Abstract
Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.
Highlights
From the Departments of Pediatrics, Cell Biology, and Physiology, Washington Uniuersity Schoolof Medicine, St
Rearranged al-AT molecule that possesses chemo- Noninhibitory homologues, such as corticosteroid-binding attractant activities, mediates an increase in synthesis globulin andthyroid hormone-binding globulin, bindtheir of al-AT by mononuclear phagocytes and hepatocytes, hormone ligands by a serpin-like mechanism [3], whereas and is more rapidly cleared from the circulation than ovalbumin and angiotensinogen may serve as substrates for is the native al-ATmolecule
In the current study,we show that a penta- Since the major physiological target of al-ATis neutrophil peptide domain in the carboxyl terminal fragment of elastase, an enzyme capable of degrading many connective al-AT is sufficient for tissue matrix constituents, this serpin is thought to function binding to theSEC receptor
Summary
Evidence That Pentapeptide370374 of al-ATIs Recognized by the SEC Receptor-Our previous studies have localized the receptor-binding domain of a,-AT-elastase complexes to amino acids 359-374 of al-AT [13]. We examined the effect of specific amino acid substitutions on competitive binding of the pentapeptide105C (Fig. IC).In this series of experiments, we first showed that there was no significant difference in the competitive binding of peptide FVFLM, which is identical with al-AT sequence 370-374, and peptide 105C (FVYLI). We showed that a Phe to Ala substitution at residue 370 only minimally affects the competitive binding efficacy of pentapeptide FVFLM, but a Metto Ala substitution at residue 374 completely abrogates binding Taken together, these data indicate that binding of al-AT pentapeptide FVFLM to HepG2 cells is sequencespecific in that it is altered by substitutions, deletions, and scrambling of the sequence. The results indicate that pentapeptide FVFLM inhibits binding of al-AT-1251-trypsincomplexes to HepG2 cells as effectively as unlabeled a,-AT-trypsin complexes, peptide 105Y, and peptide 105C. Higher degrees of inhibition were achieved by higher concentrations of competitors in other experiments
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