Identity of Urinary Trypsin Inhibitor-binding Protein to Link Protein

Abstract

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs Urinary trypsin inhibitor (UTI)1 is a Kunitz-type protease inhibitor that is responsible for the inhibition of several proteases in serum and urine as well as in amniotic fluid (1). We (2-4) and others (5, 6) have found that UTI can directly bind to neoplastic cells as well as to non-neoplastic cells via cell-associated UTI-binding proteins (UTI-BPs) or specific UTI receptors. We recently reported that one of the proteins of the UTI-BP family is a pericellular matrix-associated glycoprotein of ϳ40 kDa (UTI-BP 40 ) that is thought to be very similar to human link protein (LP) (4). Our previous finding demonstrated that UTI may be able to bind hyaluronic acid via the LP molecule since UTI fails to directly bind hyaluronic acid (7). Link protein is present on a wide variety of cells, including skin and fibroblasts (8), chondrocytes (9), chondrosarcoma cells (10 -13), synovial cells (14), aorta (15), trachea (16), and hepatocytes (17-19). Characteristics of the LP molecule have been studied by a number of different laboratories (20, 21), and it has been shown to mediate the interaction between proteoglycans and hyaluronic acid (22, 23), a characteristic that may allow it to demonstrate pericellular matrix formation and stabilization (hyaluronic acid-rich matrix formation) (17). Several studies have suggested that a proteoglycan tandem repeat (PTR), found in most of the hyaluronic acid-binding molecules including LP and aggrecan, acts as a functional site of interaction with hyaluronic acid (24 -26).This study was undertaken to define more clearly the relationship between proteins of the UTI-BP family and the LP molecule in the human chondrosarcoma cell line HCS-2/8. For this, we first purified proteins of the UTI-BP family from HCS-2/8 cells on a large scale. Sequencing of tryptic fragments of UTI-BP 40 , chromatographic and electrophoretic examination, and comparison of UTI-binding properties have revealed the identity of UTI-BP 40 to LP. We then tested a variety of antibodies raised against LP and the hyaluronic acid-binding region (HA-BR) of aggrecan proteoglycan to determine whether anti-LP and anti-HA-BR antibodies cross-react with proteins of the UTI-BP family. In addition, the domain-specific antibodies to LP synthetic peptides were used as probes in structural analyses of the LP molecule. Finally, we studied the binding and competition effect of UTI fragments or LP subdomains on the solid-phase binding in attempts to localize ligand sites in the UTI structure and the binding part of the LP molecule. MATERIALS AND METHODSCells and Culture Conditions-The human chondrosarcoma cell line HCS-2/8 (provided by one of us (M. T.)) (27, 28) was grown and cultured as described previously (29 -31). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM HEPES buffer (Life Technologies, Inc.), 2.5 mM glucosamine, 3 mM glutamine (Life...

A Kunitz-type Protease Inhibitor, Bikunin, Inhibits Ovarian Cancer Cell Invasion by Blocking the Calcium-dependent Transforming Growth Factor-β1 Signaling Cascade

“…The differences in bikunin sensitivity could be the result of variability in drug metabolism, a higher dependence on calciumsensitive functions for cell invasiveness in certain types of cells, or a variability of the amounts of bikunin receptors on tumor cell membrane (37,38). There appeared to be a specific interaction between bikunin and the tumor cell surface.…”

Genetic Down-regulation of Phosphoinositide 3-Kinase by Bikunin Correlates with Suppression of Invasion and Metastasis in Human Ovarian Cancer HRA Cells

“…2 Furthermore, we showed that there is a direct correlation between bikunin overexpression and the reduced metastatic potential of primary tumor biopsies (12) and tumor cell lines as reflected by their in vitro potential to invade through a Matrigel TM barrier. 2 We have shown that bikunin is part of the negative invasive program as evaluated by their invasion and uPA/uPAR synthesis (13)(14)(15)(16).…”

Kunitz-type Protease Inhibitor Bikunin Disrupts Phorbol Ester-induced Oligomerization of CD44 Variant Isoforms Containing Epitope v9 and Subsequently Suppresses Expression of Urokinase-type Plasminogen Activator in Human Chondrosarcoma Cells

“…We have established that LP can interact with the NH 2 -terminal domain of bik, while bik-R is able to recognize the C4S side chain of bik (15,17). Analysis of binding of native bik and deglycosylated bik to the cells showed that the low affinity binding site is LP and the high affinity site is bik-R (17).…”

“…To more precisely map the regions in the bik that can interact with LP and bik-R, various truncated proteins were tested in the solid-phase binding and ligand blot assays (15,17). We have established that LP can interact with the NH 2 -terminal domain of bik, while bik-R is able to recognize the C4S side chain of bik (15,17).…”

“…We have been studying the function and mechanism of bik on suppression of the invasive capacity of tumor cells. During the search for novel proteins interacting with bik, we identified at least two types of cell-associated binding protein; the 40-kDa link protein (LP), one of the hyaluronic acid (HA)-binding proteins, as a cell-associated bik-binding protein, and a 45-kDa specific receptor for bik (bik-R), which is a membraneassociated unidentified molecule, as a putative bik receptor (bik-R) (13)(14)(15)(16)(17). We postulate that LP is the principal extracellular matrix-binding protein and acts as ligand sink for bik.…”