Molecular Structures of Isolevuglandin-Protein Cross-Links.
Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS-PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11-17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]LGE2with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide-pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11-17) tends to spontaneously oligomerize.
Department of Chemistry, Case Western Reserve University , Cleveland, Ohio 44106, United States.
Wenzhao
W
Bi
Cole Eye Institute, Cleveland Clinic Foundation , Cleveland, Ohio 44195, United States.
Geeng-Fu
GF
Jang
Cole Eye Institute, Cleveland Clinic Foundation , Cleveland, Ohio 44195, United States.
Lei
L
Zhang
Cole Eye Institute, Cleveland Clinic Foundation , Cleveland, Ohio 44195, United States.
John W
JW
Crabb
Department of Chemistry, Case Western Reserve University , Cleveland, Ohio 44106, United States.
James
J
Laird
Department of Chemistry, Case Western Reserve University , Cleveland, Ohio 44106, United States.
Mikhail
M
Linetsky
Department of Chemistry, Case Western Reserve University , Cleveland, Ohio 44106, United States.
Robert G
RG
Salomon
D000818:Animals; D002154:Calpain / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure
D003432:Cross-Linking Reagents; D005231:Fatty Acids, Unsaturated; C526910:iso(4)levuglandin E2; D002154:Calpain