Abstract
Maspin (mammary serine protease inhibitor) was originally identified as a tumor suppressor protein in human breast epithelial cells and is a member of the serine proteases inhibitor (serpin) superfamily. It inhibits tumor cell motility and angiogenesis, and although predominantly cytoplasmic, it is also localized to the cell surface. In this study we have investigated the use of the yeast two-hybrid interaction trap to identify novel maspin targets. A target human fibroblast cDNA library was screened, and the alpha-2 chain of type I collagen was identified as a potential interactant. Binding studies with isolated proteins showed interaction between recombinant maspin and types I and III collagen but not other collagen subtypes, a profile strikingly similar to mouse pigment epithelium-derived factor (caspin), which is similarly down-regulated in murine adenocarcinoma tumors and is a potent inhibitor of angiogenesis. Kinetic analysis using an IAsys resonant mirror biosensor determined the dissociation constant of maspin for collagen type I to be 0.63 microm. Further two-hybrid interactions with maspin truncation constructs suggest that collagen binding is localized to amino acids 84-112 of maspin, which aligns with the collagen-binding region of colligin. A direct interaction between exogenous or cell surface maspin and extracellular matrix collagen may contribute to a cell adhesion role in the prevention of tumor cell migration and angiogenesis.
Highlights
Mammary serine protease inhibitor (Maspin) belongs to the serpin superfamily of proteins and can be included in the ovalbumin subfamily, which appear to be mostly intracellular [5, 6]
In this study we have investigated the use of the yeast two-hybrid interaction trap to identify novel maspin targets
A direct interaction between exogenous or cell surface maspin and extracellular matrix collagen may contribute to a cell adhesion role in the prevention of tumor cell migration and angiogenesis
Summary
Materials—All components of the two-hybrid system including vectors pEG202 and pJG4 –5, WI-38 fetal fibroblast cDNA “target” library in pJG4 –5, Saccharomyces cerevisiae EGY48, Escherichia coli strain KC8, and the polyclonal anti-LexA antibody were kindly donated by Luke O’Neill (Trinity College Dublin). The product was cloned into the yeast two-hybrid bait vector pEG202 using EcoRI and SalI restriction sites and propagated in E. coli XL-1 Blue cells. The bait-containing vector pEG202 and the library-containing target vector pJG4 –5 were sequentially transformed into the two-hybrid yeast strain EGY48 and grown on medium lacking leucine to select for potential interactors. Yeast colonies passing the dual selection screen were carried forward as potential interactor clones Unique clones within this pool were detected by PCR amplification of the target inserts using the following primers to the vector arms of pJG4 –5: (primer C, 5Ј-CCAGCCTCTTGCTGAGTGGAGATG-3Ј, and primer D, 5Ј-GACAAGCCGACAACCTTGATTGGAG3Ј). Following stringent washing with 150 mM NaCl and 50 mM Tris-Cl, pH 7.6, to remove unbound ligand, membranes were subsequently immunoblotted for maspin as described above.
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