was supported by the National Institutes of Health, the W.M. Keck Foundation and the Skaggs Institute for Chemical Biology at Scripps.