Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases released from neutrophils.

Abstract

Human neutrophils, when stimulated with phorbol myristate acetate or fMet-Leu-Phe in the presence or absence of cytochalasin B, released metalloproteinases that catalytically inactivated the plasma serine proteinase inhibitor, a1-antitrypsin. Inactivation, measured as loss of elastase inhibitory capacity, was accompanied by cleavage of a M, 4,000 peptide from the COOH-terminus. Cleavage of a1-antitrypsin by cell supernatants was inhibited by EDTA, o-phenanthroline, and DTT, but not by inhibitors of serine or thiol proteinases. Gelatinase and collagenase were separated from the medium of stimulated neutrophils. Both preparations cleaved and inactivated a,-antitrypsin, with cleavage occurring close to the reactive center, at the Phe-Leu bond between positions P7 and P6. Cleavage by purified gelatinase was very slow and could account for only a minor fraction of the activity of neutrophil supernatants. The collagenase preparation was more active. However, the unusual cleavage site, and the ability of fMet-Leu-Phe-stimulated neutrophils to cleave a1-antitrypsin without releasing collagenase, suggests that collagenase is not responsible for cleavage by the cells, which, by implication, is due to an as yet uncharacterized metalloenzyme. Our results demonstrate that by releasing metalloproteinases, neutrophils could proteolytically inactivate a,-antitrypsin at sites of inflammation. This provides an alternative to the previously documented mechanism of inactivation by neutrophil-derived oxidants.

Inactivation of human plasma α1‐proteinase inhibitor by human PMN leucocyte collagenase

“…For this reason the investigation of oxidative [3,4] or proteolytic [5][6][7][8][9][10][11][12][13] inactivation of oq-PI by bacterial or human proteinases becomes an important feature in the regulation of elastic fiber turnover of the lung.…”

“…The discovery that a metalloproteinase secreted from PMN leucocytes inactivates crt-PI [12,13] led to a new understanding of the pathogenesis of pulmonary emphysema. We now conclude that the proteolytic inactivation of oq-PI is due to PMN leucocyte collagenase.…”

“…It was shown during the last two decades that O~l-PI is susceptible to proteolytic inactivation by different proteolytic enzymes of bacterial [5,6] and human origin [9,[11][12][13] (for details: see Table I). The interesting fact that secreted PMN leucocyte collagenase can catalytically cleave and inactivate Cel-PI in the Table I Cleavage sites of the proteolytic inactivation of human Otl-Pl by different proteolytic enzymes Residue no.…”

“…Cleavage site I: Cathepsin L [9], Serratia marcescens metalloproteinase [6] Cleavage sitell: Pseudomonas aeruginosa [7], macrophage elastase [11], PMN collagenase (secondary product) (this paper) Cleavage site II1: Cathepsin L [9], Staphylococcus aureus cysteine and serine proteinase [5] Cleavage site IV: Staphylococcus aureus metalloproteinase [5], secreted PMN metalloproteinase [12,13], PMN collagenase (primary product) (this paper) Cleavage site V: Crotalus adamanteus proteinase II [8] proteinase-sensitive region may contribute to a persisting activity of PMN leucocyte elastase by lowering the level of intact cet-PI. Although the physiological significance of c~l-PI inactivation by PMN leucocyte collagenase has yet to be determined, it could possibly play an important role in the extracellular turnover of cel-PI, since the enzyme is easily secreted upon specific stimuli [23,24] and is optimally active at physiologic pH, It has recently been reported that proteolytically inactivated cel-PI has chemotactic properties for polymorphonuclear leucocytes [25] and may thus enhance the invasion of cells.…”

Compromised inhibition of human lung lavage cell elastases

“…In contrast, neutrophil elastase, which tas no methionine at its active site, is relatively less affected I-,y oxidants [15]. Since pro-matrix metalloproteinases can be activated both by serine proteinases such as neutrophil elastase and by cell-derived oxidants [16], such metalloproteinases could inactivate a-l-proteinase inhibitor [17] and enable continued neutrophil elastase activity. Thus, overall, elastinolysis would be amplified.…”