Specific processing of tenascin-C by the metalloprotease meprinbeta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinbeta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinbeta. In chicken tenascin-C, meprinbeta processed all three major splicing variants by removal of 10kDa N-terminal and 38kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15kDa) and two C-terminal fragments (40 and 55kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinbeta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinbeta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinbeta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinbeta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinbeta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607

Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of beta- and alpha(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of beta-casein being faster than that of alpha(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of beta- and alpha(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from alpha(s1)- and beta-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^

MASP-1 of the complement system promotes clotting via prothrombin activation.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

MASP-1 Induced Clotting--The First Model of Prothrombin Activation by MASP-1.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.

REGULATION OF CYTOSKELETAL ASSEMBLY: A STUDY OF THE INTERACTION OF FILAMIN AND F-ACTIN

Filamin is a high molecular weight (2 x 250,000) actin crosslinking protein found in a wide variety of cells and tissues. The most striking feature of filamin is its ability to crosslink F-actin filaments and cause ATP-independent gelation and contraction of F-actin solutions. The gelation of actin filaments by filamin involves binding to actin and crosslinking of the filaments by filamin self-association. In order to understand the role of filamin-actin interactions in the regulation of cytoskeletal assembly, two approaches were used. First, the structural relationship between self-association and actin-binding was examined using proteolytic fragments of filamin. Treatment of filamin with papain generated two major fragments, 90Kd and 180Kd. Upon incubation of the papain digest with F-actin and centrifugation at 100,000 x g, only the 180Kd fragment co-sedimented with F-actin. The binding of the 180Kd fragment, P180, was similar to native filamin in its sensitivity to ionic strength. Analytical gel filtration studies indicated that, unlike native filamin, P180 was monomeric and did not self-associate. Thermolysin treatment of P180 produced a 170Kd fragment, PT170, which no longer bound and co-sedimented with F-actin. These results suggested that filamin contained a discrete actin-binding domain. In order to locate the actin-binding domain, affinity purified antibodies to the papain and thermolysin sensitive regions of filamin were used in conjunction with filamin fragments generated by digestion with S. aureus V8 protease and elastase. The results indicated that the papain and thermolysin cleavage sites were close together, and, most likely, within 10Kd of one another. Taken together, these data suggest that filamin contains a discrete, internal actin-binding domain. The second approach was to use the non-crosslinking fragment P180 to develop a quantitative assay of filamin-actin binding. The binding of ('14)C-carboxyalkylated P180 was examined using the co-sedimentation assay. ('14)C-P180 binding to actin was equivalent to that of unlabelled P180 and exhibited comparable sensitivity of binding to changes in ionic strength. Within 5 min. of incubation the process had reached equilibrium. The specificity of binding was shown by the lack of binding of ('14)C-PT170. The binding of ('14)C-P180 was found to be a reversible and saturable process, with a K(,d) of 2 x 10('-7) M. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^

A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Construction of a Z-DNA-specific restriction endonuclease

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Zα) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Zα nuclease, which is a chimera of Zα and FokI nuclease. Purified Zα nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Zα nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Zα binds Z-DNA in an environment similar to that in a cell. Zα nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.

TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.