Sulfation patterns in heparin and heparan sulfate: effects on the proliferation of bovine pulmonary artery smooth muscle cells

Heparin's (HP's) antiproliferative effect on smooth muscle cells is potentially important in defining new approaches to treat pulmonary hypertension. The commercially available HP and heparan sulfate (HS) are structurally heterogenous polymers. In order to examine which sulfonate groups are required for endogenous antiproliferative activity, we prepared the following six chemically modified porcine mucosal HP and HS, which fell into three groups. One group consisted of fully O-sulfonated-N-acetylated, the second group consisted of de-N-sulfonated and re-N-acetylated, and the third group consisted of 6-O-desulfonated HP and HS derivatives. These six preparations were assayed for their antiproliferative potency on bovine pulmonary artery smooth muscle cells. The results of this assay show that (a) over-O-sulfonation of both HP and HS increases antiproliferative activity, (b) substitution of hexosamine with N-acetyl diminishes antiproliferative activity in both HP and HS, and (c) 6-O-desulfonation of HP and HS diminishes antiproliferative potency. Surprisingly, the type of uronic acid residue present at a given level of sulfation is unimportant for antiproliferative potency. In conclusion, only the level of O- and N-sulfo group substitution correlates well with HP and HS antiproliferative activity.     

Synthesis and antiproliferative activity of C- and N-terminal analogues of culicinin D

Culicinin D (1), a 10 amino acid peptaibol containing several unusual residues, has been shown to exhibit potent anticancer activity. Previous work in our group towards developing a structure-activity relationship (SAR) for this peptaibol has concentrated on replacement of the synthetically challenging AHMOD (3) and AMD (4) residues, resulting in the discovery of analogues with equivalent or better potency and simplified synthesis. The SAR of this peptaibol is extended in this work by investigating the effect of the N-terminal lipid tail and C-terminal amino alcohol, revealing the key contribution of each of these moieties on antiproliferative activity in a panel of breast and lung cancer cell lines.     

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals.The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[³H]₄ and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper.The method is also useful for discrimination between glucoronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.     

Action on peptides by wheat carboxypeptidase

A kinetic analysis has been performed with purified wheat carboxypeptidase by the use of N-acyl dipeptides, Z-Gly-Pro-Leu-Gly (Z = benzyloxycarbonyl), angiotensin II and bradykinin. The values of k_cat were dramatically influenced by amino acid residues occupying the penultimate position from the carboxyl terminus of substrates. The structure of the substrate did not appreciably affect the K_m values.     

Modifications of Diflunisal and Meclofenamate Carboxyl Groups Affect Their Allosteric Effects on GABAA Receptor Ligand Binding

Gamma-aminobutyric acid type A receptors (GABA_AR) are allosterically modulated by the nonsteroidal anti-inflammatory drugs diflunisal and fenamates. The carboxyl group of these compounds is charged at physiological pH and therefore penetration of the compounds into the brain is low. In the present study we have transformed the carboxyl group of diflunisal and meclofenamate into non-ionizable functional groups and analyzed the effects of the modifications on stimulation of [³H]muscimol binding and on potentiation of γ-aminobutyric acid-induced displacement of 4′-ethenyl-4-n-[2,3-³H]propylbicycloorthobenzoate. N-Butylamide derivative of diflunisal modulated radioligand binding with equal or higher potency than the parent compound, while diflunisalamide showed reduced allosteric effect as compared to diflunisal. Amide derivative of meclofenamate equally affected radioligand binding parameters, while both diflunisal and meclofenamate methyl esters were less active than the parent compounds. Our study clearly demonstrates that an intact carboxyl group in diflunisal and meclofenamate is not indispensable for their positive GABA_AR modulation. Further derivatization of the compound might yield compounds with higher selectivity for GABA_ARs that could be utilized in drug development.     

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex_0-9 HexNAc_2-4 Hex_0-3 Pent_0-3 Fuc_0-3. A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.     

Preparation and immunochemical properties of methoxypolyethylene glycol-coupled and N-carboxymethylated derivatives of ragweed pollen allergen, antigen E.

