Epilysin (MMP-28) is deposited to the basolateral extracellular matrix of epithelial cells.

Epilysin (MMP-28) is a conserved member of the matrix metalloproteinase (MMP) family. It is expressed in various normal tissues, and induced in wounds and in developing and regenerating nerves. Epilysin induces TGF-beta mediated epithelial to mesenchymal transition, but its other functions are largely unknown. We have characterized the localization of both catalytically active and mutated inactive, overexpressed epilysin in established epithelial cell lines. We found that epilysin was localized abundantly to the basolateral side of the cells and associated with the extracellular matrix (ECM) as verified by immunoblotting and confocal microscopy. Overexpression of epilysin in MDCK cells resulted in a drastic reduction of basolateral ECM, as observed by the disappearance of collagen type IV, laminin and fibronectin. Cultivation of epilysin expressing MDCK cells in defined serum free medium resulted in the restoration of these proteins to the ECM. The levels of fibronectin and collagen IV were, however, reduced in epilysin expressing cells under the serum free conditions, and degradation fragments of collagen IV were detected supporting the activation of proteolysis by epilysin. Epilysin was observed in its unprocessed 50 kDa active form in the ECM of MDCK cells under serum free conditions whereas in cells cultured in serum containing it was processed to the 48 kDa form. Current results indicate that epilysin associates with the basolateral ECM of cultured epithelial cells, where it plausibly plays a role in the regulation of matrix composition and turnover.

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library.

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.