Elemer Moczar

Fatty acid peptide derivatives as model compounds to protect elastin against degradation by elastases

Peptide sequences which fit the extended binding sites of porcine pancreatic elastase and human leukocyte elastase were covalently coupled to oleic acid. These compounds behave as competitive inhibitors towards both elastases. The coupling of fatty acid moiety to the peptide greatly decreases its inhibitor constant (Ki) vs human leukocyte elastase (Ki for Oleoyl(Ala)2ProValine: 3.0 (10(-6)M). It is less active on porcine pancreatic elastase (Ki for Oleoyl(Ala)2ProAlanine: 3.8 10(-4)M). The modifications of the carboxylic end group of the peptide to an aldehyde further greatly enhanced the inhibition capacity of the compound towards leukocyte elastase (Ki for Oleoyl(Ala)2ProAlaninal: 0.7 microM). Oleoyl peptide derivatives were seen to bind in a saturable fashion to purified insoluble elastin, and decreased the susceptibility of the macromolecule to hydrolysis by both pancreatic and leukocyte elastases. As stoichiometric quantities of elastase (vs inhibitor) could not desorb 3H-oleoyl(Ala)2Pro-Val bound to insoluble elastin, it is postulated that oleoyl peptide derivatives may act as bifunctional agents. This contention was further strengthened by the comparison of the adsorption curves of elastase to untreated insoluble elastin and elastin saturated with oleoyl peptide derivatives respectively. It was shown finally that Oleoyl(Ala)2Pro-Valine was also capable of inhibiting elastases in their adsorbed form to insoluble elastin.

Inhibition of the human leukocyte endopeptidases elastase and cathepsin G and of porcine pancreatic elastase by N-oleoyl derivatives of heparin

N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.

Mapping and specific detection of glycopeptides on thin-layer plates

Abstract A thin-layer electrophoretic method is used for the separation of the glycopeptides. Specific detection methods permit the differentiation of the sugar constituents. Carbohydrate-containing and carbohydrate-free peptides are distinguished by the successive application of ninhydrin and orcinol-sulphuric acid reagents on to the thin-layer peptide maps of glycoprotein hydrolysates.

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.

Cell surface glycosaminoglycans of rat rhabdomyosarcoma lines with different metastatic potentials and of non-malignant rat myoblasts

Glycosaminoglycans of cultured nickel-induced rat rhabdomyosarcoma cell lines with different metastatic potentials and of non-malignant myoblasts, grown in the presence or in the absence of hydrocortisone, were studied comparatively. The newly formed [3H]glucosamine-labelled cell surface proteoglycans and glycosaminoglycans were separated by ion exchange chromatography and partially characterized. The overall incorporation of the label in the glycosaminoglycan fractions and the average molecular weight of the heparan and of the chondroitin sulfate proteoglycans was lower in the malignant cells than in the non-malignant L6 myoblasts. The strongly metastatic 9-4/0 parental line and the 6 subline were relatively richer in chondroitin sulfate and poorer in dermatan sulfate labels than the very weakly metastatic 8 subline and the L6 myoblasts. Hyaluronic acid and heparan sulfate labels were inversely related to the metastatic capacity of the cell lines studied. Hydrocortisone treatment induced an increase in the cell surface chondroitin and dermatan sulfate labels in the case of the strongly metastatic lines, and a decrease of the same parameters in the case of the weakly metastatic 8 line.

Design of potent lipophilic-peptide inhibitors of human neutrophil elastase: In vitro and in vivo studies

Abstract In keeping with the presence of an extended hydrophobic binding site in human neutrophil elastase (HNE), a series of lipophilic peptide derivatives were synthesized. In vitro studies, using an oligopeptide substrate for HNE, showed that lipidic amino acid derivatives 1b , 1c and 1d were potent HNE inhibitors; the lipophilic amino acid trimer conjugate ld (IC 50 = 1.8 × 10 −10 ) was a more powerful inhibitor than the dimer conjugate lc ( IC 50 = 2.8 × 10 −10 ) which was more potent than the monomer conjugate lb ( IC 50 = 2.9 × 10 −9 ). When injected intradermally to rabbit dermis, compound ld protected skin elastic fibres against degradation by HNE.

Cell surface glycosaminoglycans of a tumor cell line and its DNA transfected variant differing in their lung colonizing potential

A highly lung-colonizing cell line RMS/82 was obtained by DNA transfection from a low lung-colonizing line RMS/8, a clone of a rat rhabdomyosarcoma cell line. The cells were metabolically labeled with3H-glucosamine and35S-sulfate. The newly synthesized pericellular glycosaminoglycans and the ability of the cells from the two lines to degrade extracellular matrix components were studied comparatively. The following conclusions were obtained: 1) Thein vitro proliferation rate is not a determinant in the modulation of the colonizing potential of these cells; 2) The strongly colonizing RMS/82 cells release more radioactivity from the radiolabeled extracellular matrix than their weakly colonizing counterparts; 3) The cells with a high colonizing potential incorporated less radioactivity into the cell surface glycosaminoglycans, and exhibited a lower heparan sulfate to chondroitin sulfate ratio than the weakly colonizing RMS/8 line.

Inhibition of human leucocyte elastase by fatty acyl-benzisothiasolinone, 1,1-dioxide conjugates (fatty acyl-saccharins)

Derivatives of benzisothiazolinone 1,1-dioxide (saccharin) N-acetylated with aliphatic and aromatic substituted aliphatic acyl groups were prepared. The inhibitory activity of the compounds was assayed against human leucocyte elastase (EC 3.4.21.37) and several other proteases. The IC50 values for inhibition of the human leucocyte elastase decreased with increasing length of the acyl residue, and reached a minimum value at C16 (2 microM). This phenomenon and the decrease of the inhibition by surfactants or by saturation of the enzyme with palmitic acid, indicates that in addition to acylation, hydrophobic interactions are also involved in the inhibition of this proteinase by compounds substituted with acyl groups containing at least 12 carbon atoms. The inhibitory activity of N-palmitoyl-benzisothiazolinone 1,1-dioxide (palmitoyl-saccharin) is about 14 times higher toward human leucocyte elastase than for thrombin (EC 3.4.21.5), and several hundred times, compared to porcine pancreatic elastase (EC 3.4.21.36) and to plasmin (EC 3.4.21.7). Fatty acylated saccharin derivatives were seen to bind in a saturable fashion to insoluble elastin, and decreased the susceptibility of this protein to hydrolysis by human leucocyte elastase.

Galactosylated glycan expression and macrophage sensitivity of lewis lung tumor cells with different metastatic phenotype

SummaryBiochemical and cytochemical analysis of Lewis lung tumor variants revealed that the low metastatic cells contained more galactose/N-acetylgalactosamine residues in a high-molecular-mass (15–20 kDa) mixedN- andO-glycan fraction than the highly metastatic ones. It was also found that the highly metastatic variant was less sensitive to macrophage cytotoxicity in vitro. The cytotoxicity against the low metastatic target cells was inhibited by asialofetuin (10–20 μM), and, to a small degree — and at much higher concentration — by lactose, while galactose and other monosaccharides were ineffective. We suppose that complex galactosylated tumor cell membrane glycans could play a role in the antitumoral cytotoxicity of macrophages.