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NIEHS Investigator Advances Understanding of Heparin Biosynthesis

By Brian Chorley
December 2008

Lars Pedersen
Along with its own research, Pedersen's group develops three-dimensional structural characterization using molecular biology, biochemistry and X-ray crystallographic techniques for other NIEHS researchers. (Photo courtesy of Steve McCaw)

NIEHS Structure and Function Research Group Leader Lars Pedersen, Ph.D, and colleagues at the University of North Carolina (UNC) Chapel Hill have succeeded in creating a group of recombinant enzymes that synthesize novel varieties of heparan sulfate with unique biological functions - offering potential for advancing future research and for developing new therapeutic applications. The findings ( Exit NIEHS of their research, which was funded in part by NIEHS, appeared online November 20 ahead of print on the Proceedings of the National Academy of Sciences (PNAS) web site.

Heparin is a naturally occurring molecule produced in many living organisms. The mechanisms governing heparin biosynthesis are highly conserved among species, lending credence to the idea that heparin and heparin-like molecules are critical for life.

In the medical world, heparin is primarily utilized as an anticoagulant. One of the oldest drugs still commonly used today, heparin is widely available, cheap to manufacture, and easy to store and administer.

In a natural context, heparin and heparin-like molecules participate in development, inflammatory response and normal physiology. In addition to heparin's use as an anticoagulant, many scientists believe that there is great therapeutic potential for heparin-like molecules. Current research has suggested its use for symptomatic treatment of asthma, cancer and inflammatory bowel syndrome, as well as other conditions.

Heparin, a highly sulfated form of heparan sulfate, is a linear polysaccharide. Its differential sulfation patterns determine both form and function. Much of today's commercially available heparin is a heterogeneous mixture of heparin moieties. The focus of current heparin research is to discover the specific therapeutic qualities, as well as deleterious side effects, of the various forms of the compound.

Synthesis is dependent on a number of specialized enzymes, including a group of sulfotransferases. In their report, Pedersen and colleagues dissected the enzymatic process of the heparan sulfate 2-O-sulfotransferase (2OST). 2OST is known to sulfate two specific moieties of the heparan sulfate chain. One variant is more common in nature than the other, and the biological role of the rare sulfated form is not well understood.

In the study, the researchers report finally solving the crystal structure of the catalytic portion of 2OST using a recently developed technique of structurally guided mutagenesis. "The structure of this enzyme was something we have been going after for a long time," Pedersen said.

By comparing and contrasting known structures of other sulfotransferases, the researchers identified regions important for enzymatic activity, complex formation and substrate specificity. This information aided the team in developing a host of 2OST mutant enzymes. The mutants possess a range of activity levels and substrate specificities. One mutant form, to the delight of Pedersen and the research team, demonstrated almost complete propensity to manufacture the rare sulfated unit on the heparan sulfate polysaccharide chain.

Pedersen explained that there are likely potential benefits in producing homogenous preparations of heparin, such as those manufactured by recombinant biosynthetic enzymes. For example, a known side effect of general heparin treatment is a condition called thrombocytopenia, or a dangerously low platelet count.

Research with homogenous preparations of heparin may be able to identify variants which have reduced side effects yet retain anticoagulant properties. Additionally, some variants that do not function as an anticoagulant may prove to be most effective for other therapeutic uses. "You don't necessarily want to give [heparin that functions as an] anticoagulant as cancer therapy," Pedersen summarized.

In addition to Pedersen, members of the UNC Eshelman School of Pharmacy's Division of Medicinal Chemistry and Natural Products participated in the research. The UNC team included principal investigator, Jian Liu, Ph.D, as well as co-authors Heather Bethea and Ding Xu. The research was funded by the American Heart Association, National Institutes of Health and the Division of Intramural Research at NIEHS.

Citation: Bethea HN, Xu D, Liu J, Pedersen LC. ( Exit NIEHS 2008. Redirecting the substrate specificity of heparan sulfate 2-O-sulfotransferase by structurally guided mutagenesis. Proc Natl Acad Sci U S A. Nov 20. [Epub ahead of print]

(Brian Chorley, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)

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