Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

Transarterial interventions in civilian gunshot wound injury: experience from a level-1 trauma center.

PURPOSE: To assess the effectiveness of trans-arterial vascular interventions in treatment of civilian gunshot wounds (GSW). MATERIALS AND METHODS: A retrospective review was performed at a level-1 trauma center to include 46 consecutive adults admitted due to GSW related hemorrhage and treated with endovascular interventions from July 2018 to July 2022. Patient demographics and procedural metrics were retrieved. Primary outcomes of interest include technical success and in-hospital mortality. Factors of mortality were assessed using a logistic regression model. RESULTS: Twenty-one patients were brought to the endovascular suite directly (endovascular group) from the trauma bay and 25 patients after treatment in the operating room (OR group). The OR group had higher hemodynamic instability (48.0% vs 19.0%, p = 0.040), lower hemoglobin (12.9 vs 10.1, p = 0.001) and platelet counts (235.2 vs 155.1, p = 0.003), and worse Acute Physiology and Chronic Health Evaluation (APACHE) score (4.1 vs 10.2, p < 0.0001) at the time of initial presentation. Technical success was achieved in all 40 cases in which targeted embolization was attempted (100%). Empiric embolization was performed in 6/46 (13.0%) patients based on computed tomographic angiogram (CTA) and operative findings. Stent-grafts were placed in 3 patients for subclavian artery injuries. Availability of pre-intervention CTA was associated with shorter fluoroscopy time (19.8 ± 12.1 vs 30.7 ± 18.6 min, p = 0.030). A total of 41 patients were discharged in stable condition (89.1%). Hollow organ injury was associated with mortality (p = 0.039). CONCLUSION: Endovascular embolization and stenting were effective in managing hemorrhage due to GSW in a carefully selected population. Hollow organ injury was a statistically significant predictor of mortality. Pre-intervention CTA enabled targeted, shorter and equally effective procedures.

Modulating antibody effector functions by Fc glycoengineering.

Antibody based drugs, including IgG monoclonal antibodies, are an expanding class of therapeutics widely employed to treat cancer, autoimmune and infectious diseases. IgG antibodies have a conserved N-glycosylation site at Asn297 that bears complex type N-glycans which, along with other less conserved N- and O-glycosylation sites, fine-tune effector functions, complement activation, and half-life of antibodies. Fucosylation, galactosylation, sialylation, bisection and mannosylation all generate glycoforms that interact in a specific manner with different cellular antibody receptors and are linked to a distinct functional profile. Antibodies, including those employed in clinical settings, are generated with a mixture of glycoforms attached to them, which has an impact on their efficacy, stability and effector functions. It is therefore of great interest to produce antibodies containing only tailored glycoforms with specific effects associated with them. To this end, several antibody engineering strategies have been developed, including the usage of engineered mammalian cell lines, in vitro and in vivo glycoengineering.

Non-Antigenic Modulation of Antigen Receptor (TCR) Cß-FG Loop Modulates Signalling: Implications of External Factors Influencing T-Cell Responses.

T-cell recognition of antigens is complex, leading to biochemical and cellular events that impart both specific and targeted immune responses. The end result is an array of cytokines that facilitate the direction and intensity of the immune reaction-such as T-cell proliferation, differentiation, macrophage activation, and B-cell isotype switching-all of which may be necessary and appropriate to eliminate the antigen and induce adaptive immunity. Using in silico docking to identify small molecules that putatively bind to the T-cell Cß-FG loop, we have shown in vitro using an antigen presentation assay that T-cell signalling is altered. The idea of modulating T-cell signalling independently of antigens by directly targeting the FG loop is novel and warrants further study.

Mechanism of glycoform specificity and in vivo protection by an anti-afucosylated IgG nanobody.

Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-ß-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-ß-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.

Mechanism of glycoform specificity and protection against antibody dependent enhancement by an anti-afucosylated IgG nanobody.

Immunoglobulin G (IgG) antibodies contain a single, complex N -glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

The role of adjuvants in overcoming antibacterial resistance due to enzymatic drug modification.

