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Interactions between the matricellular protein BM-40 and fibrillar collagens type I, II and III

BM-40, also known as osteonectin and SPARC, is a multifunctional glycoprotein belonging to the matricellular group of proteins. It modulates cellular interactions with the extracellular matrix by its binding to structural matrix proteins, such as collagens. BM-40 interacts with the collagen molecule through its EC domain in a Ca2+-dependent manner, in a region of collagen which has not yet been mapped. Aim of this work is therefore the identification of the region(s) on type I, II and III collagens responsible for this interaction. For this purpose we used both CNBr peptides with a natural sequence derived from bovine type I and II collagens, and recombinant fragments from human type III procollagen. Using solid phase assays, ELISA, we studied the interaction between peptides from type I collagen and BM-40. We found that just 2 peptides are able to interact in a triple-helical specific manner, alpha1(I) CB7 and alpha2(I) CB 3,5; these peptides overlap in an homologous region at the C-terminus of the collagen molecule. Also for type II collagen we have identified at least one peptide able to interact with BM-40, alpha1(II) CB10, that is the homologous of the CB7 and CB3,5 from type I. Besides, we studied the interaction of the complex procollagen/BM-40 in electron microscopy with the technique of the rotary shadowing. The data obtained for collagens type I, II and III, as well as confirming the results obtained in ELISA, suggest that another binding site is present on the collagen molecules and is located upstream in a more central region. For type III collagen, we confirmed this result also by cloning two recombinant fragments that were including just the putative binding regions of the triple helix. These recombinant peptides were first characterized and then their ability to bind BM-40 was tested in ELISA.

Mostra/Nascondi contenuto.
CHAPTER 1 INTRODUCTION

Tesi di Dottorato

Dipartimento: Dipartimento di Biochimica

Autore: Camilla Giudici Contatta »

Composta da 94 pagine.

 

Questa tesi ha raggiunto 292 click dal 12/10/2004.

 

Consultata integralmente una volta.

Disponibile in PDF, la consultazione è esclusivamente in formato digitale.