Shark Protein Structure Solved

By Jason Socrates Bardi

A team of scientists at The Scripps Research Institute and at the University of Maryland School of Medicine has determined the structure of an antibody isolated from Ginglymostoma cirratum—a nurse shark.

"This represents the first structure to be determined of an antibody from a non-mammalian species in complex with its antigen," says Professor Ian Wilson, who is an investigator in the Skaggs Institute for Chemical Biology at Scripps Research.

Sharks are the most primitive animals known to have antibodies, and the antibody structure provides insight into how their immune system works. The shark antibody binding region is smaller than human antibody binding regions, and its structure suggests that the adaptive immune system of sharks operates slightly differently than those of humans and other mammals.

Furthermore, the shark antibody may provide a useful template for antibodies engineered for human therapeutic uses.

The structure, which is being published in the journal Science, was determined by Assistant Professor Robyn Stanfield and Wilson at Scripps Research Institute, in collaboration with Research Fellow Helen Dooley and Professor Martin F. Flajnik at the University of Maryland School of Medicine.

The Adaptive Immune System

Also called immunoglobulins, antibodies are proteins produced by B cells of the adaptive immune system, and they are designed by nature to recognize a wide range of foreign pathogens. After a bacterium, virus, or other pathogen enters the bloodstream, antibodies target antigens—proteins, carbohydrate molecules, and other pieces of the pathogen—specific to that foreign invader. These antibodies then alert the immune system to the presence of the invaders and attract lethal "effector" immune cells to the site of infection.

The scientists determined the structure of the shark antibody using the technique in structural biology known as x-ray crystallography, which involves making crystals of ordered arrays of protein and then blasting the frozen crystals with x-ray radiation. The atoms in the protein crystals cause the x-rays to diffract, and the scientists collect and analyze the pattern of diffraction to solve the atomic-level structure of the proteins.

Such structures often reveal details about proteins that cannot otherwise be predicted. In this case, the structure of a shark antibody reveals details of how sharks develop their antibody repertoire.

The antibody repertoire is one of the most interesting aspects of the immune systems of vertebrate animals. It is an essential factor enabling these immune systems to recognize and combat a great number of foreign pathogens.

In humans, this diverse repertoire is generated during B cell development in the bone marrow. There, developing B cells rearrange their antibody genes into one of more than a million possible combinations, and every mature B cell produces its own single antibody of unique specificity. The sum total of these antibodies comprises the repertoire.

The key to the difference in specificity lies in what are termed "complementarity determining regions" on the antibodies. These are highly variable loops at the ends of the conserved antibody framework that recognize the foreign antigen. Human antibodies have six complementarity determining regions on one single tip.

But this particular shark antibody has fewer of these complementarity determining regions—only two per tip. Nevertheless, sharks are able to generate a diversity of antibodies with only these two because one of their complementarity determining regions is very long and may adopt widely different conformations, thus changing the shape and characteristics of the binding site on the antibody.

This raises interesting evolutionary questions. This particular shark antibody seems to be able to generate adequate diversity with only two complementarity determining regions, so why did sharks (and all other vertebrates, including humans) also evolve antibodies that have six complementarity determining regions?

"We don't know the answer to that one," says Flajnik. "One possibility is that sharks have both types of antibodies because they are complementary to each other."

This minimal binding domain also has important implications for the design of therapeutic antibodies. Designer antibodies, some of which have been approved by the U.S. Food and Drug Administration, are promising therapeutics for a number of human diseases ranging from rheumatoid arthritis to leukemia because they target particular cells and attract other parts of the immune system to the site.

This work is a classic example of scientific collaboration. Flajnik and his group at the University of Maryland School of Medicine have been studying the immune system of this type of shark for a number of years, and Wilson and his group at Scripps Research have a long track record of solving the structures of antibodies.

The article, " Crystal structure of a shark single domain antibody V region in complex with lysozyme" by Robyn L. Stanfield, Helen Dooley, Martin F. Flajnik, and Ian A. Wilson will appear on Science Express the early online edition of Science on August 19, 2004. Subscribers can access the magazine online at: http://www.sciencemag.org.

This work was supported by the National Institutes of Health and the Skaggs Institute for Research.

Send comments to: jasonb@scripps.edu

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Scripps Research investigators Ian Wilson and Robyn Stanfield collaborated with researchers at the University of Maryland Medical School. Photo by Kevin Fung.