The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.     

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen

Malignant human gliomas are the most common forms of primary tumors in the central nerve system. Due to their location and invasive nature, treatment so far has been mainly palliative. Thus, understanding the molecular detail of tumor transformation and progression is crucial for developing effective therapeutic strategy for this fetal tumor. Among the genetic alternations found in these tumors, p53 inactivation and PDGF/PDGFR activation represent the early events, and the loss of chromosome 10 and gene amplification and rearrangement of EGFR represent the late events. Studies with both glioma cell lines and primary tumor tissues have strongly suggested that TGF-alpha and EGFR function as an important autocrine loop in supporting proliferation of human glioma, especially in high grade glioma, since elevated TGF-alpha expression is also found in these high grade tumors. Furthermore, down regulation of the expression of TGF-alpha by antisense constructs has been shown to inhibit several types of human tumor cell growth including glioma. Other means of therapeutic approaches using this autocrine loop as a target also include the use of monoclonal antibodies and their cytotoxic conjugated. Considerable understanding of the EGFR-mediated signal transduction pathways has become available recently, which including GRB2/mSOS1 mediated MAP kinase activation; JAK/STATs pathway; PLC-gamma pathway. However, much work still needs to be done before a specific component of these pathways can be applied for effective control of tumor growth in the clinic.     

Subunit of glycosylation-inhibiting factor is an abundant protein that binds to certain glycoproteins and sugars

The adherence of Staphylococcus aureus to biomaterials used in orthopaedic surgery (polymethylmethacrylate, fresh bone, steel and titanium alloys) and to glass was studied in vitro at 1, 2, 6, 24 and 48 h of incubation. Nonslime-producing strains (72, 80 and 510) and slime-producing variants of these strains were used. An automated and fast method of ATP-bioluminiscence was applied to determine bacterial viability. The lowest adherence corresponded to polymethylmethacrylate and bone, and the highest to metals. Significant adherence was detected in all cases after 6 h and was strain dependent, being lowest for strain 72. In most cases, adherence of nonslime-producing variants was not significant compared with controls, and slime-producing were more adherent than nonslime-producing variants. These differences were maximal at 6 h or 48 h, depending on the strain and the material. The findings suggest that the appearance of slime-producing cells within a given nonslime-producing bacterial population may jeopardise postoperative immune systems and antibiotic efficacy as a consequence of biofilm formation on implants and prostheses.     

Characterization and expression of a new class of zinc finger protein that binds to silencer region of ascorbate oxidase gene

A unique A/T-rich sequence (5'-AAAAAGTAAAAA-GTAAAAAAGTAAAAAG-3), referred to as the AGTA repeat, is found in the silencer region of the pumpkin ascorbate oxidase gene. A cDNA for protein (AOBP) that binds to the AGTA repeat was isolated from pumpkin by the southwestern method. The AOBP protein has a new class of zinc/DNA-binding domain named Dof/MOA domain that is highly conserved in many plant proteins and is significantly related to those of steroid hormone receptors and GATA1. Gel retardation analysis indicated that AOBP bound to the AGTA repeat through the Dof/MOA domain. Metal chelators, 1,10-phenanthroline and EDTA, specifically inhibited the DNA binding of AOBP, indicating that metal coordination plays an important role in DNA binding of AOBP. Thus, the Dof/MOA domain acts as a zinc/DNA-binding domain in AOBP. Gel retardation analysis with mutated oligonucleotides suggested that the Dof/MOA domain recognized the AGTA core sequence. AOBP mRNA was expressed in mature tissues of pumpkin, but was expressed only in small amounts or was not expressed in growing tissues. Furthermore, the expression was auxin-independent. The expression pattern of AOBP and that of ascorbate oxidase did not show a positive correlation.     

Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423

A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting.     

