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J Biol Chem, Vol. 274, Issue 12, 8137-8142, March 19, 1999

Structural Details of Proteinase Entrapment by Human ₂-Macroglobulin Emerge from Three-dimensional Reconstructions of Fab labeled Native, Half-transformed, and Transformed Molecules

Usman Qazi, Peter G. W. Gettins^§, Dudley K. Strickland^¶, and James K. Stoops

From the  Dept of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, Texas 77030, ^§ Department of Biochemistry, University of Illinois at Chicago, Chicago, Illinois 60612, and ^¶ Department of Vascular Biology, J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed ₂-macroglobulins (₂Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each ₂M binds four Fabs, two at either end of its dimeric protomers approximately 145 Å apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 Å, whereas in the methylamine-transformed ₂M, the epitopes are at the base of its four arms, laterally separated by 160 Å. Upon thiol ester cleavage, the chisels on the native ₂M appear to split with a separation and rotation to give the four arm-like extensions on transformed ₂M. Thus, the receptor binding domains previously enclosed within the chisels are exposed. The labeled structures further indicate that the two protomeric strands that constitute the native and transformed molecules are related and reside one on each side of the major axes of these structures. The half-transformed structure shows that the two Fabs at one end of the molecule have an arrangement similar to those on the native ₂M, whereas on its transformed end, they have rotated. The rotation is associated with a partial untwisting of the strands and an enlargement of the openings to the cavity. We propose that the enlarged openings permit the entrance of the proteinase. Then cleavage of the remaining bait domains by a second proteinase occurs with its entrance into the cavity. This is followed by a retwisting of the strands to encapsulate the proteinases and expose the receptor binding domains associated with the transformed ₂M.

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