A methoxypolyethylene glycol (PEG)-coupled and several N-carboxymethylated (N-CM) derivatives of antigen E, the major allergenic protein of ragweed pollen, were prepared. The PEG derivative contained seven residues of PEG groups (residue weight about 2100) per molecule of protein and the groups were linked to the lysyl residues of antigen via the 2,6-positions of 4-hydroxy-triazine nucleus. The maximally N-CM derivative contained, respectively, 10, 6, and 2 residues of mono-CM, di-CM, and unmodified lysyl residues per molecule of protein. The CM groups were introduced reductively on reaction with glyoxylic and sodium cyanoborohydride and the extent of mono- and dicarboxymethylation was controlled more by the concentration of cyanoborohydride than by that of glyoxylic acid. The molar allergenic activities of the PEG and the N-CM derivatives in man were, respectively, 0.02 and 0.5 of that of the native antigen. Rabbits immunized with the PEG derivative gave antibody titers about $\text{1}\text{8}$th of those obtained with animals immunized with the native antigen. However, the rabbits preimmunized with the PEG derivative gave a vigorous secondary response on challenge with the native antigen and their titers approached those of rabbits preimmunized with the native antigen. The immunogenicity of the reduced and S-carboxymethylated derivative of antigen E which has the denatured conformation was studied as a control. Rabbits immunized with the S-CM derivative gave antibody titers $\text{1}\text{34}$th of those obtained with animals immunized with the native antigen; on secondary challenge with the native antigen, these rabbits gave antibody titers about $\text{1}\text{6}$th of those of animals preimmunized with the native antigen.     

Structural studies of the O-specific side-chains of the shigella sonnei phase I lipopolysaccharide

The structure of the O-specific side-chains of the Shigella sonnei phase I lipopolysaccharide has been investigated. The side chains are composed of disaccharide repeating-units containing two uncommon sugar components, one of witch, 2-amino-2-deoxy-L-altruronic acid, has been identified previously. The other has now been identified as 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. The uronic acid, as N-acetylated α-pyranosyl residues, is linked through O-4, and the diamino sugar, as β-pyranosyl residues, is linked through O-3. The pyranosyluronic acid residue assumes the ⁴C₁ conformation in the polymer, with the carboxyl group in the axial position.     

Intramolecular catalysis of amide hydrolysis by carboxyl and metal ion/proton agents: I. Intramolecular reactions of imidazole or pyridine derivatives of maleamic acid

Kinetic studies on the hydrolysis of N-(2-imidazolylethyl)maleamic acid, N-(2-pyridylethyl)maleamic acid, and N-(2-pyridylmethyl)maleamic acid were carried out. In these maleamic acid derivatives, the amide nitrogens together with the heterocyclic nitrogens form bidentate ligands. The compounds were hydrolyzed through nucleophilic catalysis by carboxyl groups. The pH profiles for the hydrolysis at 65°C were descending sigmoids and were unaffected by the ionization of the heterocyclic nitrogens. The absence of rate enhancement due to the added divalent metal ions revealed that chelate formation by the bidentate leaving groups is not efficient. N-(2-Pyridylmethyl)maleamic acid underwent a dual intramolecular nucleophilic reaction through participation of both the carboxyl and the pyridyl groups.     

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6′ [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16β,17β-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC₅₀ = 2.1–6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC₅₀ = 3.0 μM and 7j with IC₅₀ = 2.1 μM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.     

Synthesis of some long-chain acylamidoalkyl glucosides

4-O-β-D-Galactopyranosyl-α,β-D-glucopyranosylamine (lactosylamine), β-D-gluco-, α- and β-D-manno-pyranosylamines were bound to the carbodiimide-activated groups of lysozyme. Of the 11 free carboxyl groups of the protein, ≈3 were substituted by α,β-6-lactosylamine, and ≈2 by the monohexo-sylamines. One of the 4 glycopeptides isolated from the tryptic digest of the lysozyme-lactosylamine conjugate was identical to synthetic l-N-L-leucinoyl-4-O-β-D-galactopyranosyl-β-D-glucopyranosylamine, indicating the substitution of the carboxyl group of the C-terminal leucine residue. The isolation of a glycopeptide containing the aspartic acid residue in position 117 indicates that the second α,β-lactosylamine residue is linked to the carboxyl group of this amino acid. Both of the 2 other glycopeptides contain the same free carboxyl groups (one glutamic and two aspartic acid residues in positions 35, 48, and 52, respectively). The third α,β-lactosylamine residue seems to be linked to one of these carboxyl groups.     