Antibacterial resistance is a prominent issue with monotherapy often leading to treatment failure in serious infections. Many mechanisms can lead to antibacterial resistance including deactivation of antibacterial agents by bacterial enzymes. Enzymatic drug modification confers resistance to ß-lactams, aminoglycosides, chloramphenicol, macrolides, isoniazid, rifamycins, fosfomycin and lincosamides. Novel enzyme inhibitor adjuvants have been developed in an attempt to overcome resistance to these agents, only a few of which have so far reached the market. This review discusses the different enzymatic processes that lead to deactivation of antibacterial agents and provides an update on the current and potential enzyme inhibitors that may restore bacterial susceptibility.

An experimental and theoretical charge density study of theophylline and malonic acid cocrystallization.

The pharmaceutical agent theophylline (THEO) is primarily used as a bronchodilator and is commercially available in both tablet and liquid dosage forms. THEO is highly hygroscopic, reducing its stability, overall shelf-life, and therefore usage as a drug. THEO and dicarboxylic acid cocrystals were designed by Trask et al. in an attempt to decrease the hygroscopic behaviour of THEO; cocrystallisation of THEO with malonic acid (MA) did not improve the hygroscopic stability of THEO in simulated atmospheric humidity testing. The current study employed high-resolution X-ray crystallography, and Density Functional Theory (DFT) calculations to examine the electron density distribution (EDD) changes between the cocrystal and its individual components. The EED changes identified the reasons why the THEO:MA cocrystal did not alter the hygroscopic profile of THEO. The cocrystal was equally porous, with atomic packing factors (APF) similar to those of THEO 0.73 vs. 0.71, respectively. The THEO:MA (1) cocrystal structure is held together by an array of interactions; a heterogeneous synthon between the imidazole and a carboxylic fragment stabilising the asymmetric unit, a pyrimidine-imidazole homosynthon, and an aromatic cycle stack between two THEO moieties have been identified, providing 9.7-12.9 kJ mol-1 of stability. These factors did not change the overall relative stability of the cocrystal relative to its individual THEO and MA components, as shown by cocrystal (1) and THEO being equally stable, with calculated lattice energies within 2.5 kJ mol-1 of one other. The hydrogen bond analysis and fragmented atomic charge analysis highlighted that the formation of (1) combined both the EDD of THEO and MA with no net chemical change, suggesting that the reverse reaction - (1) back to THEO and MA - is of equal potential, ultimately producing THEO hydrate formation, in agreement with the work of Trask et al. These results highlight that a review of the EDD change associated with a chemical reaction can aid in understanding cocrystal design. In addition, they indicate that cocrystal design requires further investigation before becoming a reliable process, with particular emphasis on identifying the appropriate balance of synthon engineering, weak interactions, and packing dynamics.

Exploring the excited-state charge transfer fluorescence profile of 7-hydroxycoumarin and 2-methylimidazole - a combined X-ray diffraction and theoretical approach.

This study investigated the effect of 2-methylimidazole (2-MIM) addition on the fluorescence of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate using low-cost density functional theory (DFT) and Time-Dependent DFT calculations on single crystal X-ray geometries of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate hydrate (1), 2-MIM (2), and the 1 : 1 co-crystal of (1) and (2), (3). At low concentrations (1 : 1-1 : 10) of 2-MIM, the fluorophore shows a decrease in the fluorescence intensity, but at higher concentrations (above 1 : 10) the fluorescence excitation maximum shifted from 354 nm to 405 nm, with a significant emission intensity increase. The changed excitation and emission profile at high concentrations is due to the deprotonation of the coumarin's phenolic group, which was confirmed by the increased shielding of the aromatic protons in the titration 1H NMR spectra. The experimental fluorescence data between the 1 : 1 and 1 : 10 ratios agreed with the theoretical fluorescence data, with a redshift and decreased intensity when comparing (1) and (3). The data indicated that combining the fluorophore with 2-MIM increased levels of vibronic coupling between 2-MIM and the fluorophore decreasing de-excitation efficiency. These increased vibronic changes were due to charge transfer between the fluorophore and 2-MIM in (3). The subtle movement of the proton, H(5) toward N(2') (0.07 Å) caused a significant decrease in fluorescence due to electron density distribution (EDD) changes. This was identified by comparison of the EDD in the excited (S1) and ground (S0) states plotted as an isosurface of EDD difference. For the higher concentrations, an alternative excitation pathway was explored by modifying the crystal geometry of (3) based on 1H NMR spectroscopy data to resemble excitoplexes. Theses excitoplex geometries reflected the fluorescence profile of the fluorophore with high concentrations of 2-MIM; there were dramatic changes in the theoretical fluorescence pathway, which was 100% vibronic coupling compared to 15.31% in the free fluorophore. At this concentration, the de-excitation pathway causes remodelling of the lactone ring via stretching/breaking the CO bond in the S1 causing increased fluorescence by movement of the transition dipole moment. These results reflect previous studies, but the methods used are less experimentally and computationally expensive. This study is among the first to explain charge transfer fluorescence using crystalline geometries. This study will be of interest to the fields of crystal engineering and fluorescence spectroscopy.

