Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity.

Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue C-terminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factor-driven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.     

Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.     

Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.

Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.     

Assay of protein kinase C with an N-bromosuccinimide-cleavage fragment of histone H1.

N-Bromosuccinimide cleavage of a lysine-rich histone fraction (histone III-S) yields a peptide substrate, purified by reverse-phase h.p.l.c., for the Ca2+ + phospholipid-dependent protein kinase (protein kinase C). This substrate displays no reactivity with the cyclic AMP-dependent protein kinase, and may prove useful for the detection of protein kinase C activity in crude tissue extracts.     

Partial characterisation of human and porcine adipose acidic protease activity.

1. An aspartic protease was isolated from human and porcine white adipose tissue and from isolated human adipocytes. The three preparations appeared to represent the same enzyme. 2. Electrofocusing of all three preparations revealed two bands corresponding to a pI of 3.6 and 4.4. respectively. On PAGE a single band in the same position was obtained in all three cases. 3. Both the porcine and human fractions were optimally active at pH 3.4, using acid denatured haemoglobin as substrate, and both activities were strongly inhibited by pepstatin and iodoacetate. 4. The Km values for haemoglobin for the porcine and human proteases were 0.16 and 0.14 mM respectively, whereas Vmax values of 30 and 33 units.nmol-1, respectively, were obtained.     

Anastellin,an FN3 fragment with fibronectin polymerization activity,resembles amyloid fibril precursors

Anastellin is a carboxy-terminal fragment of the first FN3 domain from human fibronectin. It is capable of polymerizing fibronectin in vitro, and it displays anti-tumor, anti-metastatic and anti-angiogenic properties in vivo. We have determined the structure of anastellin using nuclear magnetic resonance spectroscopy and identified residues critical for its activity. Anastellin exhibits dynamic fluctuations and conformational exchange in solution. Its overall topology is very similar to the corresponding region of full-length FN3 domains. However, its hydrophobic core becomes solvent-accessible and some of its beta-strands lose their protection against hydrogen bonding to beta-strands from other molecules. These features seem to be relevant for the fibronectin polymerization activity of anastellin and resemble the characteristics of amyloid fibril precursors. We suggest that this analogy is not random and may reflect similarities between fibronectin and amyloid fibril formation.     

Insulin-like growth factor characteristics of an acidic non-suppressible insulin-like activity.

The biological activities of an acidic form of non-suppressible insulin-like activity (ILA pI 4.8) have been studied. ILA pI 4.8 was isolated from Cohn fraction IV-1 of human serum by pH 5.5 ion-exchange chromatography on SP-Sephadex. Carrier-bound ILA was eluted at pH 9.7 and then sequentially gel chromatographed in 1% formic acid on Sephadex G-75 and Bio-Gel P-30. The low-Mr (7000) active material was subjected to flat bed isoelectric focusing. Overall recovery was 87 munit of insulin equivalents/100 g of Cohn fraction IV-1, with a specific activity in the range 4-10 munit/mg of protein, representing a purity of 1-6%. This material has been tested in a variety of insulin-like growth factor (IGF)/somatomedin assay systems. It stimulated, in a dose-related manner, [14C]glucose conversion into lipid by isolated rat adipocytes, 35SO4(2-) incorporation into weanling rat costal cartilage and [3H]thymidine incorporation into DNA of cultured human fibroblasts. Like IGF-I and -II, ILA pI 4.8 was able to inhibit degradation of 125I-insulin by crude homogenates of rat liver. In addition, the biological activity of ILA pI 4.8 was completely suppressible by a recently described inhibitor of IGF-I and IGF-II. ILA pI 4.8 was able to compete, in a parallel manner, with 125I-IGF-I and 125I-IGF-II and, at higher doses, with 125I-insulin in a placental radioreceptor assay. No cross-reactivity was seen in a radioimmunoassay for IGF-I and -II C-peptides, but at higher concentrations parallel displacement was observed in a somatomedin C/IGF-I radioimmunoassay using two different antisera. These data indicate that ILA pI 4.8 does possess many of the biological activities previously reported for the IGFs. Since ILA pI 4.8 does occur naturally in serum, it would appear reasonable to tentatively include it as one of the IGF/somatomedin family.     

Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif

The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.     

Synthetic insulin fragment with insulin-like biological activity.

An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.     

