James W. Schilling

Glial tumor cell adhesion is mediated by binding of the FNIII domain of receptor protein tyrosine phosphatase β (RPTPβ) to tenascin C

The extracellular domain of receptor protein tyrosine phosphatase β (RPTPβ) is composed of several domains which mediate its interactions with distinct ligands present on the surface of either neurons or glial cells. Here, we demonstrate that the fibronectin type III domain (FNIII) of RPTPβ binds to glial tumor-derived cell lines and primary astrocytes. We used affinity purification to isolate several proteins that specifically bind to the FNIII domain of RPTPβ. One of these, a 240 kDa protein that was purified from U118MG glioblastoma cell, was identified as tenascin C based on the amino acid sequence of several tryptic peptides. The interaction of RPTPβ with tenascin C was found to mediate cell adhesion. Adhesion and spreading of SF763T astrocytoma cells expressing RPTPβ on tenascin C was specifically abolished by the addition of a soluble fragment containing the FNIII domain of the receptor. RPTPβ-dependent cell adhesion was mediated by binding to the alternatively spliced FNIII repeats A1,2,4 (TnfnA1,2,4) of tenascin C. Furthermore, COS cells expressing RPTPβ adhere to TnfnA1,2,4, while the parental cells did not. These results demonstrate that the FNIII domain of RPTPβ binds to tenascin C and suggest that RPTPβ present on glial tumor cells is a primary adhesion receptor system to the extracellular matrix.

Synthesis and characterization of the Kunitz protease-inhibitor domain of the β-amyloid precursor protein

Abstract To understand the pathological process by which amyloid is deposited in Alzheimers disease, it is important to characterize the proteolytic processing events of the β-amyloid precursor protein (β-APP) from which the amyloid-forming fragment is excised. A potentially important component in β-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of β-APP. We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in β-APP processing and amyloid formation. A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein. The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized. Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr. The reKPI is as active on a molar basis as the inhibitor-containing β-APP when assayed for inhibition of trypsin activity. Together these data suggest that reKPI protein is properly folded and lacking in modified aa. Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in β-APP function and metabolism, as well as in the proteolytic events involved in β-amyloid formation.