Gel-Assisted Proteome Position Integral Shift Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Caspase 3 Substrates.

Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

Fcgr2b and Fcgr3 are the major genetic factors for cartilage antibody-induced arthritis, overriding the effect of Hc encoding complement C5.

Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b-- mice but not in Fcgr3-- mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b++.Hc** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b--.Hc** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency.

A subset of type-II collagen-binding antibodies prevents experimental arthritis by inhibiting FCGR3 signaling in neutrophils.

Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1ß secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.

SpotLight Proteomics Identifies Variable Sequences of Blood Antibodies Specific Against Deamidated Human Serum Albumin.

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in the blood of patients with Alzheimer's disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

Testing the link between isoaspartate and Alzheimer's disease etiology.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (Aß) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free Aß (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with Aß and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of Aß and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.

Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution.

Chemical proteomics focuses on the drug-target-phenotype relationship for target deconvolution and elucidation of the mechanism of action-key and bottleneck in drug development and repurposing. Majorly due to the limits of using chemically modified ligands in affinity-based methods, new, unbiased, proteome-wide, and MS-based chemical proteomics approaches have been developed to perform drug target deconvolution, using full proteome profiling and no chemical modification of the studied ligand. Of note among them, thermal proteome profiling (TPP) aims to identify the target(s) by measuring the difference in melting temperatures between each identified protein in drug-treated versus vehicle-treated samples, with the thermodynamic interpretation of "protein melting" and curve fitting of all quantified proteins, at all temperatures, in each biological replicate. Including TPP, all the other chemical proteomics approaches often fail to provide target deconvolution with sufficient proteome depth, statistical power, throughput, and sustainability, which could hardly fulfill the final purpose of drug development. The proteome integral solubility alteration (PISA) assay provides no thermodynamic interpretation, but a throughput 10-100-fold compared to the other proteomics methods, high sustainability, much lower time of analysis and sample amount requirements, high confidence in results, maximal proteome coverage (~10,000 protein IDs), and up to five drugs / test molecules in one assay, with at least biological triplicates of each treatment. Each drug-treated or vehicle-treated sample is split into many fractions and exposed to a gradient of heat as solubility perturbing agent before being recomposed into one sample; each soluble fraction is isolated, then deep and quantitative proteomics is applied across all samples. The proteins interacting with the tested molecules (targets and off-targets), the activated mechanistic factors, or proteins modified during the treatment show reproducible changes in their soluble amount compared to vehicle-treated controls. As of today, the maximal multiplexing capability is 18 biological samples per PISA assay, which enables statistical robustness and flexible experimental design accommodation for fuller target deconvolution, including integration of orthogonal chemical proteomics methods in one PISA assay. Living cells for studying target engagement in vivo or, alternatively, protein extracts to identify in vitro ligand-interacting proteins can be studied, and the minimal need in sample amount unlocks target deconvolution using primary cells and their derived cultures. This protocol was validated in: J Biol Chem (2021), DOI: 10.1016/j.jbc.2021.10153 Graphical abstract.

Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry.

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome.

BACKGROUND: To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. METHODS: Samples and clinical data were obtained from (i) 25 anti-Jo1+ patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1- patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. RESULTS: Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. CONCLUSION: High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

First Immunoassay for Measuring Isoaspartate in Human Serum Albumin.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins mostly as a result of spontaneous deamidation of asparaginyl residues. An association has been found between isoAsp in human serum albumin (HSA) and Alzheimer's disease (AD). Here we report on a novel monoclonal antibody (mAb) 1A3 with excellent specificity to isoAsp in the functionally important domain of HSA. Based on 1A3 mAb, an indirect enzyme-linked immunosorbent assay (ELISA) was developed, and the isoAsp occupancy in 100 healthy plasma samples was quantified for the first time, providing the average value of (0.74 ± 0.13)%. These results suggest potential of isoAsp measurements for supplementary AD diagnostics as well as for assessing the freshness of stored donor blood and its suitability for transfusion.

System-wide identification and prioritization of enzyme substrates by thermal analysis.

Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

Capillary leakage provides nutrients and antioxidants for rapid pneumococcal proliferation in influenza-infected lower airways.

Influenza A virus (IAV)-related mortality is often due to secondary bacterial infections, primarily by pneumococci. Here, we study how IAV-modulated changes in the lungs affect bacterial replication in the lower respiratory tract (LRT). Bronchoalveolar lavages (BALs) from coinfected mice showed rapid bacterial proliferation 4 to 6 h after pneumococcal challenge. Metabolomic and quantitative proteomic analyses demonstrated capillary leakage with efflux of nutrients and antioxidants into the alveolar space. Pneumococcal adaptation to IAV-induced inflammation and redox imbalance increased the expression of the pneumococcal chaperone/protease HtrA. Presence of HtrA resulted in bacterial growth advantage in the IAV-infected LRT and protection from complement-mediated opsonophagocytosis due to capsular production. Absence of HtrA led to growth arrest in vitro that was partially restored by antioxidants. Pneumococcal ability to grow in the IAV-infected LRT depends on the nutrient-rich milieu with increased levels of antioxidants such as ascorbic acid and its ability to adapt to and cope with oxidative damage and immune clearance.

