Inhibitory Fc-Gamma IIb Receptor Signaling Induced by Multivalent IgG-Fc Is Dependent on Sialylation.

Immunoglobulin (IgG) Fc glycosylation has been shown to be important for the biological activity of antibodies. Fc sialylation is important for the anti-inflammatory activity of IgGs. However, evaluating the structure-activity relationship (SAR) of antibody Fc glycosylation has been hindered using simplified in vitro models in which antibodies are often displayed in monomeric forms. Presenting antibodies in monomeric forms may not accurately replicate the natural environment of the antibodies when binding their antigen in vivo. To address these limitations, we used different Fc-containing molecules, displaying their Fc domains in monovalent and multivalent fashion. Given the inhibitory role of Fc gamma receptor IIb (FcγRIIb) in autoimmune and inflammatory diseases, we focused on evaluating the impact of Fc sialylation on the activation of FcγRIIb. We report for the first time that in human cellular systems, sialic acid mediates the induction of FcγRIIb phosphorylation by IgG-Fc when the IgG-Fc is displayed in a multivalent fashion. This effect was observed with different types of therapeutic agents such as sialylated anti-TNFα antibodies, sialylated IVIg and sialylated recombinant multivalent Fc products. These studies represent the first report of the specific effects of Fc sialylation on FcγRIIb signaling on human immune cells and may help in the characterization of the anti-inflammatory activity of Fc-containing therapeutic candidates.

Optimizing interactions between soluble silk fibroin and capryl glucoside for design of a natural and high-performance co-surfactant system.

OBJECTIVES: Because of the strong consumer driver towards more natural or higher sustainability cosmetic products, silk fibroin was evaluated to help develop a formulation with natural and effective ingredients for personal care. In order to exploit the physical properties of silk fibroin, it was evaluated to maximize the surfactant properties of other commercial ingredients to lower surface tension and build up viscosity. A synergistic effect was seen between silk fibroin and capryl glucoside, a sugar surfactant which exhibited a natural and effective co-surfactant system. This system demonstrated better surface tension properties than sodium laureth sulphate (SLES), cocamidopropyl betaine (CAPB), rhamnolipids and sophorolipids, which led to greater foamability and cleansing properties. This system proved to also be compatible with polysaccharide viscosity modifiers to enhance the viscosity of the system. The present study comprises a systematic exploration of natural formulation development of silk proteins and other natural ingredients, which result in high performance such as enhanced foam quality, foam stability and enhanced sebum removal. All of these properties are desirable and may utilized when formulating cleaners and shampoos. METHODS: A force tensiometer, Attension Sigma 701, was used to measure the surface tension of the silk protein and its various combinations with biosurfactants and biopolymers. To measure bulk rheology, a traditional mechanical rheometer TA DHR-3 was utilized. Foaming tests and sebum removal assays were also carried out to evaluate the performance of the samples. RESULTS: Silk fibroin was evaluated to maximize the surfactant properties of other commercial systems to develop a formulation containing natural and effective ingredients for personal care. The surface activity of silk proteins was seen to be synergistically enhanced in the presence of sugar surfactants such as capryl glucoside, resulting in a surface tension at the air-water interface which is lower than either that of pure silk fibroin or pure capryl glucoside. This surface tension value is additionally lower than that obtained from currently utilized synthetic surfactants like sodium laureth sulphate (SLES) and cocamidopropyl betaine (CAPB). This reduction in surface tension demonstrated greater foamability and cleansing properties than that of the commercial systems. The very low surface tension values obtained through combinations of silk proteins and glucoside resulted in a natural and effective co-surfactant system by forming high-quality stable foams and enhancing sebum removal. The rheological performance of the silk proteins was impacted through microstructure modifications as a result of interactions with biopolymers like carrageenan. This shows that this system is compatible with polysaccharide viscosity modifiers. It was observed that both the flow curve and the absolute viscosity values were significantly impacted in the presence of carrageenan, with higher viscosity generation and significant non-Newtonian/shear thinning behaviour evolution. These results indicate that the silk fibroin can be utilized to build a high-performance natural product and significantly enhance the performance of other natural/sustainable cosmetic formulations through building synergistic interactions with other natural ingredients such as sugar surfactants and biopolymers. These properties exhibited by this system are all desirable for cleansers and shampoos within the cosmetic industry. CONCLUSION: Silk fibroin in combination with capryl glucoside outperforms other commercial surfactants that are commonly used in the industry because of its surface-active behaviour and synergy. This system is then enhanced further with polysaccharide rheological modifiers, carrageen and xanthan gum to help build up viscosity. The complex mixture of silk fibroin, sugar surfactant and biopolymer results in a formulation that is all natural, while still having high performance by achieving great foamability and enhanced sebum removal. The mixture can further be used to formulate a fully natural product such as a cleanser or shampoo while still having the same or greater effectiveness as synthetic surfactants and ingredients typically used in cosmetic formulations.

