Daniel J. Knauer

Ph.D., University of Nebraska, 1979
Human antithrombins and related serine protease inhibitors
Email address: djknauer@uci.edu

The research in my laboratory focuses on the structure and function of the super-gene family of serine protease inhibitors (SERPINS). Members of this gene family regulate several biological processes including cell division and migration, neurite extension, tumor cell metastasis, and blood coagulation. The SERPINS act as specific inhibitors of cell-surface and extracellular matrix serine proteases that participate in cascade mechanisms that participate in these biological processes. A unique feature of the SERPINS is the up-regulation of their anti-protease activity by specific glycosaminoglycans found in the extracellular matrix and in soluble forms. This activation, which is in the order of 10,000-fold, is mediated by a conformational change in the SERPIN induced by the binding of the glycosaminoglycan to a specific domain within the protein. The ultimate goal of my research is to understand the mechanism of this activation at the protein level.

Biochemical and immunological analyses have identified a highly conserved region within several of the SERPINS that apparently mediates glycosaminoglycan binding. This region consists of an alpha-helical domain with a positively charged face consisting mainly of lysine residues, which acts as an anchor for an extended ribbon that projects toward the reactive center of the molecule. We are presently expressing the genes for two different SERPINS, antithrombin III and protease nexin-l, in the Baculovirus-insect cell system. This system has allowed us to produce milligram quantities of these SERPINS that have biochemical and biological properties identical to their native counterparts. The goal of these studies is to pinpoint the charged residues within this region through site-directed mutagenesis studies utilizing single amino acid substitutions. Elucidation of the specific charged residues involved is a fundamental step in our understanding of the structure and function of this important class of regulatory molecules.

Selected Publications

Smith, J.W. and D.J. Knauer. 1987. A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence. J. Biol. Chem. 262:11964.

Gronke, R.S., D.J. Knauer, S. Veeraraghavan and J.B. Baker. 1989. A form of protease nexin I is expressed on the platelet surface during platelet activation. Blood 73:472.

Smith, J.W., N. Dey and D.J. Knauer. 1990. The heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody. Biochemistry, 29:8950.