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PubMed1997

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions.

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

Animals↗

PubMed1998

Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389.

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs in vivo in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to gamma radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates in vivo a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.

Animals↗

PubMed1995

Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display.

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.

Amino Acid Sequence↗

PubMed2003

Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.

Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates. The serine protease granzyme B was uncovered as the 32-kDa Hsp70-interacting protein using matrix-assisted laser desorption ionization time-of-flight mass peptide fingerprinting. Incubation of tumor cells with increasing concentrations of perforin-free, isolated granzyme B shows specific binding and uptake in a dose-dependent manner and results in initiation of apoptosis selectively in tumor cells presenting Hsp70 on the cell surface. Remarkably, Hsp70 cation channel activity was also determined selectively in purified phospholipid membranes of Hsp70 membrane-positive but not in membrane-negative tumor cells. The physiological role of our findings was demonstrated in primary NK cells showing elevated cytoplasmic granzyme B levels following contact with TKD. Furthermore, an increased lytic activity of Hsp70 membrane-positive tumor cells could be associated with granzyme B release by NK cells. Taken together we propose a novel perforin-independent, granzyme B-mediated apoptosis pathway for Hsp70 membrane-positive tumor cells.

Apoptosis↗

PubMed1985

Isolation of an actin-binding protein from membranes of Dictyostelium discoideum.

We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.

Actins↗

PubMed2000

Repertoire dynamics of autoreactive T cells in multiple sclerosis patients and healthy subjects: epitope spreading versus clonal persistence.

Autoantigen-specific T-lymphocytes are present in patients with autoimmune disease and in normal subjects. Little is currently known about the temporal variation (dynamics) of the immune repertoire of these autoreactive T cells. We analysed the long-term variation of the immune repertoire of T cells specific for myelin basic protein (MBP) in five untreated patients with multiple sclerosis and four normal control subjects over a mean observation period of 6 years. MBP-specific CD4(+) T-cell lines were selected with purified human MBP, and their epitope specificity was mapped with overlapping synthetic peptides. Three distinct patterns of repertoire development were observed. (i) Two patients and three control subjects maintained a broad epitope response with fluctuations over time. (ii) Two patients initially showed a focused response that broadened over the course of 6 years; this finding could be described as intramolecular epitope spreading. (iii) In one patient and one control subject, a strikingly focused response, which was directed to a cluster of nested epitopes in the MBP region 83-102, persisted over time. T-cell receptor Vbeta sequence analysis allowed us to trace individual clones of MBP-specific T cells for up to 7 years in the peripheral circulation in four of the five patients and three of the four controls, suggesting that the long-term persistence of MBP-specific T-cell clones is a common feature of the T-cell repertoire not unique to multiple sclerosis. The persisting MBP-specific T-cell clones were not detectable in the blood of one of the patients by complementarity-determining region (CDR)-3 spectratyping, indicating that their frequency does not exceed 1 in 5000 T cells. The temporal characteristics of the MBP-specific T-cell repertoire described here are relevant to therapeutic strategies targeting autoantigen-specific T cells in multiple sclerosis and other autoimmune diseases.

Adult↗

PubMed1993

Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization.

A scheme for rapidly mapping chromosome rearrangements relative to the physical map of Caenorhabditis elegans is described that is based on hybridization patterns of cloned DNA on meiotic nuclei, as visualized by fluorescent in situ hybridization. From the nearly complete physical map, DNA clones, in yeast artificial chromosomes (YACs), spanning the rearrangement breakpoint were selected. The purified YAC DNAs were first amplified by degenerate oligonucleotide-primed polymerase chain reaction, then reamplified to incorporate fluorescein dUTP or rhodamine dUTP. The site of hybridization was visualized directly (without the use of antibodies) on meiotic bivalents. This allows chromosome rearrangements to be mapped readily if the duplicated, deficient or translocated regions do not pair with a normal homologous region, because the site or sites of hybridization of the probe on meiotic prophase nuclei will be spatially distinct. The pattern, or number, of hybridization signals from probes from within, or adjacent to, the rearranged region of the genome can be predicted from the genetic constitution of the strain. Characterization of the physical extent of the genetically mapped rearrangements places genetic landmarks on the physical map, and so provides linkage between the two types of map.

Animals↗

PubMed2003

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding.

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.

Amino Acid Sequence↗

PubMed1998

Characterization of lymphocytes in the adult rat testis by flow cytometry: effects of activin and transforming growth factor beta on lymphocyte subsets in vitro.

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.

Activins↗

PubMed2000

Absorption of milk-borne insulin-like growth factor-I into portal blood of suckling rats.