Platinum(IV) complexes with ethylenediamine-N,N′-diacetate diester (R2edda) ligands: Synthesis, characterization and in vitro antitumoral activity

The novel N,N-type bidentate ligand precursors, diethyl, dipropyl esters of ethylenediamine-N,N′-diacetic acid dihydrochloride (HOOCCH₂NHCH₂CH₂NHCH₂COOH · 2HCl, H₂edda · 2HCl), and the corresponding tetrachloroplatinum(IV) complexes, [PtCl₄(R₂edda)] · H₂O (ROOCCH₂NHCH₂CH₂NHCH₂COOR, R = Me, Et, n-Pr), were synthesized. The esters coordinated as bidentate ligands via both N donor atoms. The esters, as well as the complexes, have been characterized by infrared, ¹H and ¹³C NMR spectroscopy and elemental analysis. Solid state structures of both dimethyl and diethyl ester platinum(IV) complexes have been determined by X-ray crystallography. Quantum chemical calculations were performed in order to investigate diastereoselectivity in the formation of the platinum(IV) complexes. The in vitro cytotoxic evaluation of the investigated complexes in human tumor cell lines 1411HP, H12.1 (both testicular germ cell tumors), DLD-1 (colon carcinoma), 518A2 (melanoma), A549 (lung carcinoma) and liposarcoma showed a dose-dependent antiproliferative effect in all cell lines. Remarkably, the highest cytotoxic activity was observed in the cisplatin-resistant cell line 1411HP. In addition, at higher concentrations the treatment with these complexes led to the induction of apoptosis in all cell lines except for DLD-1.     

Structure and binding properties of hen lysozyme modified at tryptophan 62

The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified by N-bromosuccinimide at Trp62 has been studied by both ¹H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine² residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pK values of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide of Micrococcus lysodeikticus. The binding of N-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme ¹H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)₃ in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)₃ was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blake et al., 1967a). On the basis of the results obtained by X-ray crystallography and ¹H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.     

Isolation and properties of Serratia proteamaculans 94 cysteine protease

A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Porphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37°C showed that a decrease in temperature by more than 30°C causes a 1.3-fold increase in the k _cat/K _m ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.     

A sulfated glucuronofucan containing both fucofuranose and fucopyranose residues from the brown alga Chordaria flagelliformis

A fucoidan fraction composed of l-fucose, sulfate, and d-glucuronic acid in a molar proportion of about 1:1:0.25 and small amount of acetyl groups was isolated from the brown alga Chordaria flagelliformis. Several modified polysaccharides were prepared from the native fucoidan using solvolytic desulfation, carboxyl reduction, and partial acid hydrolysis. Polysaccharide structures were elucidated by methylation analysis and 1D and 2D NMR spectroscopy. The fucoidan was shown to contain a backbone of 3-linked α-l-fucopyranose residues, about one-third of which are glycosylated at C-2 by α-d-glucopyranosyluronic acid residues. About half of the latter residues are glycosylated at C-4 by single α-l-fucofuranose residues or by disaccharides α-l-Fucf-(1→2)-α-l-Fucf-(1→. Fucofuranose residues are mono- and disulfated at different positions, whereas some additional sulfate groups occupy C-2 and C-4 of the backbone, the latter position being also partially acetylated.     

Structural determinants of antiproliferative activity of heparin on pulmonary artery smooth muscle cells

