Abstract
Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-gamma-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased Km, a highly increased catalytic efficiency (kcat), and a 80-180 times greater specificity constant (kcat/Km) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3 position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-gamma-inducible subunits.
Highlights
Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-␥-inducible LMP7, LMP2, and MECL1 subunits
Since most class I antigenic peptides contain at the carboxyl terminus either aromatic, branched chain, or basic amino acid residues (40), the expectation was that immunoproteasomes would have to exhibit increased ChT-L and T-L activities
Transfection experiments leading to overexpression of LMP2 and LMP7 subunits in a murine fibroblast cell line and a human lymphoblastoid T2 cell line led to a marked decrease of the ChT-L activity measured with succinyl-Leu-Leu-Val-Tyr-MCA as the substrate and to a depression of the peptidylglutamyl-peptide hydrolyzing (PGPH) activity measured with Z-Leu-Leu
Summary
Materials—Fresh frozen bovine spleens were obtained from PelFreez (Roger, AR). Isolation of the MPC was carried out according to a previously described procedure for the purification of the pituitary MPC (27). Substrates for determination of activities of components of the MPC, Z-GGL-pNA, Z-GGF-pAB, Z-(D)ALR-2NA, Z-LLE-2NA, Z-GPALA-pAB, Z-GPALG-pAB, Z-GPAGG-pAB, and Z-GPAFG-pAB were synthesized as described previously (15, 17, 18, 28). Under the above conditions most of the  subunits having a free NH2 terminus migrate ahead of the ␣ subunits with blocked NH2 termini This made it convenient to obtain NH2terminal amino acid sequences of the  subunits after one-dimensional SDS-PAGE, since a single run provided sufficient material for sequencing. Aliquots of the preincubation mixtures were withdrawn at 0, 5, 10, 20, and 30 min, and the activity was determined as described above. Determination of Casein Degradation by the MPC—Incubation mixtures contained 100 g of -casein, 20 mM of MgCl2, 5 g of MPC, and 0.05 M Tris-HCl buffer, pH 8.0, in a final volume of 100 l.
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