OBJECTIFS: En raison de la forte incitation des consommateurs vers des produits cosmétiques plus naturels ou plus durables, la fibroïne de soie a été évaluée pour aider à développer une formulation avec des ingrédients naturels et efficaces pour les soins personnels. Afin d'exploiter les propriétés physiques de la fibroïne de soie, il a été évalué pour maximiser les propriétés tensioactives d'autres ingrédients commerciaux pour abaisser la tension superficielle et augmenter la viscosité. Un effet synergique a été observé entre la fibroïne de soie et le capryl glucoside, un tensioactif de sucre qui présentait un système de co-tensioactif naturel et efficace. Ce système a démontré de meilleures propriétés de tension superficielle que le laureth sulfate de sodium (SLES), la cocamidopropyl bétaïne (CAPB), les rhamnolipides et les sophorolipides, ce qui a conduit à une plus grande moussabilité et des propriétés de nettoyage. Ce système s'est avéré également compatible avec les modificateurs de viscosité polysaccharidiques pour améliorer la viscosité du système. La présente étude comprend une exploration systématique du développement de formulation naturelle de protéines de soie et d'autres ingrédients naturels, qui se traduisent par des performances élevées telles qu'une qualité de mousse améliorée, une stabilité de la mousse et une élimination améliorée du sébum. Toutes ces propriétés sont souhaitables et peuvent être utilisées lors de la formulation de nettoyants et de shampooings. MÉTHODES: Un tensiomètre de force, Attension Sigma 701, a été utilisé pour mesurer la tension superficielle de la protéine de soie et ses diverses combinaisons avec des biosurfactants et des biopolymères. Pour mesurer la rhéologie de masse, un rhéomètre mécanique traditionnel TA DHR-3 a été utilisé. Tests de moussage et dosages d'élimination du sébum ont également été réalisés pour évaluer les performances des échantillons. RÉSULTATS: La fibroïne de soie a été évaluée pour maximiser les propriétés tensioactives d'autres systèmes commerciaux afin de développer une formulation contenant des ingrédients naturels et efficaces pour les soins personnels. L'activité de surface des protéines de soie s'est avérée être renforcée de manière synergique en présence d'agents tensioactifs de sucre tels que le capryl glucoside, entraînant une tension de surface à l'interface air-eau qui est inférieure à celle de la fibroïne de soie pure ou du capryl glucoside pur. Cette valeur de tension superficielle est en outre inférieure à celle obtenue à partir d'agents tensioactifs synthétiques actuellement utilisés comme le laureth sulfate de sodium (SLES) et la cocamidopropyl bétaïne (CAPB). Cette réduction de la tension superficielle a démontré une moussabilité et des propriétés de nettoyage supérieures à celles des systèmes commerciaux. Les très faibles valeurs de tension superficielle obtenues grâce à des combinaisons de protéines de soie et de glucoside ont abouti à un système de co-tensioactif naturel et efficace en formant des mousses stables de haute qualité et en améliorant l'élimination du sébum. Les performances rhéologiques des protéines de soie ont été affectées par des modifications de microstructure à la suite d'interactions avec des biopolymères comme le carraghénane. Cela montre que ce système est compatible avec les modificateurs de viscosité polysaccharidiques. Il a été observé que la courbe d'écoulement et les valeurs de viscosité absolue étaient significativement affectées en présence de carraghénane, avec une génération de viscosité plus élevée et une évolution significative du comportement d'amincissement non newtonien / cisaillement. Ces résultats indiquent que la fibroïne de soie peut être utilisée pour construire un produit naturel haute performance et améliorer considérablement les performances d'autres formulations cosmétiques naturelles / durables en créant des interactions synergiques avec d'autres ingrédients naturels tels que les tensioactifs de sucre et les biopolymères. Ces propriétés présentées par ce système sont toutes souhaitables pour les nettoyants et les shampooings dans l'industrie cosmétique. CONCLUSION: La fibroïne de soie en combinaison avec le capryl glucoside surpasse les autres tensioactifs du commerce couramment utilisés dans l'industrie en raison de son comportement tensioactif et de sa synergie. Ce système est ensuite amélioré avec des modificateurs rhéologiques polysaccharidiques, de la carraghénine et de la gomme xanthane pour aider à augmenter la viscosité. Le mélange complexe de fibroïne de soie, de tensioactif de sucre et de biopolymère donne une formulation entièrement naturelle, tout en conservant des performances élevées en obtenant une grande capacité de moussage et une élimination améliorée du sébum. Le mélange peut en outre être utilisé pour formuler un produit entièrement naturel tel qu'un nettoyant ou un shampooing tout en ayant toujours la même efficacité ou une plus grande efficacité que les tensioactifs synthétiques et les ingrédients généralement utilisés dans les formulations cosmétiques.

