BBA - General Subjects (v.1528, #2-3)

Matrix remodelling enzymes, the protease cascade and glycosylation by Philippe E. Van den Steen; Ghislain Opdenakker; Mark R. Wormald; Raymond A. Dwek; Pauline M. Rudd (61-73).
Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1).
Keywords: Gelatinase B/MMP-9; Tissue-type plasminogen activator; Plasminogen; TIMP-1; Collagenase/MMP-1; Urokinase; Sialic acid; Stromelysin-1/MMP-3;

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells by Maria Caterina Carta; Antonella Mattana; Marcella Camici; Simone Allegrini; Maria Grazia Tozzi; Francesco Sgarrella (74-80).
Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.
Keywords: Deoxyinosine; Deoxyribose 5-phosphate; WISH cell; Deoxynucleoside catabolism; Nucleoside transport;

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate by Achilleas D Theocharis; Marina E Tsara; Nikoletta Papageorgakopoulou; Demitrios H Vynios; Dimitrios A Theocharis (81-88).
The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs) – chondroitin sulfate (CS) and dermatan sulfate (DS) – were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.
Keywords: Scoliosis; Nasal cartilage; Glycosaminoglycan; Proteoglycan;

Cortical cell elution by sedimentation field-flow fractionation by S. Battu; W. Elyaman; J. Hugon; P.J.P. Cardot (89-96).
As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.
Keywords: Sedimentation field flow fractionation; Cortical cell; Neuron cell culture;

Hemoglobin can nitrate itself and other proteins by Agnieszka Grzelak; Aneta Balcerczyk; Agnieszka Mateja; Grzegorz Bartosz (97-100).
Incubation of human hemoglobin with nitrite and hydrogen peroxide was found to induce autonitration and nitration of another protein (bovine serum albumin), as demonstrated by detection of nitrotyrosine residues in Western blots of separated membrane proteins. Inhibition of nitration by conversion of hemoglobin into the cyanmet form demonstrates that nitration is due to the pseudoperoxidase activity of hemoglobin. Incubation of whole erythrocytes with nitrite and hydrogen peroxide induces nitration of erythrocyte membrane proteins, much stronger when cellular catalase was inhibited with azide. These results suggest that hemoglobin and other hemoproteins may contribute to the tyrosine nitration in vivo.
Keywords: Nitrite; Hemoglobin; Nitrotyrosine; Nitration; Erythrocyte; Peroxidase;

Glutathione transferase A4-4 (GST A4-4) is involved in the detoxication of lipid peroxidation products such as alkenals. The human enzyme has been heterologously expressed in Escherichia coli, but for more extensive characterization of the enzyme the expression level had to be elevated. A clone providing up to 8-fold higher yields was created, by screening an expression library with random silent mutations in the 5′ region of the cDNA encoding GST A4-4.
Keywords: Glutathione transferase A4-4; Heterologous expression; Random silent mutagenesis;

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection by Fabiana Superti; Rosa Siciliano; Barbara Rega; Francesco Giansanti; Piera Valenti; Giovanni Antonini (107-115).
Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86–258) and a small peptide (324–329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.
Keywords: Lactoferrin; Rotavirus; Sialic acid; Metal saturation; Tryptic fragment; Mass spectrometry;

Dietary mannose is used to treat glycosylation deficient patients with mutations in phosphomannose isomerase (PMI), but there is little information on mannose metabolism in model systems. We chose the mouse as a vertebrate model. Intravenous injection of [2-3H]mannose shows rapid equilibration with the extravascular pool and clearance t 1/2 of 28 min with 95% of the label catabolized via glycolysis in <2 h. Labeled glycoproteins appear in the plasma after 30 min and increase over 3 h. Various organs incorporate [2-3H]mannose into glycoproteins with similar kinetics, indicating direct transport and utilization. Liver and intestine incorporate most of the label (75%), and the majority of the liver-derived proteins eventually appear in plasma. [2-3H]Mannose-labeled liver and intestine organ cultures secrete the majority of their labeled proteins. We also studied the long-term effects of mannose supplementation in the drinking water. It did not cause bloating, diarrhea, abnormal behavior, weight gain or loss, or increase in hemoglobin glycation. Organ weights, histology, litter size, and growth of pups were normal. Water intake of mice given 20% mannose in their water was reduced to half compared to other groups. Mannose in blood increased up to 9-fold (from 100 to 900 μM) and mannose in milk up to 7-fold (from 75 to 500 μM). [2-3H]Mannose clearance, organ distribution, and uptake kinetics and hexose content of glycoproteins in organs were similar in mannose-supplemented and non-supplemented mice. Mannose supplements had little effect on the specific activity of phosphomannomutase (Man-6-P↔Man-1-P) in different organs, but specific activity of PMI in brain, intestine, muscle, heart and lung gradually increased <2-fold with increasing mannose intake. Thus, long-term mannose supplementation does not appear to have adverse effects on mannose metabolism and mice safely tolerate increased mannose with no apparent ill effects.
Keywords: Mannose; Metabolism; Transporter; Mouse; Phosphomannose isomerase; Phosphomannomutase;

