August 2, 2012

VU study reveals connective tissue’s reinforcing ‘rivet’

An ancient enzyme that can be traced back 500 million years forms the chemical “rivet” that reinforces connective tissue throughout the body, Vanderbilt University researchers reported this week.

An ancient enzyme that can be traced back 500 million years forms the chemical “rivet” that reinforces connective tissue throughout the body, Vanderbilt University researchers reported this week.

Their paper, published online July 29 in the journal Nature Chemical Biology, reveals that the enzyme peroxidasin promotes formation of the sulfilimine chemical bond, which the Vanderbilt team discovered three years ago. The researchers also suggest that excess peroxidasin activity may lead to disease.

The sulfilimine bond reinforces the collagen IV network which, in the kidney, provides the structural scaffolding for the glomerular basement membrane.

“In kidney disease, too much collagen IV is deposited and there is expansion of basement membranes,” said Gautam (Jay) Bhave, M.D., Ph.D., instructor in Medicine and lead author on the study. “Higher levels of peroxidasin would have to be present to facilitate this.”
The enzyme is upregulated in cancer tissue as well, making it an attractive candidate for further clinical studies.

Billy Hudson, Ph.D., seated, holds the data showing that peroxidasin forms the sulfilimine bond. Behind him are, from left, Gautam (Jay) Bhave, M.D., Ph.D., Roberto Vanacore, Ph.D., Paul Voziyan, Ph.D., and Vadim Pedchenko, Ph.D.

“The bond formation is an ancient system,” said Billy Hudson, Ph.D., senior author on the paper and director of the Vanderbilt Center for Matrix Biology. “The system has been preserved since the Cambrian explosion, which says it is pretty important.”

Peroxidasin catalyzes sulfilimine bond formation by producing hypohalous acids — highly reactive, potentially toxic chemical intermediates. Sodium hypochlorite, or household bleach, is a common example of a hypohalous acid.

Chris Cummings, a Ph.D. candidate in Hudson’s lab and the paper’s co-first author with Roberto Vanacore, Ph.D., led the effort to determine the chemistry behind the bond formation.

Evolution has preserved this biological mechanism, as peroxidasin may have provided primitive defense against bacterial infection in lower organisms by locally producing hypohalous acids in basement membranes.

“The enzyme can be viewed as a bleach generator that is embedded in the basement membrane in all tissues throughout the animal kingdom,” said Hudson. “The bleach is essential for genesis of tissues, but under- or over-production of bleach can cause disease.”

For example, overproduction of hypohalous acids may contribute to oxidative stress, which is associated with diseases such as atherosclerosis and diabetes. “Peroxidasin is an intrinsic part of basement membranes, where damage occurs in these diseases,” said Vanacore, assistant professor of Medicine.

Vanacore led the team that first reported the sulfilimine bond in Science magazine in 2009. The sulfur-to-nitrogen double bond links together collagen IV molecules in basement membranes, providing a scaffold-like structure for tissues.

Bhave discovered that the sulfilimine bond was formed by a peroxidase, an enzyme that accelerates electron transfer between two organic substances. Peroxidases are abundant, so to narrow his search Bhave took an unorthodox approach and turned to Google.

When he searched for “peroxidase and basement membrane,” he found a 1994 paper by John Fessler, Ph.D., and colleagues at UCLA, reporting the discovery of peroxidasin in the basement membrane of the fruit fly Drosophila.

“It was around midnight when I got the call. Jay had figured out that peroxidasin was making the sulfilimine bond,” said Hudson.

Hudson contacted the UCLA researchers, found out they were still working on peroxidasin, and brought them on as collaborators. Their fly mutants, which lacked the enzyme, had disorganized basement membranes and structural defects.

Other Vanderbilt contributors included research associate professor of Medicine Vadim Pedchenko, Ph.D., Ph.D. candidate Isi Ero-Tolliver and research assistant Mohamed Rafi.
Hudson also recruited the “Aspirnauts,” high school students from rural Arkansas and Tennessee who participate in a summer research program at Vanderbilt supported by the National Institutes of Health (NIH). The program was established in 2007 by Hudson, an Arkansas native, and his wife Julie Hudson, M.D., assistant vice chancellor for Health Affairs.

During the summers of 2009 through 2012, 43 Aspirnauts contributed to experiments showing the sulfilimine bond and the peroxidasin-based mechanism by which it forms can be traced back to the ancient organism, cnidarian. A paper on the evolutionary origin of the bond is in preparation.

The current study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the NIH (grants RO1 DK018381 and PO1 DK065123). The Aspirnaut program is supported by PO1 DK065123 and by an NIDDK Step Up grant.