Tropoelastin Interacts with Cell-surface Glycosaminoglycans via Its COOH-terminal Domain

Thomas J Broekelmann,Beth A Kozel,Hideaki Ishibashi,Claudio C Werneck,Fred W Keeley,Lijuan Zhang,Robert P Mecham

doi:10.1074/jbc.m507309200

Thomas J Broekelmann, Beth A Kozel + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m507309200

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Abstract

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

Highlights

Elastin is the major extracellular matrix protein capable of elastic recoil in tissues repeatedly subjected to cycles of reversible extension [1,2,3]
A Cell Adhesive Site Is Located at the Carboxyl Terminus of Elastin— Bovine tropoelastin and a series of tropoelastin fragments (Fig. 1A) were expressed as His6 fusion proteins and purified to homogeneity (Fig. 1B)
The tropoelastin molecule has a unique domain structure characterized by repeating hydrophobic stretches in tandem with lysine-containing sequences

Summary

Introduction

Elastin is the major extracellular matrix protein capable of elastic recoil in tissues repeatedly subjected to cycles of reversible extension [1,2,3] It functions as an insoluble polymer made up of cross-linked tropoelastin molecules enmeshed in a network of filamentous microfibrils (4 – 6). Are modified to form cross-links in the mature protein This changes the physical character of the protein from cationic to slightly anionic and hydrophobic in nature. The sequence in elastin assumed responsible for these activities is the hexapeptide VGVAPG and similar sequences [24, 25] located in the middle of the molecule These peptides are thought to bind to a 67-kDa elastin-binding protein (EBP67) that is a galactoside-binding lectin (8, 26 –28). Tropoelastin, in contrast, actively promotes cell attachment and spreading, suggesting that it interacts with a receptor that is different from that which recognizes the VGVAPG sequence

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