Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1.
Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.
Jensen
S A
SA
Department of Biochemistry G08, University of Sydney, New South Wales 2006, Australia.
Reinhardt
D P
DP
Gibson
M A
MA
Weiss
A S
AS
D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D004549:Elastin / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014333:Tropoelastin / Q000378:metabolism*; D054392:Adipokines
D003285:Contractile Proteins; D004549:Elastin; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins; D014333:Tropoelastin; C000605722:FBN1 protein, human; D054392:Adipokines; C016598:microfibrillar protein