Elastin Degradation by Matrix Metalloproteinases

Abstract

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P 1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.Elastin is the extracellular matrix protein that imparts elastic recoil to tissues. Its cross-linked nature and extreme hydrophobicity make it one of the most stable proteins in the body (1-3). A contributing factor to elastin's longevity is its relative resistance to proteolysis by all but a limited number of proteinases that are capable of degrading the mature, insoluble protein under physiological conditions. These enzymes, referred to as elastases, have a wide distribution in nature and are found in animals as well as in plants and bacteria (4, 5). Elastases are heterogeneous with differing substrate specificities and catalytic mechanisms. In fact, enzymes with elastolytic activity can be found in most of the major proteolytic families, including serine, thiol, aspartic, and metallo enzymes (4). Despite the differences in catalytic mechanisms, all of these elastases share a common specificity for cleaving peptide bonds associated with hydrophobic or aromatic amino acids.The most widely studied elastases, human neutrophil elastase (HLE) 1 and pancreatic elastase (PPE), belong to the serine proteinase family of enzymes. Neutrophil elastase is found in the azurophil granules of polymorphonuclear leukocytes and is essential for phagocytosis and defense against infection. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestine wh...

The elastic fibre network of the human lumbar anulus fibrosus: architecture, mechanical function and potential role in the progression of intervertebral disc degeneration

“…Elastin is readily degraded by the serine proteases pancreatic and neutrophil (or leukocyte) elastase, as well as several matrix metalloproteinases including MMP-2, MMP-7 (matrilysin), MMP-9 and MMP-12 (metalloelastase) [24,29,39]. In the disc, only limited descriptions of the presence and function of these enzymes currently exist.…”

Methods in elastic tissue biology: Elastin isolation and purification

“…[14,24]-Hot alkali extraction results in a protein product that has an amino acid composition that best matches the expected values for elastin. However, due to the harsh conditions, hot alkali-purified elastin shows evidence of degradation [22,25], which must be taken into account when designing experiments with the purified product. It is important to note that incubation at high temperature for longer than 50 minutes will result in extensive peptide bond cleavage.…”

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410

“…The second class was thought to be located in the alanine-rich hydrophobic cross-linked areas of elastin and to lead to catalysis (Heather et al, 1999). Based on the studies reported, the logistic kinetics model was generally adopted to describe the proteolysis process, especially for macromolecular substrates (Mecham et al, 1997). Fig.5 and Table 4 show the experimental results of the kinetics model analysis, respectively.…”

“…These proteinases require Zn atoms for activity and in contrast to the serine proteinases cleave peptide bonds on the amino-terminal side of the amino acid that determines specificity. Elastase from Bacillus thermoproteolyticus and Streptomyces fradiae are four and eight times more active than PPE (pig pancreatic elastase), respectively, making them some of the most potent elastolytic proteinases reported (Mecham et al, 1997).…”

“…Elastin displays extreme hydrophobicity and extensive cross-linking. Because of these properties, it is quite stable and persistent in tissues unless subjected to the catalytic activity of one or more of a group of proteinases, collectively termed elastases because of their ability to solubilize amorphous elastin (Mecham et al, 1997).…”