Antibodies against Phosphorylcholine and Malondialdehyde during the First Two Years of Life.

Abs against phosphorylcholine (anti-PC) and Abs against malondialdehyde (anti-MDA) may be protective in chronic inflammation, like atherosclerosis and cardiovascular disease. It is not known how they develop early in life. Ab titers were measured using ELISA in healthy women (n = 105; born into life study) and their children. Plasma samples were collected from the mothers before conception and from the children at birth as well as at 1 and 2 y after birth. Extracted Abs were compared using a proteomics de novo sequencing approach. It was observed that children were born with very low levels of IgM anti-PC, whereas IgM anti-MDA was present at birth. Both IgM anti-PC and anti-MDA increased during the first 2 y of life, but IgM anti-PC in contrast to IgM anti-MDA was still significantly lower than in the mothers. IgG anti-PC decreased after 1 y but reached similar levels as mothers' after 2 y, whereas IgG anti-MDA reached similar levels as mothers' already after 1 y. Proteomics peptide sequencing analysis indicated large peptide sequence variation without specific clone expression during the early stage of life compared with the adult stage for which specific peptide sequences dominated. IgM anti-PC levels develop much slower than anti-MDA and are still relatively low at 2 y. We hypothesize that anti-PC is developed by a combination of preprogramming and exposure to the external world, in which infectious agents may play a role. For anti-MDA, preprogramming is likely to play a major role and at an earlier stage than for anti-PC.

IgM antibodies against malondialdehyde and phosphorylcholine in different systemic rheumatic diseases.

IgM antibodies against phosphorylcholine (anti-PC) and malondialdehyde (anti-MDA) may have protective properties in cardiovascular and rheumatic diseases. We here compare these antibodies in systemic rheumatic conditions and study their properties. Anti-PC and anti-MDA was measured using ELISA in patients with SLE (374), RA (354), Mixed connective tissue disease (MCTD, 77), Systemic sclerosis (SSc, 331), Sjögren's syndrome (SjS, 324), primary antiphospholipid syndrome (PAPs, 65), undifferentiated connective tissue disease (UCTD, 118) and 515 matched healthy controls (HC). Cardiovascular score (CV) was broadly defined based on clinical disease symptoms. Anti-PC and anti-MDA peptide/protein characterization were compared using a proteomics de novo sequencing approach. anti-MDA and anti-PC were extracted from total IgM. The proportion of Treg cells was determined by flow cytometry. The maximal difference between cases and controls was shown for MCTD: significantly lower IgM Anti-PC but not anti-MDA among patients (median 49.3RU/ml vs 70.4 in healthy controls, p(t-test) = 0.0037). IgM low levels were more prevalent in MCTD, SLE, SjS, SSc and UCTD. IgM anti-PC variable region profiles were different from and more homologous than anti-MDA. Anti-PC but not anti-MDA were significantly negatively correlated with CV in the whole patient group. In contrast to IgM anti-PC, anti-MDA did not promote polarization of Tregs. Taken together, Anti-PC is decreased in MCTD and also in SLE, SjS and SSc but not in other studied diseases. Anti-PC may thus differentiate between these. In contrast, anti-MDA did not show these differences between diseases studied. Anti-PC level is negatively correlated with CV in the patient group cohort. In contrast to anti-PC, anti-MDA did not promote Treg polarization. These findings could have both diagnostic and therapeutic implications, one possibility being active or passive immunization with PC in some rheumatic conditions.

Proteome Integral Solubility Alteration: A High-Throughput Proteomics Assay for Target Deconvolution.

Various agents, including drugs as well as nonmolecular stimuli, induce alterations in the physicochemical properties of proteins in cell lysates, living cells, and organisms. These alterations can be probed by applying a stability- and solubility-modifying factor, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or agent intensity/concentration can be used. Compared to standard approaches where curves are fitted to protein solubility data acquired at different temperatures and drug concentrations, Proteome Integral Solubility Alteration (PISA) assay increases the analysis throughput by 1 to 2 orders of magnitude for an unlimited number of factor variation points in such a scheme. The consumption of the compound and biological material decreases in PISA by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.

SpotLight Proteomics-A IgG-Enrichment Phenotype Profiling Approach with Clinical Implications.

Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (Löfgrens and non-Löfgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated. High-resolution mass-spectrometry SpotLight proteomics and uni- and multivariate-statistical analyses were used for data processing. Major differences were particularly observed in control-BALF versus sarcoidosis-BALF. However, interestingly, information obtained from BALF profiles was still present (but less prominent) in matched serum profiles. By using information from orthogonal partial least squares discriminant analysis (OPLS-DA) differentiating 1) sarcoidosis-BALF and control-BALF and 2) LS-BALF vs. nonLS-BALF, control-serum and sarcoidosis-serum (p = 0.0007) as well as LS-serum and nonLS-serum (p = 0.006) could be distinguished. Noteworthy, many factors prominent in identifying controls and patients were those associated with Fc-regulation, but also features from the IgG-Fab region and novel peptide variants. Differences between phenotypes were mostly IgG-specificity related. The results support the analytical utility of SpotLight proteomics which prospectively have potential to differentiate closely related phenotypes from a simple blood test.

Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. METHODS: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. RESULTS: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. CONCLUSION: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

Patients with anti-Jo1 antibodies display a characteristic IgG Fc-glycan profile which is further enhanced in anti-Jo1 autoantibodies.

IgG Fc-glycans affect IgG function and are altered in autoimmune diseases and autoantibodies. Anti-histidyl tRNA synthetase autoantibodies (anti-Jo1) are frequent in patients with idiopathic inflammatory myopathies (IIM) and anti-synthetase syndrome (ASS) with associated interstitial lung disease (ILD). Thus, we hypothesized that the total-IgG Fc-glycans from Jo1+ versus Jo1- patients and anti-Jo1-IgG would show characteristic differences, and that particular Fc-glycan features would be associated with specific clinical manifestations. By proteomics based mass spectrometry we observed a high abundance of agalactosylated IgG1 Fc-glycans in ASS/IIM patients (n = 44) compared to healthy age matched controls (n = 24). Using intra-individual normalization of the main agalactosylated glycan (FA2) of IgG1 vs FA2-IgG2, ASS/IIM and controls were distinguished with an area under the curve (AUC) of 79 ± 6%. For Jo1+ patients (n = 19) the AUCs went up to 88 ± 6%. Bisected and afucosylated Fc-glycans were significantly lower in Jo1+ compared to Jo1- patients. Anti-Jo1-IgG enriched from eleven patients contained even significantly lower abundances of bisected, afucosylated and galactosylated forms compared to matched total-IgG. ASS and ILD diagnosis, as well as lysozyme and thrombospondin correlated with Jo1+ characteristic Fc-glycan features. These results suggest that the anti-Jo1+ patient Fc-glycan profile contains phenotype specific features which may underlie the pathogenic role of Jo1 autoantibodies.

Altered Fc galactosylation in IgG4 is a potential serum marker for chronic lung disease.

Characterising chronic lung diseases is challenging. New, less invasive diagnostics are needed to decipher disease pathologies and subphenotypes. Fc galactosylation is known to affect IgG function, and is altered in autoimmune disorders and under other pathological conditions. We tested how well Fc glycans in IgG from bronchoalveolar lavage fluid (BALF) and serum correlated, and if the Fc glycan profile could reveal pulmonary inflammation. A shotgun proteomics approach was used to profile Fc glycans in serum and BALF of controls (n=12) and sarcoidosis phenotypes (Löfgren's syndrome (LS), n=11; and non-LS, n=12). Results were further validated in severe asthma (SA) (n=20) and published rheumatoid arthritis (RA) patient data (n=13) including clinical information. Intra-individually, Fc-galactosylation status of IgG1 (R2=0.87) and IgG4 (R2=0.95) correlated well between matrixes. Following GlycoAge-index correction, the ratio between agalactosylated and digalactosylated Fc glycans of IgG4 could distinguish sarcoidosis and SA from healthy and RA subjects with a mean±se area under the curve (AUC) of 78±6%. The AUC increased to 83±6% using the more chronic lung disease types (non-LS and SA) and most strikingly, to 87±6% for the SA subgroup. The results indicate that the Fc galactosylation status of IgG4 is a potential blood test marker for chronic lung inflammation.

Streptococcal Endo-ß-N-Acetylglucosaminidase Suppresses Antibody-Mediated Inflammation In Vivo.

Endo-ß-N-acetylglucosaminidase (EndoS) is a family 18 glycosyl hydrolase secreted by Streptococcus pyogenes. Recombinant EndoS hydrolyzes the ß-1,4-di-N-acetylchitobiose core of the N-linked complex type glycan on the asparagine 297 of the γ-chains of IgG. Here, we report that EndoS and IgG hydrolyzed by EndoS induced suppression of local immune complex (IC)-mediated arthritis. A small amount (1 µg given i.v. to a mouse) of EndoS was sufficient to inhibit IgG-mediated arthritis in mice. The presence of EndoS disturbed larger IC lattice formation both in vitro and in vivo, as visualized with anti-C3b staining. Neither complement binding in vitro nor antigen-antibody binding per se were affected. Thus, EndoS could potentially be used for treating patients with IC-mediated pathology.