Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Complement receptor 1/CD35 is a receptor for mannan-binding lectin.

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.

Intercellular adhesion molecule 1 and beta2 integrins in C1q-stimulated superoxide production by human neutrophils: an example of a general regulatory mechanism governing acute inflammation.

OBJECTIVE: To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins in the production of superoxide (O2-) by C1q-stimulated human polymorphonuclear leukocytes (PMN). METHODS: PMN were pretreated with F(ab')2 fragments of monoclonal antibodies (mAb) that blocked or did not block beta2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O2- was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1. RESULTS: Blocking mAb to the shared beta2 integrin subunit, CD18, completely inhibited the O2- response triggered by immobilized C1q, while blocking mAb to the alpha subunits of the beta2 integrins each partially blocked the O2- response. PMN treated with C1q were found to activate the beta2 integrins lymphocyte function-associated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O2- production by PMN. CONCLUSION: beta2 integrin binding to an ICAM provided an essential costimulatory signal for O2-production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O2- production: a first signal that also activates PMN beta2 integrins, followed by a second, beta2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O2- would serve as a regulatory mechanism to limit the production of O2- to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing up-regulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor alpha, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.

In vitro and in vivo responses of murine granulocytes to human complement-derived, haemolytically inactive C5b67 (iC5b67).

Haemolytically inactive C5b67 (iC5b67), which was made from purified human components and decayed to a haemolytically inactive form, was evaluated as an agonist for murine leucocytes both in vitro and in vivo. In an in vitro assay, iC5b67 stimulated chemotaxis for both neutrophils purified from mouse bone marrow and splenic eosinophils of IL-5 transgenic mice. The stimulation was dose-dependent, with high dose inhibition. As with human neutrophils, iC5b67 also failed to up-regulate CR3 (CD11b/CD18) expression and to stimulate superoxide generation in murine bone marrow neutrophils, in vitro. In vivo, iC5b67 elicited an inflammatory response in a mouse model of pleuritis. A marked infiltration of neutrophils, which peaked at 4 h, was followed by an infiltration of eosinophils and mononuclear leucocytes. This inflammatory response was dose- and time-dependent. However, the protein concentration in the pleural wash fluid did not increase, indicating that iC5b67 did not induce a capillary leak. Although the infiltration of neutrophils could not be reproduced by pure C7 or human serum albumin (HSA), C5b6 did induce an influx of neutrophils. We were able to document the existence of C7, both antigenically and functionally, in pleural washes of normal mice, making it likely that the activity of C5b6 resulted from the in situ formation of C5b67 and iC5b67. The mouse model of pleuritis promises to be a useful in vivo system in which to evaluate the pro- and anti-inflammatory effects of iC5b67 that have been noted in vitro.

Partial amino acid sequence of gamma-46 gliadin.

We have determined the partial amino acid sequence (207 amino acids) of gamma-46 gliadin isolated from wheat cultivar Hardi. The molecular mass of the protein (Mr) estimated by electrospray mass spectrometry is 35191.3. The number of cysteine residues in gamma-46 gliadin was determined as a mass difference of the protein before and after reduction and alkylation with 4-vinylpyridine. It was shown that the protein has no free SH-groups, and all cysteine residues are involved in the formation of four disulfide bonds. The partial structure of gamma-46 gliadin was determined by N-terminal sequencing and sequencing of tryptic and chymotryptic peptides. The tryptic peptides were obtained by enzymatic hydrolysis of the protein, which was preliminarily reduced and immobilized at free SH-groups on thiopropyl-Sepharose 6B. The chymotryptic peptides were isolated by limited digestion of the native protein. The positions of cysteine residues, as well as surrounding amino acid sequences, are conserved in gamma-46 gliadin; this is typical of gliadins.

Complement receptor type 1 (CR1, CD35) is a receptor for C1q.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Serum amyloid P component forms a stable complex with human C5b6.

A 30,000 m.w. protein bound tightly to C5b6, which was formed by activating C7-depleted human serum with zymosan. The protein remained bound to the C5b6 complex during the isolation procedure for C5b6, including chromatography on lysine-Sepharose and an anion exchange resin. Following electrophoresis and electroblotting of the C5b6 complex to a polyvinylidene difluoride transfer membrane, the 30,000 m.w. protein was microsequenced. The 24 N-terminal amino acid sequence was determined and showed identity of the 30,000 m.w. protein with the serum amyloid P (SAP) component. The C5b6-SAP complex did not dissociate in the presence of 10 mM EDTA, which distinguishes SAP-C5b6 binding from SAP's usual Ca2+-dependent binding to other molecules. SAP, which was isolated from serum by chromatography, was able to bind to preformed C5b6, which had been assembled and isolated from purified components. Functionally, the C5b6-SAP could bind C7, and the resulting C5b67-SAP complex had only moderately lower specific hemolytic activity than that of C5b67. In addition, hemolytically inactive C5b67-SAP, such as hemolytically inactive C5b67, was chemotactically active for neutrophils, while isolated SAP had no effect on cell mobility. Because SAP reacts with other serum proteins and with cells, it is likely that the addition of SAP to terminal complement complexes may affect the fate of these complexes.

