Correlation between the human and porcine complement system: a small-angle scattering study of cross immunity and methylamine-induced conformational changes of porcine C3 and C4 proteins.

The porcine complement proteins C3 and C4 have been isolated and then characterized using small-angle scattering methods. Within the limits of experimental errors, the porcine proteins are virtually identical with the corresponding human proteins as measured in terms of mol. wt, Mr and radius of gyration, R,: Mr(C3) = 198,000, Mr(C4) = 207,000, and R(C3) = 4.4 nm, R(C4) = 4.5 nm. The C3 and C4 proteins from pigs show cross-immunity with monoclonal antibodies (mAbs) raised against human C3 and C4, respectively. Using the Fab fragments of these mAbs as markers, it is indicated that porcine C3 and C4 undergo a conformational change after reaction with methylamine. The relatively large increase in the radius of gyration observed, delta R = 1.0-1.2 nm, going from the Fab complexes to the Fab complexes of the methylamine derivatives, is similar to that observed for human C3 under similar conditions. This may indicate that methylamine cleaves a labile thiol ester bond supposed to be present within the porcine proteins and that the epitopes interacting with the Fab fragments are very similar to those of the human proteins. Porcine C3 also resembles the human analogue by forming dimers after being subjected to methylamine and dilute lauryl sulphate: Mr = 404,000 and R = 7.9 nm.

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.

Methylamine-induced conformational change of alpha 2-macroglobulin and its zinc (II) binding capacity. An X-ray scattering study.

Methylamine induces a conformational change of alpha 2-macroglobulin which is very similar to that obtained by proteinase reaction and binding. This was shown by small-angle X-ray scattering at 21 degrees C in 0.03 M Hepes buffer of pH 8.0 containing 0.15 M NaCl and 0.3 mM EDTA. When alpha 2-macroglobulin reacts with methylamine the side maximum virtually disappears from the X-ray scattering curve and the radius of gyration decreases from 7.8 nm to 7.2 nm. The X-ray data of alpha 2-macroglobulin are consistent with an open shape model similar to that deduced via electron micrographs [Schramm, H. J. and Schramm, W. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 803-812]; one projection of the model resembles the letter H; the four subunits are mainly represented as elliptical cylinders which are connected via a central, quite flat cylinder. Zinc(II) ions cause aggregation of alpha 2-macroglobulin even at such a low total zinc concentration as 12.5 microM; for 25 microM zinc(II) concentration, the average molecular mass indicates that the aggregation goes beyond the dimeric stage. Monomeric species of alpha 2-macroglobulin appear to have the capacity specifically to bind 8.0 zinc(II) ions per molecule, which corresponds to two zinc(II) ions per subunit.