A mass spectrometry approach to understanding elastin assembly

Abstract

This thesis describes a detailed investigation of the cross-linked organisation of tropoelastin, the soluble precursor of elastin in synthetic and natural elastin. A set of detailed protease cleavage and mass spectrometry methodologies were developed to probe these systems, and identify preferred cross—linking regions in tropoelastin.

Recombinant full-length human tropoelastin was chemically cross-linked to form the elastin— like synthetic elastin. The susceptibility of synthetic elastin to proteases was quantified using a novel assay that additionally served as an in vitro model of elastin destruction. The sensitivity and reproducibility of this assay facilitated measurements of the degradative ability of the serine protease human neutrophil elastase and the matrix metalloproteinase MMP-12, previously implicated in elastin destruction in vivo. It also allowed for a quantitative comparison of the relative efficiencies of MMP-12 inhibitors. This approach also led to the definition of protease conditions conducive to cleavage of elastin for subsequent cross—link mapping studies, which had previously been substantially inaccessible.

Protease cleavage followed by a combination of electrospray and matrix assisted laser desorption ionisation mass spectrometry, followed by software filtering and detailed analysis of synthetic elastin determined that the region spanning domains 17—27 of tropoelastin was enriched in intermolecular interactions during coacervation. Distinct intramolecular links were separately observed in the N-terminal third of the molecule. These links identified a strong preference for specific lysines within domains, with a marked delineation in the use and exclusion of closely spaced lysines.

By modifying the choice of protease and defining a specific cleavage profile for pepsin under controlled conditions, a subset of the domains that were chemically cross—linked in synthetic elastin were identified in cross-links derived from multiple bovine and human elastin sources. Linear cross-linking in domains 19—25 and in the region from domain 4 —14 was seen in bovine elastin from aorta and lung. Fewer links were identified in human lung and skin samples, which showed similarity to the bovine profiles and were enriched for domain regions spanning 12—15 and domain 25.

There is a clear preference for specific cross—linking domains in coacervation as identified in synthetic elastin, and in bovine and human elastin from multiple tissues. Some of the lysines exposed to chemical cross—linking in synthetic elastin are involved in linear cross-linking in bovine and human elastin. These links are subdivided into regions enriched for intramolecular and separate intermolecular linkages, collectively providing the most comprehensive understanding of elastin assembly currently available.