It is well established that high doses of monomeric immunoglobulin G (IgG) purified from pooled human plasma [intravenous immunoglobulin (IVIG)] confer anti-inflammatory activity in a variety of autoimmune settings. However, exactly how those effects are mediated is not clear because of the heterogeneity of IVIG. Recent studies have demonstrated that the anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Here we determine the precise glycan requirements for this anti-inflammatory activity, allowing us to engineer an appropriate IgG1 Fc fragment, and thus generate a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency. This therapeutic molecule precisely defines the biologically active component of IVIG and helps guide development of an IVIG replacement with improved activity and availability.

This report is the first in a series of three focused on establishing congruent strategies for carbohydrate sequencing. The reports are divided into (i) analytical considerations that account for all aspects of small oligomer structure by MS n disassembly, (ii) database support using an ion fragment library and associated tools for highthroughput analysis, and (iii) a concluding algorithm for defining oligosaccharide topology from MS n disassembly pathways. The analytical contribution of this first report explores the limits of structural detail exposed by ion trap mass spectrometry with samples prepared as methyl derivatives and analyzed as metal ion adducts. This data mining effort focuses on correlating the fragments of small oligomers to stereospecific glycan structures, an outcome attributed to a combination of metal ion adduction and analyte conformation. Facile glycosidic cleavage introduces a point of lability (pyranosyl-1-ene) that upon collisional activation initiates subsequent ring fragmentation. Product masses and ion intensities vary with interresidue linkage, branching position, and monomer stereochemistry. Excessive fragmentation is the property of small oligomers where collisional energy within a smaller number of oscillators dissipates through extensive fragmentation. The procedures discussed in this report are unified into a singular strategy using an ion trap mass spectrometer with the sensitivity expected for electron multiplier detection. Although a small set of structures have been discussed, the basic principles considered are fully congruent, with ample opportunities for expansion.The proliferation of reports attributing biological function to oligosaccaharide epitopes continues unabated. To fully appreciate their specific biological roles, an improved accounting of carbohydrate structural details needs greater scrutiny and more judicious reporting. Unfortunately, a comprehensive strategy for carbohydrate sequencing is lacking. Over the past decades, the absence of an integrated strategy to fully define a carbohydrate sequence has been given little attention due to overwhelming and direct functional lineage of nucleic acid and protein polymers. Even selective strategies to assign components of structure show little focus toward congruency. The reporting of partial sequences, assumed motifs, sequence arrays devoid of linkage and branching definition, and multiple structures enclosed in brackets are currently acceptable conclusions in published reports. The general acceptance of such reports diminishes the driving force for the development of improved and comprehensive methodologies.A sequence definition should provide all the components of structure necessary for reporting or synthesis. Structural components should include monomer identification, positions of interresidue linkage, and array topology, where a linkage definition includes anomericity, and array topologies describe linear and branching sequence information. When considering molecular glycosylation, sites of conjugatio...

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clari®ed, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The b-amyloid fragment (Ab) of the amyloid precursor protein induces calcium in¯ux, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium in¯ux and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Ab neurotoxicity. HC potentiated the Ab-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-Lcysteine blocked apoptosis following cotreatment with HC and Ab, indicating that apoptosis is associated with oxidative stress. These ®ndings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Ab.