Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin.
The carboxy-terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is approximately 0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysine-derived cross-link in this region, which would alter the extractability and antigenicity of the carboxy-terminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.
Broekelmann
Thomas J
TJ
Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Ciliberto
Christopher H
CH
Shifren
Adrian
A
Mecham
Robert P
RP
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004549:Elastin / Q000737:chemistry; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014333:Tropoelastin / Q000737:chemistry*
D003432:Cross-Linking Reagents; D000939:Epitopes; D010455:Peptides; D011108:Polymers; D014333:Tropoelastin; D004549:Elastin