Mechanism of cooperative N-glycan processing by the multi-modular endoglycosidase EndoE.

Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-ß-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-ß-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.

Sculpting therapeutic monoclonal antibody N-glycans using endoglycosidases.

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-ß-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity. We discuss the molecular mechanism by which ENGases recognize different N-glycans and protein substrates, especially those that are specific for IgG antibodies, in order to rationalize the glycoengineering of immunotherapeutic antibodies, which increase the impact on the treatment of myriad diseases.

Management of Recurrent Myocarditis Due to Desmoplakin Cardiomyopathy: Diagnostic and Therapeutic Challenges.

We present the case of a 25-year-old woman with desmoplakin cardiomyopathy-related myocarditis. Her high-sensitivity troponin and symptoms improved with pulse steroid therapy and mycophenolate mofetil. The literature lacks data to effectively guide the management of recurrent myocarditis in desmoplakin cardiomyopathy. (Level of Difficulty: Advanced.).

A Guide to In Silico Drug Design.

The drug discovery process is a rocky path that is full of challenges, with the result that very few candidates progress from hit compound to a commercially available product, often due to factors, such as poor binding affinity, off-target effects, or physicochemical properties, such as solubility or stability. This process is further complicated by high research and development costs and time requirements. It is thus important to optimise every step of the process in order to maximise the chances of success. As a result of the recent advancements in computer power and technology, computer-aided drug design (CADD) has become an integral part of modern drug discovery to guide and accelerate the process. In this review, we present an overview of the important CADD methods and applications, such as in silico structure prediction, refinement, modelling and target validation, that are commonly used in this area.

Development of a Swine Polytrauma Model in the Absence of Fluid Resuscitation.

BACKGROUND: In locations in which access to resuscitative therapy may be limited, treating polytraumatized patients present a challenge. There is a pressing need for adjuncts that can be delivered in these settings. To assess these adjuncts, a model representative of this clinical scenario is necessary. We aimed to develop a hemorrhage and polytrauma model in the absence of fluid resuscitation. MATERIALS AND METHODS: This study consisted of two parts: pulmonary contusion dose-finding (n = 6) and polytrauma with evaluation of varying hemorrhage volumes (n = 6). We applied three, six, or nine nonpenetrating captive bolt-gun discharges to the dose-finding group and obtained computed tomography (CT) images. We segmented images to assess contusion volumes. We subjected the second group to tibial fracture, pulmonary contusion, and controlled hemorrhage of 20%, 30%, or 40% and observed for 3 hours or until death. We used Kaplan-Meier analysis to assess survival. We also assessed hemodynamic and metabolic parameters. RESULTS: Contusion volumes for three, six, and nine nonpenetrating captive bolt-gun discharges were 24 ± 28, 50 ± 31, and 63 ± 77 cm3, respectively (p = .679). Animals receiving at least six discharges suffered concomitant parenchymal laceration, whereas one of two swine subjected to three discharges had lacerations. Mortality was 100% at 12 and 115 minutes in the 40% and 30% hemorrhage groups, respectively, and 50% at 3 hours in the 20% group. CONCLUSION: This study characterizes a titratable hemorrhage and polytrauma model in the absence of fluid resuscitation. This model can be useful in evaluating resuscitative adjuncts that can be delivered in areas remote to healthcare access.

Understanding Hygroscopicity of Theophylline via a Novel Cocrystal Polymorph: A Charge Density Study.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.