Antonio Baici

Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases.

The tissue inhibitors of matrix metalloproteinases (MMPs), TIMP‐1 and TIMP‐2, are also angiogenesis inhibitors. Cathepsin B and MMPs are found at sites of neovascularization in pathologies such as cancer and osteoarthritis. Treatment of TIMP‐1, TIMP‐2, and of a mixture of both inhibitors from human articular chondrocytes with cathepsin B resulted in their fragmentation, whereby they lost their MMP‐inhibitory and anti‐angiogenic activities. Our data suggest that, besides directly participating in tissue destruction, cathepsin B can be harmful for two further reasons: it raises the activity of the MMPs also in the absence of mechanisms up‐regulating these enzymes, and it stimulates angiogenesis. This is a prerequisite for blood vessel invasion in a variety of pathological situations of which cancer and osteoarthritis are prominent examples.

The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin

Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig-κ-variable region, an Ig-κ enhancer, and the exon encoding the Ig-κ constant region (Cκ) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with Cκ. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.

Inhibition of human elastase from polymorphonuclear leucocytes by a glycosaminoglycan polysulfate (Arteparon

Abstract Human lysosomal elastase from polymorphonuclear leucocytes is inhibited by the glycosaminoglycan polysulfate Arteparon. The inhibition is of a mixed type: hyperbolic uncompetitive. The interaction between inhibitor and enzyme occurs via electrostatic forces, and the binding is very tight ( K i ranges from 10 −8 to 10 −7 M). Depending on the chain length of the polysulfated glycosaminoglycan, two, three or five enzyme molecules can be tightly bound by a single inhibitor molecule. These findings suggest a possible therapeutic role of Arteparon as an inhibitor of elastase, a potent mediator of connective tissue breakdown, since the enzyme is inhibited by a drug concentration as small as 1 μg/ml or less.

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin‐deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 × 104 M−1 • s−1 was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7–8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C‐termi‐nal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/ K+‐ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.—Reif, R., Sales, S., Hettwer, S., Dreier, B., Gisler, C., Wolfel, J., Luscher, D., Zurlinden, A., Stephan, A., Ahmed, S., Baici, A., Ledermann, B., Kunz, B., Sonderegger, P. Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation. FASEB J. 21, 3468–3478 (2007)

Cathepsin B in osteoarthritis: zonal variation of enzyme activity in human femoral head cartilage.

OBJECTIVES--To determine the quantitative topographical distribution of cathepsin B in human femoral head cartilage by measuring the zonal variation of enzyme activity in specimens taken from various anatomical regions of normal and osteoarthritic (OA) tissues, and to correlate this parameter with the severity of the OA lesions. METHODS--OA articular cartilage was obtained at surgery for total hip replacement and control cartilage obtained at postmortem. Cylinders of full thickness cartilage with underlying bone were retrieved with a biopsy trephine. Sections of cartilage were produced by cryocutting the tissue as slices parallel to the articular surface and assayed for cathepsin B with a specific, highly sensitive fluorogenic substrate. The severity of the OA lesions was graded according to the histopathological-histochemical method of Mankin. RESULTS--Zonal cathepsin B activity of normal cartilage was uniform and low in all regions of the femoral head. In apparently intact OA cartilage and in severely degraded tissue the zonal distribution and the amounts of enzyme were similar to control values. At sites with active disease, cathepsin B activity was much greater than in controls and its irregular zonal distribution correlated with tissue degeneration, hypercellularity, or cloning of chondrocytes as determined histochemically. Particularly high enzyme levels were observed at sites with regenerating cartilage, where some zonal peaks attained 20-fold activity with respect to controls. CONCLUSION--Cathepsin B may play a role in sustaining the chronicity of OA, not as an initiator, but rather as a perpetuator of the disease and as an antagonist of regeneration.

Interaction of site specific hirudin variants with α-thrombin

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitors reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site‐directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (K i = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher K i value for their complexes with thrombin. The observed effects are mainly due to increased k off rate constants.

Interaction between Human Cathepsins K, L, and S and Elastins MECHANISM OF ELASTINOLYSIS AND INHIBITION BY MACROMOLECULAR INHIBITORS

Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins.

Analysis of glycosaminoglycans in human serum after oral administration of chondroitin sulfate

SummaryChondroitin sulfate was administered orally to six healthy volunteers, six patients with rheumatoid arthritis and six patients with osteoarthritis. Blood was collected at intervals before and after treatment and the glycosaminoglycan concentration was analyzed in serum using a sensitive assay based on the metachromatic reaction with 1,9-dimethylmethylene blue. The glycosaminoglycan concentration in serum before and after ingestion of chondroitin sulfate was statistically unchanged in all of the subjects studied. We suggest that chondroprotection by orally administered chondroitin sulfate is a biologically and pharmacologically unfounded theory. Any possible benefit to osteoarthritic patients after ingestion of chondroitin sulfate should be sought at the gastrointestinal rather than at the plasmatic or articular cartilage level.

Interaction between human leukocyte elastase and chondroitin sulfate

Chondroitin sulfate (Structum) interacts with human leukocyte elastase, a potent mediator of articular cartilage degradation, producing a partial inhibition of the enzyme activity (60% at saturation). Kinetically, the inhibition mechanism can be classified as simple intersecting, hyperbolic noncompetitive and is almost identical to that found earlier for similar compounds. The best inhibitory activity of chondroitin sulfate was found in fractions having at the same time a high proportion of chondroitin-6-sulfate relative to the corresponding 4-isomer and a high molecular mass. Thus, a fraction with high Mr and containing 92% of isomer 6 inhibited leukocyte elastase with Ki = 1.8 micrograms/ml, whereas a fraction with low Mr and almost equal composition of the 4- and 6-isomer had Ki = 140 micrograms/ml. Ki for unfractionated chondroitin sulfate was 3.4 micrograms/ml. It is suggested, that the modulation of the extracellular activity of cartilage-degrading enzymes by cartilage-derived factors may explain, at least in part, the beneficial effects of some therapeutically used chondroprotective agents.

Inhibition of human elastase from polymorphonuclear leucocytes by gold sodium thiomalate and pentosan polysulfate (SP-54®)

Abstract Human lysosoma) elastase, the serine proteinase from the azurophil granules of polymorphonuclear leucocytes, is inhibited by gold thiomalate and pentosan polysulfate (SP-54®). The kinetic mechanism of the inhibition was studied using succinyl-alanyl-alanyl-prolyl-valyl-4-methyl-7-coumarylamide and (t-butyloxycarbonyl-alanyl-p-nitrophenylester as subsrates. The degree of inhibition was also tested using insoluble elastin as substrate. Independent of the substrate, the maximal inhibition of elastase by gold thiomalate and pentosan polysulfate was 40% and 60%, respectively. Pentosan polysulfate behaved as a simple intersecting, hyperbolic, non-competitive inhibitor and k′i. the dissociation constant of the E-S-I complex, was 1.8 × 10−7M. The interaction between inhibitor and enzyme is driven by electrostatic forces. Gold thiomalate showed a hyperbolic mixed type inhibition (intersecting, slope-hyperbolic, intercept-hyperbolic, non-competitive inhibition) with k′i = 5.4 × 10−5M. The overall kinetic mechanism of lysosomal elastase conforms to that of other serine proteinases. With both the ester and peptide substrates the rate limiting step of the reaction has been identified with the formation of the acyl-enzyme.