Development of an antipeptide antibody that binds to the C-terminal region of human CYP1B1

An antipeptide antibody was raised against a 14-mer synthetic peptide (CDFRANPNEPA KMN) corresponding to the amino acid sequence from 491 to 504 of human cytochrome P-450 (CYP)1B1. Rabbit-derived antisera demonstrated the ability to induce moderately high antibody titers (>1:10(5)) as judged by enzyme-linked immunosorbent assay. In Western blot analysis, the purified antibody recognized a single protein band (estimated as 56 kDa) in microsomes prepared from human and rodent tissues. No significant cross-reactivity to either human CYP1A1 or human CYP1A2 protein was detected. Titration studies using recombinant human CYP1B1 and an enhanced chemiluminescence-based detection method demonstrated a minimal detection sensitivity for this antiserum at about 0.34 ng/band in 8 x 7-cm minigels. The immunoprecipitation and immunoinhibition results indicate that this antisera recognizes the nondenatured human CYP1B1 protein but does not inhibit its enzyme activity. Using this antibody, CYP1B1 protein was detected in nine different human tissues and in cultured cells induced by various chemicals. This highly specific, highly sensitive antibody provides an important tool to study tissue distribution and cellular expression levels of CYP1B1, with negligible cross-reactivity from the other members of the CYP1 family.     

Taurine depletion in the intact animal stimulates in vitro phosphorylation of an approximately 44-kDa protein present in the mitochondrial fraction of the rat heart

Sprague-Dawley rats were treated with 1.5% guanidinoethanesulfonic (GES) acid in their water in order to deplete the taurine levels partially in cardiac tissue. After 6 weeks of GES treatment, in vitro phosphorylation of a approximately 44 kDa protein present in the mitochondrial fraction of the rat heart was increased by 85%. The increase in the in vitro phosphorylation of the specific approximately 44 kDa protein after GES treatment was reversed when the animals were subsequently given 1.5% taurine in their drinking water for an additional 6 weeks. Taurine (1.5%) alone for a period of 6 weeks had no effect on the phosphorylation of the approximately 44 kDa protein. These results suggest that taurine has a regulatory role in the phosphorylation of a specific protein in cardiac tissue.     

Characterization of an RNA-a protein complex isolated from the RNA bacteriophage M12

The properties of an RNA-A protein complex isolated from the RNA bacteriophage M12 are described. The molar ratio of RNA to A protein in the complex is estimated to be 1:1. In sucrose gradients, the complex sediments like free RNA molecules. In contrast to RNA alone, which can only infect spheroplasts, the RNA-A protein complex infects intact Escherichia coli cells and produces infectious progeny particles like the original phage. Evidence is presented that the infection of the host cells by the complex takes place via F pili. All of the infectivity disappears if the ionic bonds of RNA to A protein in the complex are dissociated in 0.5 M sodium chloride buffer at 37 degrees C. Furthermore, the kinetics of complex dissociation and loss of infectivity are the same, implying that the binding of A protein to the RNA is a prerequisite for infectivity on intact host cells.     

Characterization of an ochratoxin-A-dedifferentiated and cloned renal epithelial cell line

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with predominant nephrotoxic action. OTA impairs postproximal renal electrolyte handling and increases the incidence of renal adenoma and carcinoma. Furthermore, it is supposed to be involved in the pathogenesis of different forms of human renal diseases. Previously we have shown that OTA activates extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the C7 clone but not in the C11 clone of renal epithelial MDCK cells. Here we show that nanomolar concentrations of OTA lead to stable and irreversible phenotypical and genotypical alterations, resulting in sustained dedifferentiation of MDCK-C7 cells but not of MDCK-C11 cells. Dedifferentiated MDCK-C7 cells (OTA-C7 cells) display a distinct morphology from the parent cell line (spindle-shape, pleiomorphic, narrow intercellular spaces, increased cell size) and show a reduced proliferation rate and numerical chromosomal aberrations. Functionally, OTA-C7 cells are characterized by a dramatic reduction of transepithelial electrolyte transport and the complete loss of responsiveness to the mineralocorticoid hormone aldosterone. Our data provide further evidence that OTA can lead to cell dedifferentiation and eventually to transformation of cloned quiescent cells. The changes in phenotype due to this dedifferentiation could explain some of the OTA-induced changes in renal function.     

Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.     

Expression and characterization of a 70-kDa fragment of the insulin receptor that binds insulin. Minimizing ligand binding domain of the insulin receptor

In order to characterize regions of the insulin receptor that are essential for ligand binding and possibly identify a smaller insulin-binding fragment of the receptor, we have used site-directed mutagenesis to construct a series of insulin receptor deletion mutants. From 112 to 246 amino acids were deleted from the alpha-subunit region comprising amino acids 469-729. The receptor constructs were expressed as soluble insulin receptor IgG fusion proteins in baby hamster kidney cells and were characterized in binding assays by immunoblotting and chemical cross-linking with radiolabeled insulin. The shortest receptor fragment identified was a free monomeric alpha-subunit deleted of amino acids 469-703 and 718-729 (exon 11); the mass of this receptor fragment was found by mass spectrometry to be 70 kDa. This small insulin receptor fragment bound insulin with an affinity (Kd) of 4.4 nM, which is similar to what was found for the full-length ectodomain of the insulin receptor (5.0 nM). Cross-linking experiments confirmed that the 70-kDa receptor fragment specifically bound insulin. In summary we have minimized the insulin binding domain of the insulin receptor by identifying a 70-kDa fragment of the ectodomain that retains insulin binding affinity making this an interesting candidate for detailed structural analysis.     

Molecular characterization of an anchor protein (AKAPCE) that binds the RI subunit (RCE) of type I protein kinase A from Caenorhabditis elegans

Classical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in the cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In Caenorhabditis elegans, a single type I PKA mediates all aspects of cAMP signaling. We have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE) of C. elegans PKAICE. AKAPCE is a novel, highly acidic RING finger protein composed of 1,280 amino acids. It binds RI-like RCE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE.RCE complexes. The RCE-binding site was mapped to a segment of 20 amino acids in an N-terminal region of AKAPCE. Several hydrophobic residues in the binding site align with essential Leu and Ile residues in the RII-selective tethering domain of prototypic mammalian AKAPs. However, the RCE-binding region in AKAPCE diverges sharply from consensus RII-binding sites by inclusion of three aromatic amino acids, exclusion of a highly conserved Leu or Ile at position 8 and replacement of C-terminal hydrophobic amino acids with basic residues. AKAPCE.RCE complexes accumulate in intact cells.     

Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin

Moraxella (Branhamella) catarrhalis is a gram-negative human mucosal pathogen, which primarily causes otitis media in young children. However, this bacterium is also a common cause of lower respiratory tract infections in adults with underlying lung disease. Our previous data have shown that M. catarrhalis expresses iron-repressible outer membrane proteins in response to iron limitation. We have extended these observations to demonstrate that one of these proteins, termed outer membrane protein (OMP) B1, binds human transferrin. Using a newly developed monoclonal antibody to OMP B1, we determined that this protein is conserved in the iron-stressed outer membranes of all clinical isolates of M. catarrhalis tested to date. Furthermore, our data have confirmed that children infected with M. catarrhalis have immunoglobulin G antibodies to OMP B1 in their convalescent sera. These current data suggest that OMP B1 is immunogenic and expressed in vivo and may be involved in an iron uptake mechanism utilized by M. catarrhalis.     

