Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown.
Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs)-in particular SBDPs of 150 kDa (SBDP150) and 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of αII-spectrin model proteins by caspase-3 in detail with gel electrophoresis, fluorescence and mass spectrometry methods. Amongst the predicted and reported sites, our results show that caspase-3 cleaves after residues D1185 and D1478, but not after residues D888, D1340 and D1475. We also found that the cleavage at these two sites is independent of each other. It may be possible to inhibit one site without affecting the other site. Cleavage after residue D1185 in intact αII-spectrin leads to SBDP150, and cleavage after D1478 site leads to SBDP120. Our results also show that the cleavage after the D1185 residue is unusually efficient, with a kcat/KM value of 40,000 M(-1) s(-1), and the cleavage after the D1478 site is more similar to most of the other reported caspase-3 substrates, with a kcat/KM value of 3000 M(-1) s(-1). We believe that this study lays out a methodology and foundation to study caspase-3 catalyzed spectrin breakdown to provide quantitative information. Molecular understanding may lead to better understanding of brain injuries and more precise and specific biomarker development.
Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, MC 111, Chicago, IL 60607, USA.
Marta A
MA
Witek
L W-M
LW
Fung
D000595:Amino Acid Sequence; D055162:Biocatalysis; D053148:Caspase 3 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D013049:Spectrin / Q000378:metabolism*
D013049:Spectrin; D053148:Caspase 3
20mM piperazine-N,N′-bis-(2-ethanesulfonic acid) with 100mM NaCl, 10mM DTT, 1mM EDTA, 0.1% CHAPS, and 10% sucrose at pH 7.2; Brain injury biomarker; Caspase-3; D10-D11(w)(′); D10-D11(w-1)('); D10-D11(w-1)(′); D10-D11(w-2)('); D10–D11; D10–D11 with all its tryptophan residues except W1106 and W1192 replaced with phenylalanine residues; D10–D11 with all its tryptophan residues except W1192 and W1215 replaced with phenylalanine residues; D10–D11 with all its tryptophan residues except W1192 replaced with phenylalanine residues; D10–D13; D12–D13; D13; D13 with W1533F mutation; D13(w)(′); Nonerythroid (brain) spectrin; SBDP; SBDP120; SBDP150; SBDP37; Spectrin breakdown product; [casp-3]; a recombinant protein consisting of residues 1087–1344 of αII-spectrin plus GS as the first two residues; a recombinant protein consisting of residues 1087–1556 of αII-spectrin plus GS as the first two residues; a recombinant protein consisting of residues 1335–1556 of αII-spectrin plus GS as the first two residues; a recombinant protein consisting of residues 1441–1556 of αII-spectrin plus GS as the first two residues; a recombinant protein consisting of residues 780–1344 of αII-spectrin plus GS as the first two residues; caspase activity buffer; spectrin breakdown product; the SBDP with an electrophoretic mass of 120kDa; the SBDP with an electrophoretic mass of 150kDa; the SBDP with an electrophoretic mass of 37kDa; the concentration of recombinant caspase-3