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G Salvesen

Publications and source records attributed to G Salvesen.

At least 37 records · Page 2Linked to original sources
PubMed1991

Stepwise activation mechanisms of the precursors of matrix metalloproteinases 1 (tissue collagenase) and 3 (stromelysin).

Two zymogens of matrix metalloproteinases (MMPs), proMMP-1 (tissue procollagenase) and proMMP-3 (prostromelysin) were isolated from the culture medium of human rheumatoid synovial fibroplasts and their activation mechanisms by proteinases and 4-aminophenylmercuric acetate (APMA) were studied by kinetic and sequence analyses. Both zymogens were activated by unique stepwise activation mechanisms through which sequential processing events occur in the propeptide regions. The initial cleavage sites attacked by activator proteinases are located in the middle of the propeptides at Glu33-Lys-Arg-Arg-Asn37 in proMMP-1 and Phe34-Val-Arg-Arg-Lys-Asp39 in proMMP-3. The initial products of proMMP-1 generated by proteinases then undergo further autocleavage of the Thr64-Leu65 bond. The treatment of proMMP-1 and proMMP-3 with APMA results in the intramolecular cleavage of the Val67-Met68 and Glu68-Val69 bonds, respectively. The removal of a portion of propeptides results in conformational changes around the Gln80-Phe81 and His82-Phe83 bonds in respective intermediates of MMP-1 and MMP-3 and render them to rapid specific cleavage by MMP-3 to generate stable, fully active enzymes.

Amino Acid Sequence↗
PubMed1991

Zymogen activation specificity and genomic structures of human neutrophil elastase and cathepsin G reveal a new branch of the chymotrypsinogen superfamily of serine proteinases.

On the basis of amino acid sequences inferred from the genes encoding human neutrophil elastase and cathepsin G, it is likely that both are synthesized as precursors containing N- and C-terminal peptide extensions. We show that these extensions are removed about 90 min after onset of synthesis of these proteins in the U937 cell line. Removal of these extensions causes activation of the proteinases, and it is likely that the N-terminal extension of each enzyme serves as a zymogen activation peptide. Elastase and cathepsin G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Zymogen activation results from cleavage following a glutamic acid residue, a specificity opposite to most other serine proteinase zymogens. The specificity is likely to be shared, however, by neutrophil proteinase 3, rat mast cell proteinase II, and most members of the granzyme group of proteinases present in cytotoxic T-lymphocyte granules. The conservation in zymogen activation specificity between these leukocyte proteinase homologs is mirrored by the preservation of a discrete genomic organization. This suggests that most of the leukocyte serine proteinases evolved from a common ancestor distinct from the main branches of the chymotrypsinogen superfamily of serine proteinases.

Amino Acid Sequence↗
PubMed1990

Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties.

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.

Amino Acid Sequence↗
PubMed1990

Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin).

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.

Amino Acid Sequence↗
PubMed1990

Alpha-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system.

Significant primary sequence homology between the alpha-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus alpha-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus alpha-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus alpha-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general "trapping" mechanism shared by most alpha-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus alpha-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus alpha-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the alpha-macroglobulin family. Because both properties are contained in one molecule, we propose the name "limac" to describe this Limulus alpha-macroglobulin complement-like protein.

Amino Acid Sequence↗
PubMed1990

Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate.

The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl)mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37 degrees C resulted in the formation of MMP-3 of Mr = 45,000 by cleaving of the His82-Phe83 bond. Since this bond is unlikely to be cleaved by these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of Mr = 45,000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of Mr = 53,000 and two minor forms of Mr = 49,000 and 47,000. The 53,000 Mr intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45,000 Mr form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of Mr = 46,000 generated by APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of Mr = 45,000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to activate proMMP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

An unusual specificity in the activation of neutrophil serine proteinase zymogens.

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.

Amino Acid Sequence↗
PubMed1990

Evaluation of the rapid plasma elimination of recombinant alpha 1-proteinase inhibitor: synthesis of polyethylene glycol conjugates with improved therapeutic potential.

Plasma-derived alpha 1-proteinase inhibitor (alpha 1PlI) is used for replacement therapy in patients with emphysema who have a deficiency of the protein. Future therapy with alpha 1PI with reactive site mutants of this inhibitor will probably require the use of recombinant-derived alpha 1PI (r alpha 1PI). However, the pharmacologic efficacy of r alpha 1PI in humans has been hindered, since r alpha 1PI is rapidly cleared from the circulation of experimental animals. Our studies of the metabolism of r alpha 1PI in mice demonstrate that the rapid clearance of r alpha 1PI is due to renal filtration. However, after preventing renal filtration by ligation of the renal arteries, we find that r alpha 1PI is metabolized in a manner similar to alpha 1PI. Conjugation of r alpha 1PI to polyethylene glycol of Mr approximately 4000 (PEG-4) greatly slows the clearance of r alpha 1PI. The PEG-4 r alpha 1PI conjugate is metabolized in a manner similar to alpha 1PI and has similar kinetic properties before and after oxidation with N-chlorosuccinimide. Moreover, proteinase complexes of PEG-4-r alpha 1PI are catabolized by the same hepatocyte receptor that binds alpha 1PI-proteinase complexes. The results of these studies lay the groundwork for the synthesis of pharmacologically effective r alpha 1PI derivatives with acceptable plasma retention times.

Animals↗
PubMed1989

Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma. Polypeptide chain stoichiometry and assembly by glycan.

