Complement Deposition and IgG Binding on Stored Red Blood Cells Are Independent of Storage Time.

BACKGROUND: In the Netherlands, red blood cells (RBCs) are allowed to be stored up to 35 days at 2-6 °C in saline-adenine-glucose-mannitol (SAGM). During storage, RBCs undergo several changes that are collectively known as storage lesion. We investigated to what extent complement deposition and antibody binding occurred during RBC storage and investigated phagocytic uptake in vitro. METHODS: RBCs were stored for different lengths of time at 2-6 °C in SAGM. Complement deposition and antibody binding were assessed upon storage and after incubation with serum. M1- and M2-type macrophages were generated from blood monocytes to investigate RBC phagocytosis. RESULTS: No complement deposition was directly observed on stored RBCs, while incubation of RBCs with serum resulted in variable donor-dependent C3 deposition and IgG binding, both independent of storage time. Only 1-4% phagocytosis of stored RBCs by macrophages was observed. CONCLUSION: RBCs are susceptible to complement deposition and antibody binding independent of storage time. Limited phagocytic uptake by macrophages was observed in vitro.

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies.

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

UV damage endonuclease employs a novel dual-dinucleotide flipping mechanism to recognize different DNA lesions.

Repairing damaged DNA is essential for an organism's survival. UV damage endonuclease (UVDE) is a DNA-repair enzyme that can recognize and incise different types of damaged DNA. We present the structure of Sulfolobus acidocaldarius UVDE on its own and in a pre-catalytic complex with UV-damaged DNA containing a 6-4 photoproduct showing a novel 'dual dinucleotide flip' mechanism for recognition of damaged dipyrimidines: the two purines opposite to the damaged pyrimidine bases are flipped into a dipurine-specific pocket, while the damaged bases are also flipped into another cleft.

C1-inhibitor treatment in patients with severe complement-mediated autoimmune hemolytic anemia.

Complement-mediated (CM) autoimmune hemolytic anemia (AIHA) is characterized by the destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via the lysis of donor RBCs. Because C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhances RBC transfusion efficacy. We conducted a prospective, single-center, phase 2, open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of 2 RBC units were eligible for inclusion. Four IV C1-INH doses (6000, 3000, 2000, and 1000 U) were administered with 12-hour intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation, and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and activity, peaking at 48 hours after the first dose and accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 hours. Overall, markers of hemolysis, inflammation, and complement activation remained unchanged. Five grade 3 and 1 grade 4 adverse event occurred but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent-CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato.

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a ß-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Single-walled carbon nanotubes as scaffolds to concentrate DNA for the study of DNA-protein interactions.

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1.

Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility.

TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC's binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.

Lyse or not to lyse: Clinical significance of red blood cell autoantibodies.

Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease characterized by a hemolytic anemia caused by autoantibodies against red blood cells (RBCs). These autoantibodies are routinely detected via the direct antiglobulin test (DAT). As expected, the DAT score correlates with the presence of clinical symptoms, but this correlation is far from perfect. Regularly, strongly positive DAT scores are encountered with no sign of hemolysis, while severe hemolysis can be seen even in patients with a negative DAT score. Apparently, the mere amount of antibody is not the sole predictor of disease. In this paper, we review the current literature on aspects of both the autoantibodies and the patients' clearance system that together determine the clinical significance of an anti-RBC autoantibody, ranging from antibody isotype to antibody Fc-glycosylation to alternative clearance mechanisms. From this, the ensemble of tests is inferred that in our view best assesses the main determinants for pathogenicity of autoantibodies.

Methods for quantitative detection of antibody-induced complement activation on red blood cells.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal(1). Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis(1-4). However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Engineering specificity in a dynamic protein complex with a single conserved mutation.

It has been demonstrated that the complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP) exists as a delicate equilibrium of a specific, active state and the non-specific, dynamic encounter state. An ortholog of yeast Cc, horse Cc, binds CcP but forms a much more dynamic complex, as demonstrated by NMR spectroscopy. A single conservative mutation of lysine 13 to arginine reduces the dynamics and enhances the specificity. The crystal structure of the stereospecific complex resembles the yeast Cc-CcP complex. In contrast, the K13A mutation increases the dynamic nature of the complex with CcP, showing that specificity in a redox protein complex can depend on the interactions of a single side chain in the binding interface.

Involvement of a carboxylated lysine in UV damage endonuclease.

UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5' to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post-translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X-ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal-coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.