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Chain LinksTangles of collagen IV chains (depicted here) strengthen basement membranes crucial for the structural integrity of animal tissues. A new study shows that the ropes of collagen connect at globules (arrow) via sulfur-nitrogen bonding, a chemical pairing never before seen in living animals.Courtesy of Science/AAAS

The foot bone’s connected to the leg bone, the leg bone’s connected to the knee bone — thanks in part to a newly discovered chemical bond, researchers report in the Sept. 4 Science. The bond — never before seen in living things — was found buried in the basement membrane, a tough, structural layer of cells that surrounds most tissues. A greater understanding of the links within this membrane may lead to new approaches for targeting tumors and for treating a number of diseases.

Animals from roundworms to humans have basement membranes, and the layer has long been recognized as important for a body’s structural integrity. The membranes surround tissues, tethering muscle to skin or to cartilage, and in the kidney’s case, act as a huge filter for blood. Basement membranes also play a signaling role, acting sort of like a cellular thermostat.

“If the basement membrane misbehaves, then the cell misbehaves,” comments Raghu Kalluri, chief of the matrix biology division at Harvard Medical School in Boston.

Type IV collagen, a major component of basement membranes, provides a strong, cross-linked mesh material that lends superior structural support. The collagen molecule is a triple helix, three strands twisted together like rope. One end of each ropelike chain is capped with a globule of amino acid molecules. These amino acids connect to the globule of another collagen chain, forming a meshy matrix. Though such matrices are ubiquitous in animals, the nature of the bonds connecting the globular tips had eluded scientists.

Initially, researchers thought the bond might be what’s called a disulfide bridge. Previous research had hinted that sulfur was involved. And bonds between two sulfur groups on amino acids commonly impart 3-D stability in proteins. But X-rays of the globular tips’ crystal structure ruled out the sulfur-sulfur connection some time ago, says biochemist Billy Hudson, who led the new research.

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Back story: Support in the basementView larger version | Basement membranes anchor cells, act as barriers and provide a framework for blood vessel development. The membranes also help regulate cell behavior and signaling. Line art: A. Nandy. Image: Susumu Nishinaga/Photo Researchers, Inc.

Now finer instruments reveal a sulfur-nitrogen bond, a partnership created in organic chemistry labs a handful of times but never before seen in living things.

Today’s super high-resolution mass spectrometers made the discovery possible, says Hudson, director of the Center for Matrix Biology at Vanderbilt University School of Medicine in Nashville. Previous attempts with less powerful instruments suggested that when the bond formed between the amino acid tips, one hydrogen atom departed. But the experimental error was too large to say what was happening with finality. The new analysis reduced the experimental error by a factor of 1,000. The team found that two hydrogen atoms are lost during bond formation and that sulfur and nitrogen atoms linked.

Covalent sulfur-nitrogen bonds — called sulfilimine bonds — connect the two collagen chains, linking the amino acids, methionine and a version of lysine, the team reports.

“The collagen IV network is what cells lay on, no matter if it’s an anemone or a human,” Hudson says. The bond “is the molecular fastener.” Like mortar connecting bricks in a wall, the bond provides critical reinforcement.

The bond is a strong one. Covalent bonds, where atoms share electrons, “really up by an order of magnitude the force that the material can deal with,” says James Kramer of the Northwestern University Feinberg School of Medicine in Chicago. It isn’t clear what advantages a sulfur-nitrogen bond might have over a disulfide bridge, which is known for its strength. There are already disulfide bonds in the globule that eventually connects the chains, Kramer notes, so perhaps saving a lot of exposed sulfurs for later bonding would screw up the initial folding of the globule.

Research by Kramer and colleagues revealed the critical role of collagen in holding together the nematode C. elegans. Even when the genes for collagen IV were knocked out, the worm developed properly. But when it began to move, its muscles pulled apart, detaching from the skin.

Hudson’s team first found the bond in cow placenta tissue, but he’s already been looking in other creatures. Over the summer, along with several area middle school and high school students, Hudson launched a “biochemical expedition through the animal kingdom looking for that bond,” he says. The young researchers detected the two connected globules — a telltale sign of the bond — in tissues of all the animals they’ve investigated including monkeys and sea urchins.

Perhaps the cross-link evolved in response to the mechanical stress of having an elaborate body, the researchers speculate. Their analyses comparing the amino acid sequences of the globular tips from different organisms suggest the unusual bond may have arose early in animal evolution, after the sponge and jellyfish lineages diverged.

Regardless of the bond’s evolutionary past, disrupting it could provide a means for attacking tumors, Kalluri says. In some cases, nearly half of a tumor’s weight comes from the collagen-basement membrane matrix, which provides structural rigidity. Because basement membranes surround all blood vessels, the membranes can act like train tracks that allow tumor cells to spread, he says. Perhaps collapsing those blood vessels by breaking the bonds could halt tumor growth.

The work may also inform research on Alport syndrome, an inherited condition related to collagen IV that involves loss of kidney function, and Goodpasture syndrome, a rare autoimmune disease. Goodpasture syndrome, which affects the lungs and kidneys and can be fatal, is caused in part by the immune system attacking a section of the amino acid tips the newly discovered bond connects. “This bond is uniquely involved,” Hudson says. How, precisely, remains unknown, but the new work is a step forward.

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