Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans.

α-Dystroglycan (α-DG) is a glycoprotein specifically modified with O-mannosyl glycans bearing long polysaccharides, termed matriglycans, which comprise repeating units of glucuronic acid and xylose. The matriglycan is linked to the O-mannosyl glycan core through two ribitol phosphate units that can be replaced with glycerol phosphate (GroP) units synthesized by fukutin and fukutin-related protein that transfer GroP from CDP-Gro. Here, we found that forced expression of the bacterial CDP-Gro synthase, TagD, from Bacillus subtilis could result in the overproduction of CDP-Gro in human colon carcinoma HCT116 cells. Western blot and liquid chromatography-tandem mass spectrometry analyses indicated that α-DG prepared from the TagD-expressing HCT116 cells contained abundant GroP and lacked matriglycans. Using the GroP-containing recombinant α-DG-Fc, we developed a novel monoclonal antibody, termed DG2, that reacts with several truncated glycoforms of α-DG, including GroP-terminated glycoforms lacking matriglycans; we verified the reactivity of DG2 against various types of knockout cells deficient in the biosynthesis of matriglycans. Accordingly, forced expression of TagD in HCT116 cells resulted in the reduction of matriglycans and an increase in DG2 reactivity. Collectively, our results indicate that DG2 could serve as a useful tool to determine tissue distribution and function of α-DG lacking matriglycans under physiological and pathophysiological conditions.

Distinctive and Complementary MS2 Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow.

High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies. Metal oxide has been applied to enrich phosphopeptides and sialylated glycopeptides, but its use to capture sulfated glycopeptides has not been investigated. Likewise, various complementary MS2 fragmentation modes have yet to be tested against sialylated and non-sialylated sulfoglycopeptides due to limited appropriate sample availability. In this study, we have investigated the feasibility of sequencing tryptic sulfated N-glycopeptide and its MS2 fragmentation characteristics by first optimizing the enrichment methods to allow efficient LC-MS detection and MS2 analysis by a combination of CID, HCD, ETD, and EThcD on hybrid and tribrid Orbitrap instruments. Characteristic sulfated glyco-oxonium ions and direct loss of sulfite from precursors were detected as evidences of sulfate modification. It is anticipated that the technical advances demonstrated in this study would allow a feasible extension of our sulfoglycomic analysis to sulfoglycoproteomics. Graphical Abstract ᅟ.

SELENOPROTEINS. CRL2 aids elimination of truncated selenoproteins produced by failed UGA/Sec decoding.

Selenocysteine (Sec) is translated from the codon UGA, typically a termination signal. Codon duality extends the genetic code; however, the coexistence of two competing UGA-decoding mechanisms immediately compromises proteome fidelity. Selenium availability tunes the reassignment of UGA to Sec. We report a CRL2 ubiquitin ligase-mediated protein quality-control system that specifically eliminates truncated proteins that result from reassignment failures. Exposing the peptide immediately N-terminal to Sec, a CRL2 recognition degron, promotes protein degradation. Sec incorporation destroys the degron, protecting read-through proteins from detection by CRL2. Our findings reveal a coupling between directed translation termination and proteolysis-assisted protein quality control, as well as a cellular strategy to cope with fluctuations in organismal selenium intake.

Efficient Mapping of Sulfated Glycotopes by Negative Ion Mode nanoLC-MS/MS-Based Sulfoglycomic Analysis of Permethylated Glycans.

We have previously developed the enabling techniques for sulfoglycomics based on mass spectrometry (MS) analysis of permethylated glycans, which preserves the attractive features of more reliable MS/MS sequencing compared with that performed on native glycans, while providing an easy way to separate and hence enrich the sulfated glycans. Unlike LC-MS/MS analysis of native glycans in negative ion mode that has been more widely in use, the characteristics and potential benefits of similar applications based on permethylated sulfated glycans have not been fully investigated. We report here the important features of reverse phase-based nanoLC-MS/MS analysis of permethylated sulfated glycans in negative ion mode and demonstrate that complementary sets of diagnostic fragment ions afforded can allow rapid identification of various fucosylated, sialylated, sulfated glycotopes and definitive determination of the location of sulfate in a way difficult to achieve by other means. A parallel acquisition of both higher collision energy and trap-based MS(2) coupled with a product dependent MS(3) is conceivably the most productive sulfoglycomic workflow currently possible and the manually curated fragmentation characteristics presented here will allow future developments in automating data analysis.

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells.

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.

Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen.

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.

Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities.

A fucose-containing glycoprotein fraction which stimulates spleen cell proliferation and cytokine expression has been identified from the water-soluble extract of Ganoderma lucidum. Proteomic analysis of mouse spleen cells treated with this glycoprotein fraction showed approximately 50% change of the proteome. Further studies on the activities of this glycoprotein fraction through selective proteolysis and glycosidic cleavage indicate that a fucose containing polysaccharide fraction is responsible for stimulating the expression of cytokines, especially IL-1, IL-2 and INF-gamma.