ABSTRACT
The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.
Subject(s)
Receptors, Cell Surface/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Clone Cells , Fluorescent Antibody Technique , Glycosylphosphatidylinositols/metabolism , Humans , Kinetics , L Cells , Ligands , Mice , Molecular Sequence Data , Mutagenesis , Mutagenesis, Insertional , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoric Diester Hydrolases/biosynthesis , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/pharmacology , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/isolation & purification , Receptors, Urokinase Plasminogen Activator , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell-cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down-regulation of uPAR by the uPA-PAI-1 complex was observed only from the apical membrane.
Subject(s)
Cell Membrane/chemistry , Cell Polarity , Receptors, Cell Surface/analysis , Receptors, Cell Surface/biosynthesis , 3T3 Cells , Animals , Cell Adhesion , Cell Line , Dogs , Down-Regulation , Epithelial Cells , Glycosylphosphatidylinositols , Humans , Kidney/cytology , L Cells , Mice , Molecular Weight , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , TransfectionABSTRACT
Four conserved disulfide bonds and N-linked and O-linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1-3, 2-7, 4-5 and 6-8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N-linked glycan composed only of GlcNAc and Man at residue Asn142, and two O-linked glycans near the C-terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N-linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man3 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C-terminal peptide of TvMP2 indicated the presence of five Man residues in O-linked glycans. No phosphate was found in these fungal peroxidases.
Subject(s)
Disulfides/metabolism , Peroxidases/metabolism , Polyporaceae/enzymology , Amino Acid Sequence , Glycosylation , Molecular Sequence Data , Phosphorylation , Polysaccharides/metabolism , Sequence Homology, Amino AcidABSTRACT
A veratryl alcohol oxidase (VAO) enzyme was discovered in cultures of Pleurotus ostreatus. The enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O2 to H2O2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprotein and contains FAD as a prosthetic group. The amino acid composition and carboxy- and amino-terminal sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (t1/2(55 degrees C) about 1.5 h, apparent melting temperature about 60 degrees C). The enzyme stability in 50% water/organic solvents mixtures has also been studied.
