Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors

Abstract

Hyaluronic acid (HA) is the major biopolymer of the extracellular matrix and contributes significantly to cell proliferation and migration. Human hyaluronidase hPH-20 has been identified as a tumor marker for breast and laryngeal cancer. A hPH-20-autotransporter fusion protein for cell surface display was transformed into Escherichia coli BL21 (DE3) and hPH-20 was displayed on the surface of E. coli. Enzymatic activity, however, was not detectable due to competitive inhibition by lipopolysaccharide (LPS). Finally, expression in E. coli F470, a strain missing the O-polysaccharide of LPS, yielded cells with sufficient hyaluronidase activity. 6-Palmitoyl- l-ascorbic acid (Vcpal) and two indole-carboxamides, N-(4-fluorobenzyl)-1-benzyl-1H-indole-2-carboxamide ( 1) and N-(4-chlorobenzyl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide ( 2), were tested on inhibition of hPH-20. Vcpal with a concentration of 5 μM inhibited hPH-20 to 93% at pH 7, compounds 1 and 2 showed 61% and 21% inhibition at a concentration of 50 μM. At the same inhibitor concentrations the most frequently used bovine testes hyaluronidase (BTH) was inhibited by Vcpal to a similar extent (95%), whereas compound 1 (80%) and compound 2 (66%) showed much differing inhibition. Thus it can be assumed that BTH is not applicable as an alternative to human PH-20. These results indicate that Autodisplay enables the expression of human target enzymes normally forming inclusion bodies in E. coli and accelerates inhibitor testing as shown by the example of human hyaluronidase PH-20.