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

A peptide that stimulates phosphorylation of the plant insulin-binding protein. Isolation, primary structure and cDNA cloning.

The soybean seed basic 7S globulin (Bg) is capable of binding bovine insulin and insulin-like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4-kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half-cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C-terminal glycine like a number of animal peptide hormones, but not C-terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6-kDa peptide and a C-terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin-like growth factors, the leginsulin is a possible candidate for plant peptide hormones.

[Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies]. / Obratnaia transkriptaza virusa immunodefitsita cheloveka: klonirovanie, ékspressiia v Escherichia coli, ochistka fermenta i poluchenie monoklonal'nykh antitel.

To express HIV-1 reverse transcriptase in E. coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained. The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced. The purification of reverse transcriptase was carried out. The substantial proteolysis of reverse transcriptase during purification was shown. The purified preparation is predominantly, an active protein with Mr 57 kDa. Some properties of this protein differed from the reverse transcriptase isolated from HIV.

[X-ray structural study of leucine aminopeptidase with a resolution of 4 Angstroms]. / Rentgenostrukturnoe issledovanie leitsinaminopeptidazy s razresheniem 4 A.

An X-ray crystallographic structure determination has been carried out on bovine lens leucine aminopeptidase at 4.0 A resolution by using a combination of isomorphous replacement and solvent flattening. The two heavy atom derivatives used were obtained by soaking crystals in ethyl mercury chloride, which bound at four sites, and phenyl mercury acetate, which bound at one site in the monomer. The electron density map reveals that the enzyme hexameric oligomer, arranged in 32 symmetry, has a triangular barrel appearance and dimensions, of height 88 A and maximal width 118 A in barrel equatorial plane. Each subunit in an elongated ellipsoid of approximate length 92 A. Subunits contacts have been described. From an analysis of the map each subunit appears to contain some 36% alpha-helix and is organized into two distinct globular domains. Direct location of zinc cluster and competitive inhibitor binding site are presented.

[Isolation and characteristics of insulin-binding proteins from soybeans]. / Vydelenie i kharkteristika insulinsviazyvaiushchikh belkov iz soevykh bobov.

Two soybean insulin-binding proteins were isolated using affinity chromatography on insulin-Sepharose. Both proteins have molecular mass about 39 kDa and consist of two subunits linked by disulphide bonds. According to the amino acid composition and N-terminal sequences of the subunits, these proteins, characterized by the absence of free thiol groups and sugar residues, are variants of the previously described soybean basic 7S globulin. The blotted proteins as well as their subunits were shown to bind 125I-labelled bovine insulin. For one of the proteins and insulin, dissociation constant of 4.10(-9) M was measured. The existence of plant insulin-binding proteins suggests the insulin-like regulation in the plant metabolism.

[Identification of alpha-tropomyosin of the human myocardium by two-dimensional electrophoresis, and sequencing]. / Identifikatsiia alpha-tropomiozina serdechnoi myshtsy cheloveka dvumernym élektroforezom i sekvenirovaniem.

Two-dimensional electrophoresis of total cardiac muscle extracts allows the detection of about 200 protein fractions. In preliminary studies the fraction D-10 protein was characterized in terms of relative molecular mass, isoelectric point and quantitative composition as alpha-tropomyosin. The similarity of the protein to human alpha-tropomyosin was confirmed by the results of analysis of the N-terminal sequence of the D-10 protein eluate in a gas-phase sequencer.

Monoclonal antibodies reveal structural similarity between cytoskeletal filaments and a model nucleopeptide.

The cytoskeleton is considered to be a very important cellular component, playing a role in the interactions between different organelles. However, in many cases the true biochemical functions and some of the structural aspects of cytoskeletal filaments are still unknown. Several hybridoma cell lines producing monoclonal antibodies (Mab) against a complex of linear DNA with the tridecapeptide Dns-NH2-VVTGVKTGVKTVT-CO2H have been established. (Dns is 5-methylaminonaphthalene-1-sulphonic acid.) The organization of this complex, in which the DNA is in a compact form, has been well-characterized previously by physicochemical methods and electron microscopy. A monoclonal antibody, Mab 66/9, was selected for further study on the basis of its reactivity with the immunizing DNA-peptide complex and minimal reaction with DNA alone. In immunofluorescence studies with cultured cells, this Mab did not recognize any nuclear structures, but reacted with cytoskeletal intermediate filaments and with nuclear lamins. Thus, Mab 66/9 appears to define an epitope present in the immunizing DNA-peptide complex and in cytoskeletal elements. The epitope is probably determined by the conformation of the oligopeptide since it was not detected in denatured cell lysates. Our observations provide some indirect evidence in support of the DNA-binding properties of cytoskeletal components which have been hypothesized in some recent publications.

