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.
Highlights
Development of the nervous system requires neurons to migrate and to extend axons over long distances from their sites of origin to their intended targets in the peripheral and the central nervous system
The Carboxyl Terminus of F-spondin Is Cleaved by Plasmin—To test whether F-spondin is cleaved by type plasminogen activator (tPA), we incubated conditioned medium of carboxyl-terminal Myc-tagged Fspondin (Fig. 1A) together with elements of the tPA proteolysis complex
This activity is probably mediated by the tPA that is produced by HEK293 ⌻ cells [30, 31]
Summary
The PCR products were subcloned into a suitable plasmid (cloning vector) into the restriction sites indicated in the table. Conditioned medium was collected after 2– 4 days and treated with the appropriate reagents at 37 °C for 1 h. For plasmin cleavage assays, conditioned medium was treated with plasmin (Chromogenix, Sweden) at the indicated concentrations and the chromogenic substrate specific for plasmin, S-2251 (Val-Leu-Lys-p-nitroanilide, Chromogenix) in 100 mM Tris-HCl, pH 7.4, in a final volume of 100 l, in microtiter plates for 1 h at 37 °C. Conditioned medium of transfected HEK293 ⌻ cells was incubated on the ECM for 90 min at room temperature. The reactions were stopped by adding 10 g/ml aprotinin, incubated in 96-wells plates covered with ECM, for 90 min. Proteins bound to the ECM were detected using the colorimetric reagent 3,3Ј,5,5Ј-tetramethylbenzidine (Chemicon)
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