Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.     

Determination of proteolytic enzymes by flow-injection analysis

Quantitation of proteolytic enzymes using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide has been adapted to flow-injection analysis. This procedure has been developed using two different proteases: subtilisin and chymotrypsin. For both enzymes the influence of substrate concentration on spectrophotometric response has been studied. The assay is based on the merging zones technique combined with a washing step. Results are obtained in less than 15 s and samples may be run at a rate of 90/h with good reproducibility. A linear relation between peak heights and enzyme concentrations was observed for 0-0.15 Anson unit/liter of subtilisin and for 0-30 mg/liter of a commercial preparation of chymotrypsin. The method requires only small sample volumes, and the consumption of the chromogenic substrate is reduced to a minimum by using intermittent pumping.     

Inactivation of intestinal brush-border enzymes by gossypol

Gossypol, a pigment found in cottonseed that has recently been shown to have antifertility properties, inhibited the activity of 3 intestinal brush border enzymes in a concentration-dependent manner. Suspensions of rat intestinal mucosa were incubated with various concentrations of gossypol for 45 minutes and then washed. At a concentration of 6 mg per gm mucosa, gossypol inhibited the activities of alkaline phosphatase, maltase, and sucrase by 57, 73, and 77%, respectively. Gossypol is a bifunctional agent, capable of cross-linking amino acid side chains, and its action on brush-border enzymes may be due to this mechanism. Recent investigations have demonstrated that rats fed a diet of 10-15 mg of gossypol/day/kg of body weight exhibit reduced fertility. This study suggests that a partial inhibition of brush-border enzymes may occur at doses used to cause infertility. Such a side effect should be considered in studies and treatments utilizing a gossypol diet.     

Secretion of proteolytic enzymes by three phytopathogenic microorganisms

Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts.     

Conformational homogeneity in molecular recognition by proteolytic enzymes

Crystal structures for several hundred protease-inhibitor complexes have been analysed and their superimpositions have been used to demonstrate a universal relationship between inhibitor/substrate conformation and molecular recognition by all aspartic, serine, cysteine and metallo proteases. Proteases universally recognize an extended beta strand conformation in all their peptidic (and non-peptidic) inhibitors and substrate analogues without significant exceptions. This conformational homogeneity is illustrated here for a subset of 180 protease-inhibitor structures which are displayed as (a) structural overlays of multiple inhibitors for each of eight aspartic, eight serine, six metallo and five cysteine proteases; (b) single inhibitors each bound to different proteases; and (c) Ramachandran plots of peptide or pseudo-peptide dihedral angle pairs which demonstrate beta strands (Phi -54 degrees to -173 degrees, Psi 24 degrees to 174 degrees ) like those normally found paired in proteins as beta sheets. However, unlike beta sheets, alpha and 3(10) helices, beta and gamma turns, where the folded main chain amide components are intramolecularly hydrogen bonded and thus unavailable for interaction with proteins, an inhibitor/substrate in an isolated beta strand conformation provides maximum exposure of its hydrogen bonding donors/acceptors and side chain components to a putative protease receptor. This analysis highlights the advantages of a strand conformation over other elements of secondary structure for protease recognition and may lead to generic strategies for inhibitor design.     

The versatility of proteolytic enzymes

The growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes. Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis. Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases. The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.