Modulation of the proteolytic activity of the complement protease C1s by polyanions: implications for polyanion-mediated acceleration of interaction between C1s and SERPING1.

The complement system plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical complement and lectin pathways are controlled by SERPING1 [(C1 inhibitor) serpin peptidase inhibitor, clade G, member 1], which inactivates the components C1s and MASP-2 (mannan-binding lectin serine peptidase 2). GAGs (glycosaminoglycan) and DXS (dextran sulfate) are able to significantly accelerate SERPING1-mediated inactivation of C1s, the key effector enzyme of the classical C1 complex, although the mechanism is poorly understood. In the present study we have shown that C1s can bind to DXS and heparin and that these polyanions enhanced C1s proteolytic activity at low concentrations and inhibited it at higher concentrations. The recent determination of the crystal structure of SERPING1 has given rise to the hypothesis that both the serpin (serine protease inhibitor)-polyanion and protease-polyanion interactions might be required to accelerate the association rate of SERPING1 and C1s. To determine what proportion of the acceleration was due to protease-polyanion interactions, a chimaeric mutant of alpha1-antitrypsin containing the P4-P1 residues from the SERPING1 RCL (reactive-centre loop) was produced. Like SERPING1, this molecule is able to effectively inhibit C1s, but is unable to bind polyanions. DXS exerted a biphasic effect on the association rate of C1s which correlated strongly with the effect of DXS on C1s proteolytic activity. Thus, whereas polyanions are able to bind C1s and modulate its activity, polyanion interactions with SERPING1 must also play a vital role in the mechanism by which these cofactors accelerate the C1s-SERPING1 reaction.

Elucidation of the substrate specificity of the MASP-2 protease of the lectin complement pathway and identification of the enzyme as a major physiological target of the serpin, C1-inhibitor.

Complement is a central component of host defence, but unregulated activation can contribute to disease. The system can be initiated by three pathways: classical, alternative and lectin. The classical and lectin pathways are initiated by the C1 and mannose-binding lectin (MBL) or ficolin complexes, respectively, with C1s the executioner protease of the C1 complex and MASP-2 its counterpart in the lectin complexes. These proteases in turn cleave the C4 and C2 components of the system. Here we have elucidated the cleavage specificity of MASP-2 using a randomised substrate phage display library. Apart from the crucial P1 position, the MASP-2 S2 and S3 subsites (in that order) play the greatest role in determining specificity, with Gly residues preferred at P2 and Leu or hydrophobic residues at P3. Cleavage of peptide substrates representing the known physiological cleavage sequences in C2, C4 or the serpin C1-inhibitor (a likely regulator of MASP-2) revealed that MASP-2 is up to 1000 times more catalytically active than C1s. C1-inhibitor inhibited MASP-2 50-fold faster than C1s and much faster than any other protease tested to date, implying that MASP-2 is a major physiological target of C1-inhibitor.

Differential effects of detergents, fatty acids, cations and heating on ostrich skeletal muscle 20S proteasome.

The 20S proteasome, the catalytic core of the 26S proteasome, has previously been isolated, purified and partially characterised from ostrich skeletal muscle (Thomas, A.R., Oosthuizen, V., Naude, R.J., Muramoto, K. 2002. Biol. Chem. 383, 1267-1270). Due to the apparent latency of the 20S proteasome purified from various sources, this study focuses on further characterising the ostrich enzyme in terms of the effects of selected detergents, fatty acids and cations, as well as heating at 60 degrees C, on four of its activities. Results showed that ostrich skeletal muscle 20S proteasome was affected in a non-concentration-dependent manner by the selected detergents and fatty acids. Monounsaturated fatty acids, unlike unsaturated fatty acids, showed no major effects on the activities of the ostrich enzyme. The enzyme did not show sensitivity towards monovalent cations and the only divalent cations that showed a relevant effect were Ca2+ and Mg2+. Heating at 60 degrees C for 1-2 min had a substantial activating effect only on the peptidylglutamylpeptide-hydrolase (PGPH) and caseinolytic activities. In conclusion, many of the effects by the abovementioned reagents and conditions were noticeably different to those shown on different sources of the enzyme, further demonstrating the unique kinetic characteristics of the ostrich skeletal muscle 20S proteasome.

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation.

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.

Purification and characterization of the 20S proteasome from ostrich skeletal muscle.

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700,000, pI 6.67 and a 'ladder' of 22.2-33.5 kDa bands on SDS-PAGE. The amino acid composition and amino-terminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0-11.0 and 40-70 degrees C, and stabilities between 5-12 and up to 40-60 degrees C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.