Differential effects of heparin and glucose on structural conformation of human alpha1-antitrypsin: evidence for a heparin-induced cleaved form of the inhibitor.

alpha1 Antitrypsin (alpha1AT) is the archetypal member of the serpin superfamily. Current knowledge of its inhibitory mechanism does not provide for any heparin-induced enhancement of serine proteinase inhibition. Since previous results have shown that an apparently altered alpha1AT form may be purified from the plasma of insulin-dependent diabetics by means of heparin-affinity chromatography, in the present work the possibility was tested that heparin at various concentrations modifies the structural conformation and function of human alpha1AT in the absence and presence of glucose, used at concentrations of 15 mM to mimick mild hyperglycemic conditions. Heparin was observed to bind strongly to alpha1AT, causing maximal enhancement of tryptophan fluorescence emission at 50 microg/ml, mostly in the presence of glucose. Circular dichroism spectra revealed that heparin with glucose caused the most relaxed, ordered structure of the inhibitor with increased heat stability. Modification in conformation was accompanied by loss of inhibitory activity, as demonstrated by the inability of alpha1AT to block bovine trypsin in the specific assay and by alterations of its immunological properties. However, despite inactivation, in the presence of heparin-both with and without glucose-alpha1AT was still able to bind trypsin, as revealed by inhibitor-to-proteinase complexes visible in both SDS- and nondenaturing electrophoreses. These complexes showed the same feature regardless of trypsin concentration and differed from those formed at a molar excess of the inhibitor in the absence of heparin, since they underwent rapid, intense fragmentation accompanied by complete loss of the secondary structure of the inhibitor. Even in the absence of trypsin, cleavage of alpha1AT was also observed to occur at both Val321- and Glu344- in the primary sequence of the inhibitor in the presence of 50 microg/ml heparin, with and without glucose. These results suggest that heparin binding to alpha1AT causes profound structural changes in the molecule, involving both the expulsion of the reactive site out of the molecule plane and a relaxed, heat-stable form of the inhibitor, rendered a substrate for the proteinase. Although glucose apparently does not affect alpha1AT functioning, it does enhance the effects of heparin on the alpha1AT structure. The possibility is discussed that, while heparin and glucose binding occurs at different sites on alpha1AT, glucose favors heparin binding by inducing a partially relaxed form in the inhibitor. Differences in structure and charge between the two substances account for both different individual effects on alpha1AT and the predominance of the effects of heparin.