Inter-α-trypsin Inhibitor Bound to Tumor Cells Is Cleaved into the Heavy Chains and the Light Chain on the Cell Surface

Abstract

Inter-alpha-trypsin inhibitor (ITI), a human serum protease inhibitor of molecular mass 240 kDa which may release physiological derivatives, has been shown to interact with hyaluronic acid (HA), resulting in pericellular matrix stabilization (Chen, L., Mao, S.J.T., McLean, L. R., Powers, R. W., and Larsen, W. J. (1994) J. Biol. Chem. 269, 28282-28287). The purpose of this study is to determine whether ITI binding to tumor cell surface is mediated by urinary trypsin inhibitor (UTI)-receptor or cell-associated hyaluronic acid (HA). We demonstrated specific complex formation of the heavy (H) chains of ITI with HA. Binding of the H-chains of ITI to immobilized HA was detected and quantified using colorimetric immunoassays. Binding was time-, temperature-, and concentration-dependent. However, UTI and HI-8 (the carboxyl terminus of UTI) failed to bind to immobilized HA. ITI bound to HA remained functional protease inhibiting activity. After incubation of SMT-cc1 cells with purified biotinylated ITI, biotinylated ITI is bound to the cells, dissociated, and gives rise to the H-chains and UTI on the cell surface. The cell surface receptor-bound UTI derived from ITI may be the result of the limited proteolysis on the cell surface. In the cells treated with hyaluronidase, bound H-chains disappeared from the surface of the cells, while most of the cell surface ITI derivatives was present in deglycosylated UTI (28 kDa). It is suggested that the binding of ITI to the cell surface is mediated by HA on the cells. This was confirmed by the fact that the hyaluronidase-treated cells can abolish the ITI binding. The cell surface UTI formation was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and eglin C, suggesting that elastase-like enzyme(s) may be responsible for the UTI formation. Preincubation of the cells with UTI did not decrease in exogenously added ITI on the cell surface. A model for cell surface UTI formation is proposed in which ITI binding to cells from serum used for the culture is followed by the limited proteolysis by trace amounts of active serine proteases, to form cell-surface receptor-bound UTI and the H-chains intercalated into cell surface HA. This process is subject to regulation of cell-associated UTI and of stabilization of pericellular matrix.