Vanderbilt University
Vanderbilt Institute of Chemical Biology

home research discoveries core facilities training & research opportunities seminars & events news giving contact


Discoveries Featured

Biochemical Collaboration Leads to Novel Biologically Active Lipids

By: Carol A. Rouzer, VICB Communications
Published: April 25, 2011

VICB investigator Claus Schneider and his laboratory report the structures of novel lipid signaling molecules produced through a cooperation between the cyclooxygenase and lipoxygenase pathways.

Arachidonic acid (AA) is a 20-carbon long polyunsaturated fatty acid containing four double bonds between carbons 5 and 6, 8 and 9, 11 and 12, and 14 and 15 (Figure 1).  AA is a normal component of cellular membrane phospholipids, but it also plays a unique role in vertebrate lipid metabolism. Under conditions of cell stimulation, activation of certain phospholipase enzymes  leads to release of AA from phospholipid stores, subjecting it to metabolism via a number of specialized oxygenase enzymes.  Among these are the cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) enzymes that play important roles in a wide variety of cellular functions, including inflammation and immunity, cardiovascular and renal homeostasis, and reproduction.


Figure 1. Arachidonic acid metabolism via the COX-2 and 5-LOX pathways.  COX-2 converts AA to PGH2, which is then enzymatically converted or spontaneously decomposes to PGE2 and PGD2.  5-LOX converts AA to 5S-HETE, which is then converted by COX-2 to a di-endoperoxide intermediate similar to PGH2.  The di-endoperoxide can be converted enzymatically to HKD2, or it nonenzymatically decomposes to a mixture of HKD2 and HKE2.

The COX enzymes catalyze a fascinating and complex set of reactions that lead to the addition of two molecules of oxygen (O2) to AA.  One molecule of oxygen forms an endoperoxide bridge between carbons-9 and -11 (O-O, red, Figure 1). The reaction also links carbons-8 and -12 with a single bond (dark blue, Figure 1), forming a five-membered ring.  The other molecule of O2, added at carbon-15, is rapidly reduced, leaving a hydroxyl group (OH, light blue, Figure 1) at this position.  The resulting product is called prostaglandin H2 (PGH2).  PGH2 is subject to further metabolism by a series of additional enzymes, among them prostaglandin E synthase (PGES) and prostaglandin D synthase (PGDS), which produce PGE2 and PGD2, respectively.  However, the endoperoxide group of PGH2 is unstable, and in the absence of enzymes, it undergoes rapid decomposition to a mixture of PGE2 and PGD2 (Figure 1).  The reaction catalyzed by 5-LOX is much simpler, involving the addition of oxygen at carbon-5, which is readily reduced intracellularly to form a hydroxyl group (green, Figure 1).  The product of this reaction is 5S-hydroxyeicosatetraenoic acid (5S-HETE).

COX-1 is highly specific for AA as substrate, but COX-2 is somewhat promiscuous, accepting a wider variety of fatty acid structures.  This led VICB investigator Claus Schneider and his laboratory to discover that COX-2 is capable of using the 5-LOX product, 5S-HETE, as a substrate.  The reaction of COX-2 with 5S-HETE is similar to its reaction with AA in that O2 is added across carbons-9 and -11, yielding an endoperoxide and at carbon-15, yielding a hydroxyl group.  However, instead of forming a single bond between carbons-8 and -12 as it does with AA, an oxygen atom is inserted between these two atoms of 5S-HETE, forming a second endoperoxide structure (dark blue, Figure 1).  The ability of COX-2 to metabolize 5S-HETE suggests the possibility of cooperation between the COX-2 and 5-LOX pathways, leading to new products with potentially unique biological activities.  Now, the Schneider lab reports on the fate of the di-endoperoxide formed by the action of COX-2 on 5S-HETE [Griesser et al. (2011) Proc. Natl. Acad. Sci. U.S.A., published online April 11, DOI: 10.1073/pnas.1019473108].

As in the case of PGH2, the di-endoperoxide of 5S-HETE was unstable in aqueous solution.  It decomposed to yield a mixture of two novel compounds that the Schneider lab designated hemiketal E2 (HKE2) and hemiketal D2 (HKD2), due to their similarities to PGE2 and PGD2, respectively (Figure 1). In analogy to PGH2 metabolism, the Schneider lab also discovered that one isoform of PGDS could catalyze the conversion of the di-endoperoxide to HKD2, suggesting that formation of this compound might be favored under some circumstances in vivo.

Figure 2. Peripheral blood neutrophils surrounded by red blood cells.  Neutrophils express 5-LOX. Image courtesy of Wikimedia Commons under the GNU Free Documentation License.

Not satisfied with defining a biochemical pathway in a test tube, the Schneider lab asked whether the HKs could be produced in intact cells.  Because few cells contain both COX-2 and 5-LOX simultaneously, they used human peripheral blood leukocytes, which include 5-LOX-expressing neutrophils (Figure 2) and eosphinophils in addition to monocytes (Figure 3) which can be triggered to express COX-2 by treatment with bacterial lipopolysaccharide (LPS).  Following LPS treatment, the cells were stimulated with a calcium ionophore to release AA from cellular phospholipids and activate 5-LOX.  Indeed, HKE2 and HKD2 were identified among the AA metabolites produced by the cells, accounting for approximately 1 to 5% of the total 5-LOX products of the cells.

Figure 3. Peripheral blood monocyte surrounded by red blood cells.  Monocytes can be induced to express COX-2 by exposure to bacterial lipopolysaccharide.  Image courtesy of Wikimedia Commons and the European Bioinformatics Institute under the GNU Free Documentation License.

The ability of blood leukocytes to collaborate in HK formation led the investigators to question whether the compounds possess biological activity.  One function of the COX-2 and 5-LOX pathways is to modulate the inflammatory response, which includes vascular repair and remodeling.  Thus, the Schneider lab, in collaboration with the Ambra Pozzi lab (Division of Nephrology) explored the effects of the HKs on vascular endothelial cells (Figure 4).  They discovered that the HKs stimulated the process of tubulogenesis - the formation of vessel-like structures - in a dose-dependent fashion at low concentrations.  These results suggest that HKs produced by the cooperation of leukocytes may play a role in new blood vessel formation at the site of inflammation.


Figure 4. Electron micrograph of a blood vessel capillary showing a red blood cell in the lumen and two endothelial cells that form the thin capillary wall.  Image courtesy of Wikimedia Commons under the GNU Free Documentation License.









Follow us on Twitter

Vanderbilt University School of Medicine | Vanderbilt University Medical Center | Vanderbilt University | Eskind Biomedical Library

The Vanderbilt Institute of Chemical Biology 896 Preston Building, Nashville, TN 37232-6304 866.303 VICB (8422) fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2013 by Vanderbilt University Medical Center