Engineered IgG1-Fc Molecules Define Valency Control of Cell Surface Fcγ Receptor Inhibition and Activation in Endosomes.

The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 µm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

Anti-inflammatory Activity of IgG-Fc.

Over 80 different autoimmune disorders have been identified. A common denominator across most of these disorders is the presence of pathogenic autoantibodies. The pathogenic and inflammatory nature of antibodies is well accepted, and over the last three decades, evidence in humans and rodent models has revealed that antibodies can induce anti-inflammatory activities. The discovery of the relationship between immunoglobulin G (IgG) glycovariants and disease activity in autoimmune patients has provided insight into the structural and functional characteristics of IgG associated with its pro- and anti-inflammatory activity. In this chapter, we discuss evidence of the anti-inflammatory nature of IgG and the mechanisms by which this activity is exerted. Current clinical evidence of this anti-inflammatory activity is also discussed.

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.

High-resolution physicochemical characterization of different intravenous immunoglobulin products.

Intravenous immunoglobulin (IVIg) is a complex mixture drug comprising diverse immunoglobulins and non-IgG proteins purified from the plasma of thousands of healthy donors. Approved IVIg products on the market differ regarding source of plasma, isolation process, and formulation. These products are used widely, and often interchangeably, for the treatment of immunodeficiency and autoimmune and inflammatory diseases, but their mechanisms of action in different indications are not well understood. A primary limitation to understanding the therapeutic relevance of specific components within IVIg has been the limited resolution of analytics historically implemented to characterize its complex mixture. In this study, high-resolution analytics were applied to better understand the composition of IVIg and product variations. We characterized three approved IVIg products: Gammagard®, Privigen®, and Octagam®. Differences in the distribution of molecular weight species, IgG sequence variants, isoforms, glycoforms, and the repertoire of previously reported antibody specificities were identified. We also compared the effect of aging on these products to identify changes in size distribution and posttranslational modifications. This type of characterization may provide insights into the specific factors and components of IVIg that may influence its activity and ultimately lead to optimization of IVIg products for use in autoimmune diseases.

Elucidating the interplay between IgG-Fc valency and FcγR activation for the design of immune complex inhibitors.

Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.

Fc-gamma receptors: Attractive targets for autoimmune drug discovery searching for intelligent therapeutic designs.

Autoantibody immune complexes (ICs) mediate pathogenesis in multiple autoimmune diseases via direct interference with target function, complement fixation, and interaction with Fc-gamma receptors (FcγRs). Through high avidity interactions, ICs are able to crosslink low affinity FcγRs expressed on a wide variety of effector cells, leading to secretion of pro-inflammatory mediators and inducing cytotoxicity, ultimately resulting in tissue injury. Given their relevance in numerous autoimmune diseases, FcγRs have been considered as attractive therapeutic targets for the last three decades. However, a limited number of investigational drug candidates have been developed targeting FcγRs and only a few approved therapeutics have been associated with impacting FcγRs. This review provides a historical overview of the different therapeutic approaches used to target FcγRs for the treatment of autoimmune and inflammatory diseases.

A single glycan on IgE is indispensable for initiation of anaphylaxis.

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity.

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.

Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

A simple assay to probe disease-associated enzyme activity using glycosaminoglycan-assisted synthesized gold nanoparticles.

A simple assay to probe disease-associated enzyme activity using glycosaminoglycan-assisted synthesized gold nanoparticles is reported.

The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate.

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.

Renal injury is a consistent finding in Dutch Belted rabbits experimentally infected with enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin (Stx) and causes renal disease in humans. Dutch Belted (DB) rabbits naturally infected with EHEC O153 develop hemolytic-uremic syndrome-like disease. The aims of this study were to experimentally reproduce O153-induced renal disease in DB rabbits and investigate bacterial and host factors involved in pathogenesis. The pathogenicity of E. coli O157:H7 was also investigated in rabbits. The stx1AB region of O153 was sequenced. By use of liquid chromatography-tandem mass spectrometry, we identified homologs of the Stx receptor, globotriaosylceramide (Gb3), in rabbit kidney extracts. Infected rabbits developed clinical signs and intestinal and kidney lesions. Renal pathological changes consisted of intimal swelling, perivascular edema, erythrocyte fragmentation, capillary thickening, luminal constriction, leukocytic infiltration, mesangial deposits, and changes in Bowman's capsule and space. Sequence analysis of a approximately 7-kb region of the O153 chromosome indicated homology to the Stx1-producing bacteriophage H19B. Our findings indicate that DB rabbits are suitable for the study of the renal manifestations of EHEC infection in humans.

Biochemical characterization of the chondroitinase ABC I active site.

cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.

Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.

GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

Effects of glycosylation on peptide conformation: a synergistic experimental and computational study.

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.