[The use of monoclonal antibodies against insulin for isolation of proteins inhibiting the cell growth]. / Ispol'zovanie monoklonal'nykh antitel k insulinu dlia vydeleniia belkov, ingibiruiushchikh kletochnyi rost.

Extracts of pig kidneys or germinated soya beans after preliminary steps were affinity chromatographied on Sepharose containing cyanogen bromide immobilized monoclonal antibodies to pig insulin. The material bound to the affinity column was separated by HPLC resulting in one homogeneous protein from each source. Both proteins have been shown to inhibit DNA synthesis in cultured embryonic human fibroblasts and VERO fibroblasts. The effect of pig kidney protein was potentiated by insulin. Soya and pig proteins were characterized by the following parameters: molecular weights of 8.5 and 10.3 kD, apparent constants of dissociation with rat liver plasma membranes of 4.7 x 10(-8) M and 9.8 X 10(-8) M, respectively. The soya proteins competed for the binding sites on plasma membranes with insulin whereas the pig protein did not. The N-terminal amino acid sequences of 20 residues were determined for both proteins. Comparison of these sequences with known protein sequences was performed. A 30-40% primary structure homology of the studied fragment of soya bean protein with the fragments of some oncogenic viruses proteins and transforming proteins was revealed.

[Isolation and study of the properties of an insulin-binding protein from the blood serum]. / Vydelenie i izuchenie svoistv insulinsviazyvaiushchego belka syvorotki krovi.

A question of the existence of blood proteins binding unmodified insulin has remained open for many years. This paper is devoted to the description of a method of isolation and characteristics of the insulin-binding protein (IBP) isolated from rat and human blood sera. The level of IBP in the serum was 0.12-0.15 mg/ml. The protein was referred to IgG basing on the data of electrophoresis under denaturing conditions, immunoelectrophoresis and immunoenzymatic analysis. The constant of protein association with insulin varied with regard to a source within 1.5.10(-7) to 2.5.10(7) M-1. The studied protein bound both 125I-modified and unmodified insulin preparations. The calculation based on IBP parameters, showed that no less than half of the blood insulin must be in a bound condition. The capacity of such insulin-binding system is rather great and is capable of leveling down sharp changes in a blood insulin concentration.

Histone crosslinking patterns indicate dynamic binding of histone H1 in chromatin.

Crosslinking of histone H1 molecules to each other and to the core histones with bifunctional reagents in mouse liver nuclei and chromatin was compared with that under the conditions of random 'contacts' between these molecules. The patterns of crosslinking of the H1 subfractions (H1A, H1B, and H10) to each other in nuclei, chromatin and in solution at different ionic strengths due to random collisions were essentially the same. Moreover, the contacts between the H1 molecules were qualitatively the same in nuclei, chromatin and in solution also at the level of the chymotryptic halves of the H1 molecules. The contacts between the H1 molecules and the core histones in nuclei were similar to those obtained in chromatin at 70 mM NaCl, when H1 molecules readily migrate, and at 0.6 M NaCl, when H1 molecules are dissociated from chromatin. We conclude that spatial arrangement of H1 subfractions and mutual orientation of H1 molecules in isolated nuclei are random-like at least in terms of cross-linking. The static and dynamic models of histone H1 binding to chromatin compatible with the known data are considered. Although unequivocal verification of the models is not possible at present, the dynamic models do correspond better to recent data on the location of the histone H1 in nuclei and chromatin.

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes.

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.

[Localization of histone H1 in chromatin. Cross-linking of the N- and C-terminal halves of the molecule with bifunctional reagents]. / Raspolozhenie gistona H1 v khromatine. Sshivanie bifunktsional'nym reagentom N- i C-kontsevykh polovin molekuly.

Mutual arrangement of histone H1 molecules was studied in calf thymus nuclei, extended chromatin and chromatin, isolated and kept in 8 M urea. Histone H1 dimers crosslinked with methyl 4-mercaptobutyrimidate were digested with chymotrypsin and crosslinked fragments obtained were analysed by diagonal gel electrophoresis. In all chromatins tested the N- and C-terminal parts of the H1 molecules were crosslinked in all possible combinations, i.e. C-C, C-N and N-N. These and related data obtained earlier indicate, that the proximity of histone H1 molecules in chromatin is determined by the structure of nucleosomal chain itself and not by chromatin superstructure. The results also suggest that the H1A and H1B subfractions of histone H1 are interspersed in extended nucleosomal chains.