Localization of O-glycans in MUC1 glycoproteins using electron-capture dissociation fragmentation mass spectrometry

Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cellderived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one-and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc) 2 -Hex 2 -(NeuAc) 2 being the most complex glycan detected on Thr 194 in both cellular and secreted apoE. Four additional glycans were identified on apoE(283-299), and using ␤-elimination/alkylation by methylamine in vitro, we identified Ser 290 as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry. Molecular & Cellular Proteomics 9: 1968 -1981, 2010.

Section: Determination Of Location Of Second Site Of O-glycosylation Inmentioning

confidence: 98%

Glycosylation and Sialylation of Macrophage-derived Human Apolipoprotein E Analyzed by SDS-PAGE and Mass Spectrometry

Lee

Kockx

Raftery

et al. 2010

“…Additionally, the exact glycosylation site of identified peptides containing several Ser/Thr residues cannot be predicted due to the lack of a consensus sequence for mucin-type O-glycosylation. The alternative fragmentation techniques electron capture dissociation (ECD) (38,39) and electron transfer dissociation (ETD) (40) have been introduced for site-specific analysis of CID-labile PTMs but characterization of protein O-glycosylations using ECD/ETD have generally been limited to synthetic glycopeptides or single glycoproteins (41)(42)(43)(44)(45) (46). We have previously developed a sialic acid specific capture-and-release protocol for the enrichment of both N-and O-glycosylated peptides from sialylated glycoproteins in biological samples using hydrazide chemistry (37).…”

mentioning

confidence: 99%

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

Halim

Nilsson

Rüetschi

et al. 2012

141

151

“…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)(24)(25)(26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23,26,27).…”

mentioning

confidence: 99%

“…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)(24)(25)(26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23,26,27).…”

mentioning

confidence: 99%

Clustered O-Glycans of IgA1

Takahashi

Wall

Suzuki

et al. 2010

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-Schö nlein 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).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.