Inhibition of Human Neutrophil Elastase by Polyguanylic Acid and Other Synthetic RNA Homopolymers

Abstract

Human neutrophils contain large amounts of a neutral serine protease, human neutrophil elastase (HNE), which has been implicated as a mediator of acute and chronic lung injury. We found that this enzyme is effectively inhibited, at physiological ionic strength, by several synthetic non-base-paired polyribonucleotides. Among the most active of these is polyguanylic acid (poly G). Inhibitory activity is greatest with high-molecular-weight poly G fractions, but poly G fractions even as low as 60K M_r (app) are effective. Both amidolysis of synthetic elastase substrates, such as succinyl-ala-ala-ala-p-nitroanilide, and proteolysis of elastin are blocked. Poly G inhibits elastin proteolysis even when subsequently added to mixtures of elastin and HNE that have first been preincubated together for 10 min. Under these conditions, polyribosylribitol phosphate, a polyanion derived from Haemophilus influenzae capsular polysaccharide, is not inhibitory. Complex formation between HNE and poly G is dependent on ionic rather than covalent interactions, since it is blocked by 0.6 M NaCl but not by inactivation of the enzyme's catalytic-site serine residue with diisopropylfluorophosphate. However, nonspecific ionic interactions alone cannot explain complex formation, since pancreatic elastase and cathepsin G, an even more basic serine protease from human neutrophils, do not form complexes with poly G, even at low ionic strength. Moreover, in the presence of the amphiphiles taurocholic acid and glycocholic acid, HNE is much less effectively blocked by poly G. Peptide chloromethyl ketone-inactivated HNE (which has its extended substrate-binding pocket occupied by the peptidyl inactivator) also fails to form complexes with poly G. These results indicate that HNE may utilize both hydrophobic and ionic binding sites to couple with poly G, and suggest that these sites may be close to or within the extended substrate-binding pocket of the enzyme.