Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19–20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1–4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1–4V62 and FH1–4I62 bind immobilized C3b with similar affinities (KD = 10–14 μm), whereas FH1–4I62 is slightly more effective than FH1–4V62 as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1–4V62 binds to C3b with comparable affinity (KD ∼12 μm) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1–4V62, binds poorly to immobilized C3b (KD >35 μm) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.

Structural knowledge of interactions amongst the~40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Fö rster resonance energy transfer (FRET) was used to investigate the 10 lM K D (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under nearphysiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.

No abstract

No abstract

The Agilent 4200 TapeStation system provides automated, fast, and reliable DNA and RNA electrophoresis for up to 96 samples using prepackaged reagents and minimal manual handling. The Agilent D5000 ScreenTape and High Sensitivity D5000 ScreenTape assays have been developed for the separation and analysis of DNA fragments from 100 bp to 5,000 bp, a size range that complements and resides between the Agilent D1000 ScreenTape and the Genomic DNA ScreenTape assays. The 4200 TapeStation system and the DNA ScreenTape assays can be used at several steps of the Next Generation Sequencing (NGS) workflow. With the emergence of methodologies, such as the use of Transposomes, NGS library sizes are tending to increase beyond 1,000 bp, even for the short-read NGS technologies. This Poster focuses on the performance of both D5000 ScreenTape assays with respect to the accuracy and precision of quantification and sizing, as well as the sensitivity of these assays. Data analysis for quantification and molarity determination was compared against the corresponding assay for the Agilent 2100 Bioanalyzer system. Additionally, performance of both the D5000 and High Sensitivity D5000 assays on the 4200 TapeStation was compared to the 2200 TapeStation system. Citation Format: Rainer Nitsche, Lidia Prieto-Lafuente, Claire MacDonald, Isabell Pechtl. Performance of the Agilent D5000 and high sensitivity D5000 ScreenTape assays for the Agilent 4200 TapeStation system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3635.