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Una L. Kelly, Jin-Dong Ding, Kent Y. Feng, Haixiang Jiang, Michael Frank, Catherine Bowes Rickman; Concentration and Composition Changes in Heparan Sulfate Increase Complement Activation Through Modulation of C3bBb Convertase Formation and This Effect is Modified by Factor H. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2301.
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Allotypic variations in complement factor H (CFH) increase the risk of developing age-related macular degeneration (AMD), yet there has been little evidence of significant functional differences between the disease-associated allotypic variant of CFH, V62/H402, and the protective variant, I62/Y402. This study tests the hypothesis that AMD risk conferred by CFH is modulated by components of the extracellular matrix of Bruch’s membrane, particularly heparan sulfate glycosaminoglycans (HS GAGs), and that allotypic differences in CFH will only be detectable if CFH, C3, and GAG interactions are studied together in functional assays.
Two assays were used in this study: (1) a convertase assembly assay that detects the conversion of C3 to C3b, and Factor B to Bb and (2) a fluorescence-based assay that measures the extent of unfolding of C3 in the presence of HS-GAGs. Electron microscopy was used to detect age-dependent changes in HS in human eyes.
We found that HS binds to C3 and C3b, changing their conformation such that factor B can no longer bind to C3b or undergo factor D mediated cleavage to form the convertase C3bBb, thereby blocking complement activation. We established the minimum HS concentration threshold for this effect and found that it is dependent on the HS source and chemical modifications. We also show that CFH competes with C3 for binding to HS and that this alters the concentration threshold for C3 denaturation by HS. In addition, in human and in mouse, HS distribution in Bruch’s membrane changes with age.
We have recently shown that HS accelerates the rate of factor I-mediated cleavage of C3b (Kelly et al. J.Immunol. 2010). This study extends the effects HS to C3bBb formation, reinforcing the importance of the GAG composition in the Bruch’s membrane/choroid region of the eye. Taken together, our results suggest that alterations in the GAG composition that occur as a part of aging potentially make this tissue more vulnerable to damaging complement activation.
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