Characterization of the 2H12 antigen as a nonshuttling human isoelectric variant of the nucleolar protein B23

It has become obvious that a better understanding of the nucleolar compartment should encompass the elucidation of structural and functional relationships between its molecular constituents. Using a mouse monoclonal antibody referred to as 2H12, we have identified a human epitope that appears to be implicated in the regulatory events governing the elaboration and stabilization of the nucleolar architecture. By immunofluorescence and immunoblotting, the 2H12 monoclonal was shown to be directed against a nucleolar protein with a relative mobility of 38-40 kDa and an isoelectric point of 5.1 that is present in human cells, regardless of their proliferation state. No reactivity was detected in cells from other species, implying that the targeted epitope could be unique to humans. Investigation of the fate of the epitope throughout the cell cycle led to evidence that its immunoreactivity was phosphodependent and suggested that the disassembly and reassembly of the nucleolar apparatus during cell division is accompanied by dephosphorylation/phosphorylation modifications at this site. In a series of double immunofluorescence experiments and two-dimensional immunoblotting analyses, it was demonstrated that the 2H12 antigen corresponds to an isoelectric variant of the human nucleolar protein B23 that is most prominent during interphase. Tightly associated with the nuclear matrix, this human B23 isoelectric variant did not shuttle between the nucleus and the cytoplasm but remained sequestered within the human nucleolus during mobility assays in human-murine heterokaryons.     

Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12

Natural killer cell and T cell subsets express at their cell surface a repertoire of receptors for MHC class I molecules, the natural killer cell receptors (NKRs). NKRs are characterized by the existence of inhibitory and activating isoforms, which are encoded by highly homologous but separate genes present in the same locus. Inhibitory isoforms express an intracytoplasmic immunoreceptor tyrosine-based inhibition motif, whereas activating isoforms lack any immunoreceptor tyrosine-based inhibition motif but harbor a charged amino acid residue in their transmembrane domain. We previously characterized KARAP (killer cell activating receptor-associated protein), a novel disulfide-linked tyrosine-phosphorylated dimer that selectively associates with the activating NKR isoforms. We report here the identification of the mouse KARAP gene, its localization on chromosome 7 and its genomic organization in five exons. Point mutation and transfection studies revealed that KARAP is a novel signaling transmembrane subunit whose transduction function depends on the integrity of an intracytoplasmic immunoreceptor tyrosine-based activation motif. In contrast to previous members of the immunoreceptor tyrosine-based activation motif polypeptide family, KARAP is ubiquitously expressed on hematopoietic and nonhematopoietic cells, suggesting its association with a broad range of activating receptors in a variety of tissues.     

Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.     

Characterization of group B streptococcal invasion of human chorion and amnion epithelial cells In vitro

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779-784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171-177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.     

Isolation and characterization of a 14.5-kDa trichloroacetic-acid-soluble translational inhibitor protein from human monocytes that is upregulated upon cellular differentiation

A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to protein sequencing. The full-length cDNA of the protein was cloned and sequenced from a lambda gt11 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057c/YJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation-dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.     

Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4

CDC37, an essential gene in Saccharomyces cerevisiae, interacts genetically with multiple protein kinases and is required for production of Cdc28p/cyclin complexes through an unknown mechanism. We have identified mammalian p50Cdc37 as a protein kinase-targeting subunit of the molecular chaperone Hsp90. Previously, p50 was observed in complexes with pp60v-src and Raf-1, but its identity and function have remained elusive. In mouse fibroblasts, a primary target of Cdc37 is Cdk4. This kinase is activated by D-type cyclins and functions in passage through G1. In insect cells, Cdc37 is sufficient to target Hsp90 to Cdk4 and both in vitro and in vivo, Cdc37/Hsp90 associates preferentially with the fraction of Cdk4 not bound to D-type cyclins. Cdc37 is coexpressed with cyclin Dl in cells undergoing programmed proliferation in vivo, consistent with a positive role in cell cycle progression. Pharmacological inactivation of Cdc37/Hsp90 function decreases the half-life of newly synthesized Cdk4, indicating a role for Cdc37/Hsp90 in Cdk4 stabilization. This study suggests a general role for p50Cdc37 in signaling pathways dependent on intrinsically unstable protein kinases and reveals a previously unrecognized chaperone-dependent step in the production of Cdk4/cyclin D complexes.