In a biomembrane modeling system, reverse micelles, somatic ACE forms dimers via carbohydrate-mediated interaction, providing evidence for the existence of a carbohydrate-recognizing domain on the ACE molecule. We localized this putative region on the N-domain of ACE using monoclonal antibodies (mAbs) to seven different epitopes of ACE. Two mAbs, 9B9 and 3G8, directed to distinct, but overlapping, epitopes of the N-domain of ACE shielded the CRD. Only "simple" ACE-antibody complexes were found in the system. Five mAbs allowed the formation of "double" antibody-ACE-ACE-antibody complexes via carbohydrate-mediated interactions. The results were confirmed using the ACE N- and C-domains. Testicular ACE was unable to form carbohydrate-mediated ACE dimers in the reverse micelles, while the N-domain of ACE, obtained by limited proteolysis of the parent full-length ACE, retained the ability to form dimers. Furthermore, mAb 3G8, which blocked ACE dimerization in micelles, significantly inhibited ACE shedding from the surface of ACE-expressing cells. Galactose prevented ACE dimerization in reverse micelles and also affected antibody-induced ACE shedding in an epitope-dependent manner. Restricted glycosylation of somatic ACE, obtained by the treatment of CHO-ACE cells with the glucosidase inhibitor N-butyldeoxynojirimycin, significantly increased the rate of basal ACE shedding and altered antibody-induced ACE shedding. A chemical cross-linking approach was used to show that ACE is present (at least in part) as noncovalently linked dimers on the surface of CHO-ACE cells. These results suggest a possible link between putative ACE dimerization on the cell surface and the proteolytic cleavage (shedding) of ACE.
The regulation of the Sso II restriction-modification system from Shigella sonnei was studied in vivo and in vitro . In lacZ fusion experiments, Sso II methyltransferase (M. Sso II) was found to repress its own synthesis but stimulate expression of the cognate restriction endonuclease (ENase). The N-terminal 72 amino acids of M. Sso II, predicted to form a helix-turn-helix (HTH) motif, was found to be responsible for the specific DNA-binding and regulatory function of M. Sso II. Similar HTH motifs are predicted in the N-terminus of a number of 5-methylcytosine methyltransferases, particularly M. Eco RII, M.dcm and M. Msp I, of which the ability to regulate autogenously has been proposed. In vitro, the binding of M. Sso II to its target DNA was investigated using a mobility shift assay. M. Sso II forms a specific and stable complex with a 140 bp DNA fragment containing the promoter region of Sso II R-M system. The dissociation constant (Kd) was determined to be 1.5x10(-8) M. DNaseI footprinting experiments demonstrated that M. Sso II protects a 48-52 bp region immediately upstream of the M. Sso II coding sequence which includes the predicted -10 promoter sequence of M. Sso II and the -10 and -35 sequences of R. Sso II.
Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential. This work examines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), termed “nanozyme.” The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer, poly(L-lysine)-poly(ethyleneglycol), followed by covalent cross-linking of the complexes with 3,3′-dithiobis(sulfosuccinimidylpropionate) sodium salt. The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with immunogenic uveitis. Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity; however, the nanozyme was much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as the intensities of corneal and iris edema, hyperemia of conjunctiva, lens opacity, fibrin clots, and the protein content in aqueous humor. Clinical findings were confirmed by histological data. Thus, SOD1-containing nanozyme is potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.
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