Characterization of .alpha.2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

Abstract

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.

Reaction of proteinases with .alpha.2-macroglobulin: evidence for alternate reaction pathways in the inhibition of trypsin

“…However, the remaining two a2M subunits are readily cleaved (Strickland et al, 1988) when incubated with an excess of trypsin. In Figure 4, the conversion of complex VI to complex VIII accounts for this nonproductive cleavage of a2M, which has been observed in previous studies examining the reaction of immobilized trypsin with a2M (Bjork, 1984) and in studies analyzing the reaction of plasmin (Steiner et al, 1987;Pochon, 1987;Roche & Pizzo, 1987) or thrombin (Straight & McKee, 1984;Steiner et al, 1985) with a2M. The majority of complex VI appears to be converted to complex VIII via this pathway; however, since a small amount of proteinase is incorporated into these complexes (Strickland et al, 1988), a pathway (complex VI to complex V) has been included in the model allowing for incorporation of additional proteinase into the binary complex.…”

Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of .alpha.2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

“…These results are in dramatic contrast to those obtained following treatment of native a2M with CH3NH2. In this case, the bait regions are not highly succeptible to proteolytic cleavage (Gonias & Pizzo, 1983b;Roche & Pizzo, 1987), and the conformation of the inhibitor is dramatically altered as determined by changes in the TNS emission spectrum (Strickland & Bhattacharya, 1984), mobility in nondenaturing PAGE (Barrett et al, 1979), and exposure of the receptor recognition sites (Imber & Pizzo, 1981). These results demonstrate quite conclusively that treatment with DSP effectively prevents thiolester bond cleavage dependent conformational changes in a2M.…”

Separation and localization of the four cysteine-949 residues in human .alpha.2-macroglobulin using fluorescence energy transfer

“…The central location required by the present studies is also attractive from the point of view of being able to accomodate a number of the properties of a2M. Thus, since the bait region is close to the thiol ester (Gettins et al, 1988), a central location for the bait regions could account for the slow cleavage of the remaining pair of bait regions by protease in binary complex with a2M (Roche & Pizzo, 1987;Strickland et al, 1988), if there is indeed a wide solvent channel connecting the two protease binding sites. Such a channel has been suggested from lowangle, low-resolution, X-ray scattering studies on a2M in solution (Osterberg & Pap, 1983).…”