The polypeptide chain composition of protein material referred to in the literature as "inter-alpha-trypsin inhibitor" was investigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but susceptible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein migrated with inter-alpha mobility on agarose gel electrophoresis and is designated inter-alpha-trypsin inhibitor, whereas the 125,000-Da protein migrated with pre-alpha mobility, and we designate it pre-alpha-trypsin inhibitor. Analysis of the proteins, the separated chains, and proteolytic derivatives thereof revealed that each protein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-alpha-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify the confusion surrounding the composition of plasma proteins referred to as inter-alpha-trypsin inhibitor.

Alpha-Globulins↗
PubMed1989

Genomic organization and chromosomal localization of the human cathepsin G gene.

Cathepsin G is a 26,000-Da serine protease that is found in the azurophil granules of neutrophils and monocytes. The cathepsin G gene is expressed at high levels in U937 promonocytic cells, but is down-regulated with phorbol-induced differentiation. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic fibroblast library using cathepsin G cDNA, and obtained two lambda clones that contained the cathepsin G locus. The cathepsin G gene spans 2.7 kilobase pairs of genomic DNA and consists of 5 exons and 4 introns. The genomic organization of cathepsin G is similar to that of human neutrophil elastase, rat mast cell protease II, murine adipsin, and murine cytotoxic T-cell serine proteases, with protease catalytic residues located near the borders of exons 2, 3, and 5. Using in situ hybridization techniques, we localized cathepsin G to chromosome 14q11.2, a site that is near the alpha/delta T-cell receptor complex. Cathepsin G transcription is abolished in U937 nuclei with 2 micrograms/ml alpha-amanitin, indicating that this gene is probably transcribed by RNA polymerase II. The 5' end of the cathepsin G gene was defined by primer extension and S1 nuclease protection assays. A TATA box is found at position -29, and a CAAT box is found at -69 with respect to the transcription initiation site. Having defined the genomic structure and chromosomal location of cathepsin G, we are now attempting to identify the DNA elements in or near this gene that mediate its tissue and development-specific pattern of expression.

Amino Acid Sequence↗
PubMed1989

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3.

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.

Acute-Phase Proteins↗
PubMed1989

Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites.

The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.

Amino Acid Sequence↗
PubMed1989

The human neutrophil elastase gene. Analysis of the nucleotide sequence reveals three distinct classes of repetitive DNA.

DNA sequence analysis reveals the gene encoding human neutrophil elastase to be contained on a 6-kb EcoRI fragment. The gene contains five exons and closely resembles rat mast cell proteinase II and mouse adipsin in its exon structure and intron splice phase. Non-coding regions are very rich in repetitive DNA, containing seven Alu-like segments, three distinct clustered direct repeats with monomer lengths of 53 (six repeats), 23 (three repeats) and 41 (ten repeats) nucleotides, and a 200-nucleotide AT-rich region. Protein sequence analysis, inferred from the coding regions of the gene, indicates that neutrophil elastase may contain an unusual activation peptide similar to that found in the other major neutrophil serine proteinase, cathepsin G.

DNA↗
PubMed1988

In vivo catabolism of alpha 1-antichymotrypsin is mediated by the Serpin receptor which binds alpha 1-proteinase inhibitor, antithrombin III and heparin cofactor II.

The in vivo catabolism of 125I-labeled alpha 1-antichymotrypsin was studied in our previously described mouse model. Native alpha 1-antichymotrypsin cleared with an apparent t1/2 of 85 min, but alpha 1-antichymotrypsin in complex with chymotrypsin or cathepsin G cleared with a t1/2 of 12 min. Clearance of the complex was blocked by a large molar excess of unlabeled complexes of proteinases with either alpha 1-antichymotrypsin or alpha 1-proteinase inhibitor. These studies indicate that the clearance of alpha 1-antichymotrypsin-proteinase complexes utilizes the same pathway as complexes with the homologous inhibitor alpha 1-proteinase inhibitor. Previous studies have demonstrated that this pathway is also responsible for the catabolism of two other serine proteinase inhibitors, antithrombin III and heparin cofactor II. This pathway is thus responsible for removing several proteinases involved in coagulation and inflammation from the circulation, thereby decreasing the likelihood of adventitious proteolysis.

Antithrombin III↗
PubMed1988

Neutrophil elastase and cathepsin G: structure, function, and biological control.

When neutrophils invade inflamed areas of the body to remove either dead or foreign components they inadvertently release potent enzymes which can, if not properly controlled, cause severe damage to healthy tissue. This can lead to a myriad of diseases including emphysema, rheumatoid arthritis, and glomuerlopnephritis, all of which are really problems of abnormal connective tissue turnover due to uncontrolled protelysis by neutrophil elastase and cathepsin G. An important step in elucidating the functions of both elastase and cathepsin G has been made by virtue of the fact that the amino acid sequence of each has been determined. Furthermore, the crystal structure of one, neutrophil elastase, is now understood. With this knowledge in mind and with the potential for a similar understanding of the mechanism of action of cathepsin G, it should soon be possible to produce synthetic inhibitors of each enzyme which can act as adjunct inhibitors to those naturally circulating in the blood or present in other tissues. As a result there is great hope for reducing the severity of injury produced by these enzymes and, therefore, in decreasing the risk for development of the debilitating diseases associated with abnormal proteolysis by neutrophil proteinases.

Amino Acid Sequence↗