In addition to its anticoagulant properties, heparin (HP), a complex polysaccharide covalently linked to a protein core, inhibits proliferation of several cell types including pulmonary artery smooth muscle cells (PASMCs). Commercial lots of HP exhibit varying degrees of antiproliferative activity on PASMCs that may due to structural differences in the lots. Fractionation of a potent antiproliferative HP preparation into high and low molecular weight components does not alter the antiproliferative effect on PASMCs, suggesting that the size of HP is not the major determinant of this biological activity. The protein core of HP obtained by cleaving the carbohydrate-protein linkage has no growth inhibition on PASMCs, demonstrating that the antiproliferative activity resides in the glycosaminoglycan component. Basic sugar residues of glucosamine can be replaced with another basic sugar, i.e., galactosamine, without affecting growth inhibition of PASMCs. N-sulfonate groups on these sugar residues of HP are not essential for growth inhibition. However, O-sulfonate groups on both sugar residues are essential for the antiproliferative activity on PASMCs. In whole HP, in contrast to an earlier finding based on a synthetic pentasaccharide of HP, 3-O-sulfonation is not critical for the antiproliferative activity against PASMCs. The amounts and distribution of sulfonate groups on both sugar residues of the glycosaminoglycan chain are the major determinant of antiproliferative activity.     

Oxidation of mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass spectrometry

The hydroxyl radicals are widely implicated in oxidation of carbohydrates during biological and industrial processes being responsible for their structural modifications and causing functional damage. The identification of intermediate oxidation products is hampered by a lack of reliable sensible methods for their detection. In this study, the oxidation of two models of galactomannans (Man₃ and GalMan₂) has been studied in reaction with hydroxyl radical generated by Fenton reaction. The oxidation patterns were assessed using preparative ligand-exchange/size-exclusion chromatography (LEX/SEC) coupled with tandem electrospray mass spectrometry (ESI-MS/MS). This allowed the identification of derived oligosaccharides (OS) containing hexuronic, hexonic, pentonic and erythronic acid residues and neutral OS bearing hydroperoxy, hydrated carbonyl moieties and residues from pyranosyl ring cleavage. The depolymerization products have been also detected upon oxidation of oligomers. This study allowed developing a simple, effective ‘fingerprinting’ protocol for detecting the damage done to mannans by oxidative radicals.     

Molecular organization and antiproliferative domains of arterial tissue heparan sulfate.

Heparan sulfate isolated from mammalian arterial tissue inhibits the growth of homologous arterial smooth muscle cells when added to subconfluent cell cultures at a concentration of 50 to 100 micrograms/ml culture medium. Disintegration of the heparan sulfate molecule by hydrazinolysis that deacetylates N-acetylglucosaminyl residues and by subsequent treatment with nitrous acid at pH 3.9 results in the formation of a mixture of oligosaccharides which was further resolved into sulfate-enriched oligosaccharides with antiproliferative activity in an in vitro bioassay system. A decasaccharide and dodeca/tetradecasaccharide fraction had a significantly higher antiproliferative effect on arterial smooth muscle cells than the native heparan sulfate molecule. The antiproliferative oligosaccharides have a sulfate content of 0.9 to 1.2 sulfate groups/disaccharide unit and consist of 60 to 70% monosulfated, disulfated, and trisulfated disaccharide units. Up to 32% of the sulfate groups were in 2-position of the uronic acid. In contrast, nitrous acid degradation of heparan sulfate at pH 1.5, which cleaves glycosidic linkages of N-sulfoglucosaminyl residues, results in the formation of sulfate-poor or sulfate-free oligosaccharides without antiproliferative potency. The results indicate that (a) heparan sulfate has a heterogeneous molecular organization where sulfate-rich domains are separated by sulfate-poor sequences and that (b) the antiproliferative activity of heparan sulfate resides in domains enriched with 2-O-sulfated uronic acid residues.     

The Active Site on the Phytotoxin of Corynebacterium sepedonicum

Corynebacterium sepedonicum produces an extracellular phytotoxic glycopeptide that possesses a capacity to wilt plant cuttings. It has been previously demonstrated that the integrity of some of the membranes of the host cells is destroyed, suggesting the possibility that a biologically active site is present on the toxin molecule. The toxin was chemically altered in the following ways and then tested for biological activity: (a) the NH₂-terminal group on the peptide portion of the toxin was blocked by the dansylation technique; (b) the OH groups on the sugar and amino acid residues as well as the NH groups on the amino acid residues were blocked by exhaustive methylation; (c) the COO⁻ groups were converted to their respective methyl esters; (d) the peptide moiety was removed by pronase digestion. Experimental results indicate that the carboxyl groups of the nonpeptide portion of the molecule are responsible for the biological activity of the toxin. Other experiments showed that the toxin does not affect the membranes of animal cells.