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O-linked N-acetylglucosamine (O-GlcNAc) was discovered during studies using bovine milk galactosyltransferase to ‘map’ the surface topography on cells of the murine immune system (Torres and Hart, 1984). Later, O-GlcNAc was shown to reside almost exclusively in the nucleus and cytoplasm (Kearse and Hart, 1991b), and to be present in eukaryotes ranging from trypanosomes, yeast, plants, to man, as well as in viruses (Hart et al. 1989; Hart et al. 1994a; Greis and Hart, 1994). We now know that O-GlcNAc is an exceedingly abundant post-translational modification of specific serine/threonine residues of many important nuclear and cytoplasmic proteins (Haltiwanger et al.1992b). Figure 1 lists the O-GlcNAc-bearing proteins identified to date. O-GlcNAc is attached as a monosaccharide and is generally not further modified. The O-GlcNAc turn-over rates are typically many-times that of the polypeptide backbone on which it is found (Chou et al.1992a; Hart and Roquemore, unpublished). The saccharide is attached at sites similar to those also used by the ‘growth-factor’, proline-directed family of kinases (Roach, 1991; Taylor and Adams, 1992). O-GlcNAc-bearing proteins have a diverse range of functions, but are characterized by several common features: 1) They all are also phosphorylated. 2) They typically form specific and regulated multimeric associations with other polypeptides. 3) In several cases, O-GlcNAcylation and phosphorylation appear to be reciprocal events.

While probing murine lymphocyte cell surfaces with bovine milk galactosyltrans-ferase, we discovered a new form of protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine moieties (O-GlcNAc) (Torres and Hart 1984). Surprisingly, O-GlcNAc is highly abundant (lymphocytes have > 1.5 × 108 molecules/cell), and is exclusively localized in nucleoplasmic and cytoplasmic compartments of the cell (Kearse and Hart 1991b). Based upon probing with galactosyltransferase (Whiteheart et al. 1989), a myriad of proteins in both the nucleus and cytoplasm are modified by O-GlcNAc. O-GlcNAc has been found in eukaryotes from yeast to man (Haltiwanger et al. 1992b; Hart et al. 1989), but has not yet been detected in prokaryotes. Identified O-GlcNAc-bearing proteins are listed in Table 1. These intracellular glycoproteins have a diverse range of functions, including transcription regulatory factors, enzymes, nuclear pore proteins, cytoskeletal proteins, and viral proteins. However, all of these glycoproteins are also phosphoproteins that form reversible multimeric complexes, depending upon their phosphorylation states, thus suggesting that O-GlcNAc may playa role in mediating/modulating regulated and reversible protein subunit interactions.

We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. © 2015 by The American Society of Human Genetics. All rights reserved.

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC50 curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were