O-glycosylation potential of lepidopteran insect cell lines.
(57/19351)
The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line. (+info)
Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress.
(58/19351)
Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress. (+info)
Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains.
(59/19351)
The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion. (+info)
Molecular cloning, characterization, and expression of the M antigen of Histoplasma capsulatum.
(60/19351)
The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2, 187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity. (+info)
Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor.
(61/19351)
Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation. (+info)
A differential requirement for the COOH-terminal region of the epidermal growth factor (EGF) receptor in amphiregulin and EGF mitogenic signaling.
(62/19351)
The epidermal growth factor receptor (EGFR) mediates the actions of a family of bioactive peptides that include epidermal growth factor (EGF) and amphiregulin (AR). Here we have studied AR and EGF mitogenic signaling in EGFR-devoid NR6 fibroblasts that ectopically express either wild type EGFR (WT) or a truncated EGFR that lacks the three major sites of autophosphorylation (c'1000). COOH-terminal truncation of the EGFR significantly impairs the ability of AR to (i) stimulate DNA synthesis, (ii) elicit Elk-1 transactivation, and (iii) generate sustained enzymatic activation of mitogen-activated protein kinase. EGFR truncation had no significant effect on AR binding to receptor but did result in defective GRB2 adaptor function. In contrast, EGFR truncation did not impair EGF mitogenic signaling, and in c'1000 cells EGF was able to stimulate the association of ErbB2 with GRB2 and SHC. Elk-1 transactivation was monitored when either ErbB2 or a truncated dominant-negative ErbB2 mutant (ErbB2-(1-813)) was overexpressed in cells. Overexpression of full-length ErbB2 resulted in a strong constitutive transactivation of Elk-1 in c'1000 but only slightly stimulated Elk-1 in WT or parental NR6 cells. Conversely, overexpression of ErbB2-(1-813) inhibited EGF-stimulated Elk-1 transactivation in c'1000 but not in WT cells. Thus, the cytoplasmic tail of the EGFR plays a critical role in AR mitogenic signaling but is dispensable for EGF, since EGF-activated truncated EGFRs can signal through ErbB2. (+info)
Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein.
(63/19351)
The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family. (+info)
A statistical analysis of N- and O-glycan linkage conformations from crystallographic data.
(64/19351)
We have generated a database of 639 glycosidic linkage structures by an exhaustive survey of the available crystallographic data for isolated oligosaccharides, glycoproteins, and glycan-binding proteins. For isolated oligosaccharides there is relatively little crystallographic data available. A much larger number of glycoprotein and glycan-binding protein structures have now been solved in which two or more linked monosaccharides can be resolved. In the majority of these cases, only a few residues can be seen. Using the 639 glycosidic linkage structures, we have identified one or more distinct conformers for all the linkages. The O5-C1-O-C(x)' torsion angles for all these distinct conformers appear to be determined chiefly by the exo-anomeric effect. The Manalpha1-6Man linkage appears to be less restrained than the others, showing a wide degree of dispersion outside the ranges of the defined conformers. The identification of distinct conformers for glyco-sidic linkages allows "average" glycan structures to be modeled and also allows the easy identification of distorted glycosidic linkages. Such an analysis shows that the interactions between IgG Fc and its own N-linked glycan result in severe distortion of the terminal Galbeta1-4GlcNAc linkage only, indicating the strong interactions that must be present between the Gal residue and the protein surface. The applicability of this crystallographic based analysis to glycan structures in solution is discussed. This database of linkagestructures should be a very useful reference tool in three-dimensional structure determinations. (+info)