Generation of a mature streptococcal cysteine proteinase is dependent on cell wall-anchored M1 protein

In the present study, we have generated a mutant strain of Streptococcus pyogenes, MC25, which lacks M protein on its surface, and we demonstrate that this strain is unable to generate a mature 28 kDa cysteine proteinase. Furthermore, we show that S. pyogenes bacteria of M1 serotype are dependent on cell wall-anchored M protein to cleave the secreted zymogen into a mature cysteine proteinase. We also show that MC25 secretes a 40 kDa zymogen, having a conformation different from that secreted by wild-type bacteria. We provide data showing that the cleavage site is not blocked but, presumably, the active site is. This suggests that M protein, when anchored to the cell wall, is involved in the unfolding of the zymogen and generation of a mature cysteine proteinase that can be activated under reducing conditions. Our data add new aspects to the interaction between two important virulence factors of S. pyogenes, the streptococcal cysteine proteinase and M protein.     

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein

Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion-exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1.     

Human hepatocytes metabolizing ethanol generate a non-polar chemotactic factor for human neutrophils

When human hepatocytes were incubated with low concentrations of ethanol they general chemotactic activity for human neutrophils. Generation of chemotactic activity was dependent upon duration of incubation and concentration of ethanol used. Production of chemotactic activity by ethanol-treated hepatocytes was inhibited completely in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole. PMN isolated from rats, in contrast, do not respond chemotactically to the factor released by homologous cells. Preliminary studies indicated that the chemotactic factor is non-polar in nature (perhaps related to leukotriene B4). These results indicate that human hepatocytes, when exposed to ethanol, generate chemotactic factor(s) for human PMN. The occurrence of this phenomenon may explain, in part, the PMN infiltrates observed in human liver during the course of acute alcoholic hepatitis.     

Lymphocytes treated with natural alfa-interferon produce a chemotactic factor for human neutrophils

Lymphocytes stimulated with alfa-interferon released a chemotactic factor for neutrophils. The process was not inhibited by cycloheximide, whereas mepacrine completely inhibited release of the chemotactic activity. The chemotactic factor was resistant to storage, heat treatment and proteolysis. Recombinant alfa-interferon did not stimulate lymphocytes to release a neutrophil chemotactic factor.     

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.     

Urotensin II is a new chemotactic factor for UT receptor-expressing monocytes

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-kappaB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.     

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.     

Demonstration of a receptor on rabbit neutrophils for chemotactic peptides

[³H]formylNorleu-Leu-Phe, a potent leucocyte chemoattractant, binds specifically to a site on rabbit neutrophils with an affinity of 1.5 × 10^?9 M. Other acylated peptide chemoattractants displace this binding at concentrations closely related to those levels required to elicit chemotaxis. The binding fulfills the major criteria for demonstration of specific receptor sites and indicates that a natural bacterial product and synthetic formyl-peptides produce chemotaxis by the same receptor mechanism.     

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes

Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.     

The macrophage-derived neutrophil chemotactic factor, MNCF: a lectin with TNF-alpha-like activities on neutrophils

The macrophage-derived neutrophil chemotactic factor (MNCF) is an α-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-α the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-α effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.     

Glutathione activation of a cysteine proteinase from Schistosoma mansoni

A cysteine proteinase isolated from Schistosoma mansoni adults requires reduction by thiols for activation. The proteinase is located in the parasite digestive tract where it degrades hemoglobin released from host red blood cells. Reduced glutathione (GSH) has been shown to be effective in activation. Total glutathione concentration and the GSH/oxidized glutathione (GSSG) ratio were measured in whole blood lysate (3.2 mM, 284), serum (24 microM, 9.8) and material collected from the parasite digestive tract (4.2 mM, 137). The ratio of GSH/GSSG at which the enzyme displays half-maximal activity (Kox) is 1.0. Proteinase activation as a function of glutathione concentration and time was determined. The first-order reaction yielded a half-time of activation of 13 min at 5 mM. The second-order rate constant was 12.7 M-1 X min-1. The function of the proteinase and its possible regulation by glutathione activation are discussed.     

Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.     

Eukaryotic initiation factor 4GI is a poor substrate for HIV-1 proteinase

Eukaryotic initiation factor (eIF) 4GI is efficiently cleaved during picornaviral replication. eIF4GI processing has also recently been observed during HIV-1 replication. We have compared the efficiency of eIF4GI proteolysis in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L(pro)) or the HIV-1 proteinase (HIV-1(pro)). L(pro) cleaved 50% eIF4GI within 12 min whereas HIV-1(pro) required 4 h; the concentrations were 2 pg/microl (0.1 nM) for L(pro) and 60 pg/microl (2.66 nM) for HIV-1(pro). HIV-1(pro) processing of eIF4GI is therefore not quantitatively analogous to that of L(pro), suggesting that the primary function of eIF4GI cleavage in HIV-1 replication may not be protein synthesis inhibition.     

Platelet-derived growth factor is not chemotactic for human peripheral blood monocytes

PDGF is a mitogenic protein stored in platelets and released upon platelet degranulation. Recent evidence indicates that PDGF plays an important role in both physiologic and pathophysiologic processes, particularly in tumorigenesis, wound healing, pulmonary fibrosis, and atherogenesis. In addition to its mitogenic potential, it has been reported that PDGF stimulates monocyte chemotaxis. Since the recruitment of monocytes from the peripheral vasculature is an important event in vivo, the potential role of PDGF as a monocyte chemoattractant has significant biologic implications. However, we now report that homogeneous human PDGF from platelets and a recombinant PDGF-2 homodimer do not stimulate monocyte chemotaxis. In contrast to previous reports these results indicate that PDGF is not a monocyte chemoattractant.