ABSTRACT
Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.
Subject(s)
Extracellular Matrix/metabolism , Fibrinolysin/chemistry , Fibrinolysin/physiology , Growth Substances , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/metabolism , Peptides , Amino Acid Sequence , Binding Sites , Cell Division , Cell Line , Cell Movement , DNA/metabolism , Dose-Response Relationship, Drug , Extracellular Matrix Proteins , Fibrinolysin/metabolism , Gene Deletion , Humans , Molecular Sequence Data , Mutation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Reelin Protein , Sequence Homology, Amino Acid , Structure-Activity Relationship , Tissue Plasminogen Activator/metabolism , TransfectionABSTRACT
Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.
Subject(s)
Epitopes/analysis , Phosphopyruvate Hydratase/immunology , Receptors, Cell Surface/immunology , Amino Acid Sequence , Antibodies, Monoclonal , Binding Sites , Blotting, Western , Cell Line , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Models, Molecular , Molecular Sequence Data , Neutrophils/chemistry , Peptide Library , Phosphopyruvate Hydratase/analysis , Protein Conformation , Receptors, Cell Surface/analysis , Receptors, Urokinase Plasminogen Activator , Surface PropertiesABSTRACT
We have characterized a receptor for plasmin (Pli-R) from a human tumor cell line, MCF7MF. The Pli-R was purified from a MCF7 0.1% Triton X-100 solubilisate by affinity chromatography. A protein of 55-60 kDa was obtained, which bound plasminogen and plasmin specifically. Chemical cross-linking of M(r) 90 kDa [125I]-Pli to the surface of MCF7 cells with DSP results in the formation of a labelled complex of M(r) 145 kDa, suggesting a M(r) of 55-60 kDa for the receptor. Comparing Pli-R with alpha-enolase (a candidate for plasminogen receptor in U937 cells) we have found a high homology between both proteins, but not an identity.
Subject(s)
Breast Neoplasms/blood , Phosphopyruvate Hydratase/analysis , Receptors, Peptide/isolation & purification , Amino Acid Sequence , Amino Acids/analysis , Breast Neoplasms/enzymology , Cross-Linking Reagents , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Tumor Cells, CulturedABSTRACT
The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment. On unfixed cells, these methods gave the same heterogeneous patterns of surface staining, consisting of contours and grains, localized mainly at the upper nonadherent face of the tumor cells by confocal microscopy. Only a part of the cells was stained. When both receptors were characterized together, their presence was found on the same cells and they gave almost superimposable patterns in many cases, as shown by confocal microscopy. In contrast, when MCF 7 cells were fixed or permeabilized before staining, quite different patterns were observed: almost all the cells were labeled. The staining was mainly cytoplasmic and localized preferentially in the center and close to the upper face of the cells. Similar results were found with antiserum against urokinase receptor and monoclonal antivinculin antibody. It is likely that the receptor for urokinase and the receptor for plasmin have similar localizations on MCF 7 cells, thus resulting in a functional cooperation.
