A model two-component system for studying the architecture of elastin assembly in vitro.
Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.
Mithieux
Suzanne M
SM
School of Molecular Biosciences, University of Sydney, NSW 2006, Australia.
Wise
Steven G
SG
Raftery
Mark J
MJ
Starcher
Barry
B
Weiss
Anthony S
AS
D000595:Amino Acid Sequence; D003895:Desmosine / Q000737:chemistry; D004549:Elastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D010455:Peptides; D011994:Recombinant Proteins; D003895:Desmosine; D004549:Elastin; D008249:Protein-Lysine 6-Oxidase; D008239:Lysine