The C. elegans hyaluronidase: A developmentally significant enzyme with chondroitin-degrading activity at both acidic and neutral pH

Mammalian hyaluronidases degrade hyaluronan and some structurally related glycosaminoglycans. We generated a deletion mutant in the Caenorhabditis elegans orthologue of mammalian hyaluronidase, hya-1. Mutant animals are viable and grossly normal, but exhibit defects in vulval morphogenesis and egg-laying and showed increased staining with alcian blue, consistent with an accumulation of glycosaminoglycan. A hya-1::GFP reporter was expressed in a restricted pattern in somatic tissues of the animal with strongest expression in the intestine, the PLM sensory neurons and the vulva. Total protein extracts from wild-type animals exhibited chondroitin-degrading but not hyaluronan-degrading activity. Chondroitinase activities were observed at both neutral and acidic pH conditions while both neutral and acidic activities were absent in extracts from hya-1 mutant strains. We also evaluated the function of oga-1, which encodes the C. elegans orthologue of MGEA-5, a protein with hyaluronan-degrading activity in vitro. oga-1 is expressed in muscles, vulval cells and the scavenger-like coelomocytes. An oga-1 mutant strain exhibited egg-laying and vulval defects similar to those of hya-1; chondroitinase activity was unaffected in this mutant.     

Suppression of T lymphocyte functions by human C3 fragments. I. Inhibition of human T cell proliferative responses by a kallikrein cleavage fragment of human iC3b

Cleavage of human iC3b by kallikrein isolated from human plasma generates a fragment, C3d-K, which is capable of inhibiting mitogen-, antigen-, and alloantigen-induced T lymphocyte proliferation. Native C3, C3a, C3b, and C3c-K had no effect on lymphocyte proliferative responses. In addition to being a potent suppressor of mitogen- and antigen-induced proliferation, C3d-K is capable of inducing leukocytosis in both mice and rabbits. Intravenous injection of C3d-K, but not C3, C3a, C3b, or C3c-K, results in a twofold to threefold increase in the number of circulating leukocytes. Thus, C3d-K exhibits two apparently independent functions, namely suppression of T cell proliferation and leukocytosis. Cleavage of iC3b by kallikrein results in the production of only two fragments. The larger fragment, C3c-K, is 144,000 m.w. and has a chemical structure analogous to that of C3c obtained from the cleavage of C3 by trypsin or elastase. The smaller fragment, C3d-K, is 41,000 m.w. and contains the metastable binding site of C3. It is through this site located in the C3d region of the molecule that C3 attaches covalently to target cells. Analysis of the amino terminal region of C3d-K provided a sequence that fails to overlap with any sequence yet reported for other characterized C3 fragments, including C3d originally obtained from elastase digestion. A revised model of the C3 molecule is proposed, with locations of the C3e and C3d fragments assigned on the basis of chemical analyses.     

The ninth component of human complement (C9). Functional activity of the b fragment

The domain structure of human complement protein C9 was investigated by determining the functional activities of the NH2-terminal (C9a) and COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin. The two fragments were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured by dialysis against buffers containing zwitterionic detergents. The C9b fragment produced membranolytic activities in three independent assays. First, it produced single, ion-conducting channels of varying conductances in planar lipid membranes. Most of the channels had an average conductance of 11 picoSiemens and an average lifetime of about 30 s. The channels showed lipid specificity and a 3-fold preference for conducting K+ over Na+. Second, the fragment also caused specific marker release from liposomes which was inhibitable by a C9b-specific monoclonal antibody, and third, it lysed erythrocytes in the absence of a fully assembled C5b-8 complex. The isolated C9a fragment did not produce single channels in planar lipid membranes but was also effective in releasing markers from liposomes and in lysing erythrocytes. Secondary structure predictions indicate the presence of several amphiphilic, "surface-seeking" segments in the primary structure of C9 which are mainly alpha-helices in C9b and beta-sheets in C9a. These results may indicate the presence of surface-binding domains in the NH2-terminal half and channel-forming domains in the COOH-terminal portion of native, monomeric C9.     

Structural studies of an acidic galactoglucomannan from the O3 reference strain (C.D.C. 863-57) of Serratia marcescens

A partially acetylated acidic galactoglucomannan has been isolated from the lipopolysaccharide of the O3 reference strain (C.D.C. 863-57) of Serratia marcescens. By means of n.m.r. spectroscopy, methylation analysis, and degradative studies, the polymer was found to have the branched pentasaccharide repeating-unit shown. The position(s) of partial acetylation were not determined. Although the polymer is believed to confer O specificity on the parent organism, it is probably not an integral component of the lipopolysaccharide. (Formula: see text).     

On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment

The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.