Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Bristles before down: A new perspective on the functional origin of feathers

Over the course of the last two decades, the understanding of the early evolution of feathers in nonavian dinosaurs has been revolutionized. It is now recognized that early feathers had a simple form comparable in general structure to the hairs of mammals. Insight into the prevalence of simple feathers throughout the dinosaur family tree has gradually arisen in tandem with the growing evidence for endothermic dinosaur metabolisms. This has led to the generally accepted opinion that the early feather coats of dinosaurs functioned as thermo insulation. However, thermo insulation is often erroneously stated to be a likely functional explanation for the origin of feathers. The problem with this explanation is that, like mammalian hair, simple feathers could serve as insulation only when present in sufficiently high concentrations. The theory therefore necessitates the origination of feathers en masse. We advocate for a novel origin theory of feathers as bristles. Bristles are facial feathers common among modern birds that function like mammalian tactile whiskers, and are frequently simple and hair‐like in form. Bristles serve their role in low concentrations, and therefore offer a feasible first stage in feather evolution.     

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Background

A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.

Methods

A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.

Results and Discussion

The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.

Conclusion

The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.