Structural studies of proteinase entrapment by human alpha(2)-macroglobulin using 3-D electron microscopy

Usman Abdul Aziz Qazi, The University of Texas Graduate School of Biomedical Sciences at Houston


Human [special characters omitted]-macroglobulin ([special characters omitted]M; homotetramer, [special characters omitted] 720 kDa) is an essential scavenger of proteinases in the serum. Each of its four subunits has a ‘bait region’, with cleavage sequences for almost all endo-proteinases, an unusual thiol ester moiety and a receptor-binding domain (RBD). Bait region cleavage in native [special characters omitted]M ([special characters omitted]M-N) by a proteinase results in rapid thiol ester breakage, with a large-scale structural transformation, in which [special characters omitted]M uniquely entraps the proteinase in a cage-like structure and exposes receptor-binding domains for rapid endocytosis. Transformed [special characters omitted]M ([special characters omitted]M-TR) contains up to two proteinases, which remain active to small substrates. 3-D electron microscopy is optimally suited to study this unusual structural change at resolutions near (1/30) Å−1. The structural importance of the thiol esters was demonstrated by a genetically-engineered [special characters omitted]M, with the cysteines involved in thiol ester formation mutated to serines, which appeared structurally homologous to [special characters omitted]M-TR. This demonstrates that the four highly labile thiol esters alone maintain the [special characters omitted]M-N structure, while the ‘closed trap’ formed by [special characters omitted]M-TR is a more stable structural form. Half-transformed [special characters omitted]M ([special characters omitted]M-HT), with cleaved bait regions and thiol esters in only two of its four subunits, provides an important structural link between [special characters omitted]M-N and [special characters omitted]M-TR. A comparison with [special characters omitted]M-N showed the two proteinase-entrapping domains were above and below the plane bisecting the long axis. Both [special characters omitted]M-N and [special characters omitted]M-TR consist of two dense, oppositely twisted strands with significant interconnections, indicating that the structural change involves a rotation of these strands. In [special characters omitted]M-HT these strands were partially untwisted with large central openings, revealing the manner in which the proteinase enters the internal cavity of [special characters omitted]M. In reconstructions of [special characters omitted]M-N, [special characters omitted]M-HT and [special characters omitted]M-TR labeled with a monoclonal Fab, the Fabs were located on distal ends of each constitutive strand, demonstrating an anti-parallel arrangement of the subunits. Separation between the top and bottom pairs of Fabs was nearly the same on all structures, but the pairs were rotated about the long axis. Taken together, these results indicate that upon proteinase cleavage the two strands in [special characters omitted]M-N separate. The proteinase enters the structure, while the strands re-twist to encage it. In [special characters omitted]M-TR, which displays receptor-binding arms, more than two subunits are transformed as strands in the transformed half of [special characters omitted]M-HT were not separated.

Subject Area

Biophysics|Pathology|Molecular biology

Recommended Citation

Qazi, Usman Abdul Aziz, "Structural studies of proteinase entrapment by human alpha(2)-macroglobulin using 3-D electron microscopy" (1999). Texas Medical Center Dissertations (via ProQuest). AAI9951901.