The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases

Human mucus proteinase inhibitor (MPI) consists of 107 amino acids arranged in two domains showing high homology to each other. This protein is an inhibitor of different serine proteinases including trypsin, chymotrypsin, leukocyte elastase and cathepsin G. On the basis of sequence comparisons it has been suggested that the first domain inhibits trypsin, whereas the second one was thought to be active against chymotrypsin and elastase. To prove the location of the different inhibitory activities gene fragments for both domains have been cloned separately and expressed in Escherichia coli. Inhibition assays with the isolated recombinant domains showed that the second domain is active against chymotrypsin, neutrophil elastase and trypsin, whereas for the first domain only a weak activity against trypsin could be detected. These results suggest that the inhibitory activities of the native molecule towards these three proteinases are all located in the second domain.     

Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II

Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu⁵ Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).     

Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum

Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.     

Strong ionic interactions between mucins and two basic proteins, mucus proteinase inhibitor and lysozyme, in human bronchial secretions.

1. Mucins were isolated from sputum from a patient with chronic bronchitis and subjected to two different preparation procedures. 2. In the first procedure, CsBr density-gradient centrifugation gave rise to two well-separated fractions. Mucins recovered in the high-density fraction still contained mucus proteinase inhibitor (MPI) and lysozyme (LSZ). 3. Mucins were purified after a second step of CsBr density-gradient centrifugation or after gel-filtration chromatography with a buffer of high ionic strength, containing 0.5 M NaCl. 4. In the second procedure, trichloroacetic acid treatment of whole sputum followed by cation-exchange chromatography allowed the obtention of a non-retained fraction composed of mucins. 5. Gel-filtration in buffer containing 0.5 M NaCl, allowed the release of MPI and LSZ from mucins, thus confirming that interactions still occurred between those components. 6. The chemical compositions of the mucins isolated by the two above procedures were quite similar. 7. These data support the hypothesis of the existence of ionic interactions between basic amino acid residues of MPI and LSZ and acid residues of the carbohydrate chains of mucins in the secretions of the large airways. 8. These interactions could play a role in the protection of mucins against proteolysis and consequently in the maintenance of rheological properties of the mucus gel in disease.     

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.     

Study of DFF45 in its role of chaperone and inhibitor: two independent inhibitory domains of DFF40 nuclease activity.

CAD/DFF40, the nuclease responsible for DNA fragmentation during apoptosis, exists as a heterodimeric complex with DFF45/ICAD. This study determines the molecular mechanisms of regulation of DFF40 via the chaperone and inhibition activities of DFF45. We analyze proteins corresponding to the fragments (D1, D2, and D3) of DFF45 generated by cleavage at the caspase consensus sites in DFF45. Either D1 or D2, as an isolated domain, is capable of inhibiting DFF40 nuclease activity while double domain fragments D1-2 and D2-3, as well as full-length DFF45, bind to DFF40 with high affinity and are much more effective inhibitors. The chaperone activity of DFF45 resides in part in its ability to maintain DFF40 as a soluble protein. In addition, D1 of DFF45 was found to be critical for the expression of active DFF40 in vivo, suggesting a role for DFF45 in binding nascent DFF40.     

Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.     

The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity

Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.     

NMR structure of the netrin-like domain (NTR) of human type I procollagen C-proteinase enhancer defines structural consensus of NTR domains and assesses potential proteinase inhibitory activity and ligand binding

Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.     

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.     

The carboxy-terminal domain of glycoprotein N of human cytomegalovirus is required for virion morphogenesis

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.     

Latent fibronectin-degrading serine proteinase activity in N-terminal heparin-binding domain of human plasma fibronectin

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.     

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.