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from α-fetoprotein by Robert MacColl; Leslie E. Eisele; Robert F. Stack; Charles Hauer; Dilip D. Vakharia; Adina Benno; Wayne C. Kelly; Gerald J. Mizejewski (127-134).
A 34-amino-acid peptide has been chemically synthesized based on a sequence from human α-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt–peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible dd transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the dd molar extinction coefficient showed that the metal ion–ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)– and Co(II)–peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60°C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.
Keywords: α-Fetoprotein; Anti-growth and anti-cancer peptide; Zn(II)–peptide complex; Co(II) spectroscopic probe for peptide binding; Peptide conformation;

Near infra-red spectra of urea with glycine betaine or trimethylamine N-oxide are additive by Michael Lever; Kelly Randall; Erwin A Galinski (135-140).
Glycine betaine and trimethylamine-N-oxide counteract urea denaturation in solutions containing urea and the methylamine in the mole ratio of 2:1. Near infra-red difference spectra (water spectrum subtracted) of solutions containing both urea with either glycine betaine or trimethylamine-N-oxide can be predicted from the spectra of the single solutes, with r 2>0.999 both using the spectrum from 1200 to 2100 nm (where most absorbance is attributable to hydrogen bonding) and using an extended range 1000 to 2500 nm, which includes solute specific bands. Thus urea and the kosmotropes appear to interact with water independently and the counteraction cannot be attributed to specific interactions between them. The spectrum of aqueous glycine betaine can be predicted from tetramethylammonium and formate ions (r 2=0.998), suggesting that independent interactions of the quaternary amine, and of the carboxyl function, with water are dominant. The exceptional properties of glycine betaine do not arise from specific intramolecular interactions between the charged groups.
Keywords: Glycine betaine; Trimethylamine-N-oxide; Protein stabilization; Osmolyte; Urea denaturation; Compatible solute; Near infrared spectroscopy;

Characterization, chemical modifications and in vitro anticoagulant properties of an exopolysaccharide produced by Alteromonas infernus by Sylvia Colliec Jouault; Lionel Chevolot; Dominique Helley; Jacqueline Ratiskol; Andrée Bros; Corinne Sinquin; Olivier Roger; Anne-Marie Fischer (141-151).
A new low-molecular-weight ‘heparin-like’ component was obtained from an exopolysaccharide produced by a mesophilic strain found in deep-sea hydrothermal vents. Data concerning the structure of the native high-molecular-weight exopolysaccharide (106 g/mol, 10% sulfate content) are reported for the first time. Two depolymerization processes were used to obtain low-molecular-weight (24–35×103 g/mol) oversulfated fractions (sulfate content 20 or 40%). Nuclear magnetic resonance studies indicated that after sulfation (40%), the low-molecular-weight fraction obtained by free radical depolymerization was less sulfated in the 6-O-position than the fraction depolymerized by acid hydrolysis. The free radical depolymerized product also had sulfated residues in the 4-O-position and disulfated ones in the 2,3-O-positions. Moreover, the compounds generated by the free radical process were more homogeneous with respect to molecular mass. Also for the first time, the anticoagulant activity of the low-molecular-weight exopolysaccharide fractions is reported. When the fractions obtained after sulfation and depolymerization were compared with heparins, anticoagulant activity was detected in oversulfated fractions, but not in native exopolysaccharide. The free radical depolymerized fraction inhibited thrombin generation in both contact-activated and thromboplastin-activated plasma, showing a prolonged lag phase only in the contact-activated assay. Affinity co-electrophoresis studies suggested that a single population of polysaccharide chains binds to antithrombin and that only a subpopulation strongly interacts with heparin cofactor II.
Keywords: Affinity co-electrophoresis; Anticoagulant activity; Depolymerization; Heparin-like; Serpin; Sulfated polysaccharide; Sulfation;