BACKGROUND: Insulin-like growth factors (IGFs) are potent mitogens that have been implicated in control of growth and development during the perinatal period. These hormones are also present in biologically significant quantities in mammalian milks. Although one site of action of these IGFs may be at the intestinal level, current information about whether they pass intact into the circulation is conflicting. METHODS: To test the hypothesis that milk-borne IGFs are absorbed into blood in receptor-active form, suckling rats were given either recombinant human (rh)125I-IGF-I or -II (4 x 10(6) counts per minute [cpm]), and the activity present in portal and cardiac blood was examined at 5, 10, 20, and 30 minutes after ingestion for presence of appropriate molecular weight peptides in these samples. In selected samples, purified radioactive samples were tested for their ability to bind competitively to crude membranes bearing IGF receptors. RESULTS: The results of these studies indicate that rh125I-IGF-I is absorbed in receptor-active form into the portal circulation and that maximal amounts are present 20 to 30 minutes after ingestion. Estimation of the presence of intact hormone was made on the basis of the elution profile of samples when run on gel chromatography as well as reversed-phase high-performance liquid chromatography. Isolated samples from portal blood also bound competitively to placental membranes bearing IGF receptors. In contrast, rh125I-IGF-II could not be demonstrated in receptor-active form in portal blood. Chromatography showed appropriate sized peaks with greater activity in portal than cardiac samples, but competitive binding was not appreciated. CONCLUSIONS: It is likely that at least milk-borne IGF-I is absorbed intact and may exert effects on liver and other peripheral tissues. In addition, this study lends further credence to the possibility of an enterohepatic circulation for IGF-I.

Animals↗

PubMed2001

Immunogenicity and protective role of an IgA reactive 31-kDa antigen of Vibrio cholerae O139.

Immunoglobulin A (IgA) is important in protective immunity against infection by Vibrio cholerae. In this study, the immune response to and protective role of a 31-kDa antigen of V. cholerae O139, reacting with IgA antibodies present in the sera of cholera patients and common to V. cholerae strains O139 and O1 was evaluated in BALB/c mice. From the various antigens of V. cholerae O139 and V. cholerae O1 which reacted with IgA antibodies in sera of a cholera patient, a 31-kDa common antigen was selected and purified by DEAE-Sepharose CL 6B column chromatography. Oral administration of live V. cholerae O139 in BALB/c mice elicited an IgA response to the 31-kDa antigen in serum and intestinal fluid, and a proliferation of the splenic lymphocytes on stimulation with the same antigen. The cytokine profile of these splenic lymphocytes revealed a shift from a mixed Th1 and Th2 response--interleukin-10 (IL-10) and interferon-gamma--in the first week after infection to a Th2 type of response--IL-10--in the third week. In passive protection studies, hyperimmune serum to the 31-kDa antigen was able to protect infant mice against challenge with O139 and O1 strains. These results demonstrate the ability of the 31-kDa antigen of V. cholerae O139 to induce humoral and cellular immune responses in mice, and its immunoprotective nature.

Animals↗

PubMed1989

Cytochrome mutants of bradyrhizobium induced by transposon tn5.

Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa(3) and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa(3). Bacteroids from one class of mutants were totally devoid of cytochrome aa(3). Several of these strains produced effective symbioses indicating that cytochrome aa(3) is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids.

Journal Article↗

PubMed2005

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins.

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.

Amino Acid Sequence↗

PubMed2005

Photo-induced oxidative cross-linking as a method to evaluate the specificity of protein-ligand interactions.

The isolation of protein-binding synthetic molecules from combinatorial libraries or compound collections is now a common practice in chemical biology. An important, but underdeveloped, aspect of characterizing the binding properties of such molecules is their level of binding specificity. This is often evaluated by simply measuring the equilibrium binding affinity of the compound of interest with its target protein and comparing this value with its affinity to one or a few other purified proteins selected at random. These measurements may not reflect accurately the ability of the compound to seek out its target in a complex mixture of proteins such as a cell extract or serum. A more desirable alternative would be to develop solution assays that measure directly the binding of the molecule of interest to both target and competitor proteins in complex solutions. In this report, we evaluate a rapid and efficient photo-triggered cross-linking reaction for assessing binding specificity of synthetic molecules in protein mixtures. Using peptide-protein complexes, we demonstrate that this reaction provides an unbiased view of the peptide-protein contacts present in solution under a given set of conditions and thus is useful for assessing binding specificity. We also discuss the potential application of this chemistry to the related, but more difficult, problem of the identification of protein targets of bioactive molecules.

Amino Acid Sequence↗

PubMed1990

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS-PAGE and immunoblotting.

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.

Actinobacillus↗

PubMed1989

Purification of cytochrome-c oxidase retaining its pulsed form.

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.

Animals↗

PubMed1992

Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts.

In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Monéger, R. (1982) Eur. J. Biochem. 127, 255-258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein-bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.

Amino Acid Sequence↗

PubMed2007

The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase.

Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.

Aminopeptidases↗