Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.
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PMID:Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. 340 61

Binding of intact plasma fibronectin and its proteolytic fragments to glycosaminoglycans immobilized on agarose beads was systematically compared at different ionic strengths. In low ionic strength buffer, intact fibronectin bound to heparin and high sulfated heparan sulfate, but not to low sulfated heparan sulfate, dermatan sulfate, chondroitin sulfates A and C, or hyaluronic acid. Fractionation of the thermolysin digest of fibronectin on the glycosaminoglycan-Sepharoses at low ionic strength revealed that three groups of fragments, i.e. Mr = 150,000-140,000, 24,000, and 16,000 (150K-140K, 24K, and 16K) fragments, were capable of binding to glycosaminoglycans with different specificities and affinities. The 150K-140K fragments exhibited the same specificities as intact fibronectin, binding only to heparin and high sulfated heparan sulfate. However, the 24K fragment bound not only to these two glycosaminoglycans but also to low sulfated heparan sulfate and other glycosaminoglycans as well. The 16K fragments were also capable of binding to most glycosaminoglycans with lower affinity than the 24K fragment. These results suggest that the binding sites in the 24K and 16K fragments are cryptic in the intact protein, but are exposed after limited proteolysis. The binding of fibronectin and its fragments to glycosaminoglycans is dependent on the ionic strength. At physiologic ionic strength, only heparin-Sepharose could bind intact fibronectin. Similarly, only heparin-Sepharose could bind the 150K-140K and 24K fragments, but not the 16K fragments, at the same ionic condition. Other glycosaminoglycan-Sepharoses did not retain significant amounts of any of the fibronectin fragments, suggesting that the affinity of plasma fibronectin and its fragments to heparan sulfate and other glycosaminoglycans, except heparin, is not strong enough to achieve stable mono- (or di-) valent binding under physiologic conditions.
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PMID:Binding of fibronectin and its proteolytic fragments to glycosaminoglycans. Exposure of cryptic glycosaminoglycan-binding domains upon limited proteolysis. 664 86

Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of approximately 4:1, anastellin:FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.
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PMID:Domain unfolding plays a role in superfibronectin formation. 1619 31