Detection and isolation of minisatellite Pc-1 binding proteins by Hirokazu Fukuda; Takashi Sugimura; Minako Nagao; Hitoshi Nakagama (152-158).
Minisatellites (MNs) are arrays of 5–100 nucleotide repeats that are dispersed throughout the genome of vertebrates. They demonstrate alteration in tumors and in cells exposed to various carcinogens, but the molecular mechanisms underlying the induction of mutations at MNs are largely unknown. Hypervariable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have found that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the molecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB, pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid. While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bound to the complementary C-rich strand. Sequence specificities for DNA binding were revealed and one base substitution and insertion into the Pc-1 repeat unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo.
Keywords: Minisatellite; Repetitive sequence; DNA binding protein; Variable number of tandem repeats;

Mechanisms of ascorbic acid recycling in human erythrocytes by James M May; Zhi-chao Qu; Jason D Morrow (159-166).
Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of d-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM d-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by d-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75–90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of d-glucose metabolism in the pentose cycle, but that in the absence of d-glucose DHA reduction can also utilize NADH.
Keywords: Ascorbic acid; Ascorbate recycling; Pentose cycle; Diethylmaleate; Human erythrocyte;

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4–P4′ site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa ∼3–4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (∼2–3×102 M−1 s−1). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300–500-fold. A similar 104–105-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4–P4′ residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4–P4′ binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.
Keywords: Factor Xa; Prothrombin; Prothrombinase; Antithrombin; Serpin;

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers by Paolo Caliceti; Santina Maria Quarta; Francesco Maria Veronese; Gennara Cavallaro; Elisa Pedone; Gaetano Giammona (177-186).
Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27 000–34 000 Da, and the polydispersivity was in the range of 1.5–1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15–25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.
Keywords: Polyaspartamide derivative; Amphiphilic copolymer; Polymeric carrier; Pharmacokinetics; Biodistribution;

Production of EGF–collagen chimeric protein which shows the mitogenic activity by Masahiro Hayashi; Masahiro Tomita; Katsutoshi Yoshizato (187-195).
Collagen has been utilized as a natural biomaterial because of its high biocompatibility, adhesiveness to cells and tissues, and biodegradability. The present study developed a recombinant technology to confer a mitogenic activity on type III collagen by fusing it to epidermal growth factor (EGF) at the collagen’s N-terminus. The chimeric protein of EGF–collagen was synthesized in insect cells by the baculovirus-insect cell expression system. The fusion protein was shown to hold the triple helical conformation of collagen and the mitogenic activity of EGF. It was also demonstrated that the chimeric protein can be immobilized on tissue culture dishes as a fibrous form and in collagen fibrils without abolishing the original mitogenic activity of EGF. This fusion protein can be utilized as a biocompatible, biodegradable, and adhesive fibrous mitogen for a variety of purposes in the area of tissue engineering.
Keywords: Recombinant protein; Fusion protein; Type III collagen; Biomaterial; Growth factor;

A serum fucolectin isolated and characterized from sea bass Dicentrarchus labrax by Matteo Cammarata; Mirella Vazzana; Cinzia Chinnici; Nicolò Parrinello (196-202).
A lectin specific for fucose and galactose was isolated by affinity chromatography on Sepharose CL-6B from the serum of Dicentrarchus labrax. The hemagglutinating activity against rabbit erythrocytes was calcium-independent, and reached its maximum at 37°C. Two protein components were found in the hemagglutinating fractions eluted from the Sepharose column. Only the 34 kDa component (DLL2) eluted from the polyacrylamide gels (SDS–PAGE) showed agglutinating activity against rabbit erythrocytes. SDS–PAGE, in non-reducing conditions, revealed a single 66 kDa protein that reacted with antibodies to the 34 kDa component. Therefore, a dimeric structure stabilized by disulfide bonds can be proposed. The Ca2+-independent fucose-binding specificity, a significant amino acid sequence homology of the N-terminal trait, and cross-reaction of eel fucolectin with antibodies to DLL2 suggest that this lectin may be included in the recently identified fucolectin family.
Keywords: Fish; Serum; Hemagglutinin; Fucolectin; Dicentrarchus labrax;

Contents Vol.1528 (205-206).

Announcement (207).