N-glycosylation on Oryza sativa root germin-like protein 1 is conserved but not required for stability or activity.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Diminished Ost3-dependent N-glycosylation of the BiP nucleotide exchange factor Sil1 is an adaptive response to reductive ER stress.

BiP (Kar2 in yeast) is an essential Hsp70 chaperone and master regulator of endoplasmic reticulum (ER) function. BiP's activity is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, Sil1 and Lhs1. Both Sil1 and Lhs1 are glycoproteins, but how N-glycosylation regulates their function is not known. Here, we show that N-glycosylation of Sil1, but not of Lhs1, is diminished upon reductive stress. N-glycosylation of Sil1 is predominantly Ost3-dependent and requires a functional Ost3 CxxC thioredoxin motif. N-glycosylation of Lhs1 is largely Ost3-independent and independent of the CxxC motif. Unglycosylated Sil1 is not only functional but is more effective at rescuing loss of Lhs1 activity than N-glycosylated Sil1. Furthermore, substitution of the redox active cysteine pair C52 and C57 in the N terminus of Sil1 results in the Doa10-dependent ERAD of this mutant protein. We propose that reductive stress in the ER inhibits the Ost3-dependent N-glycosylation of Sil1, which regulates specific BiP functions appropriate to the needs of the ER under reductive stress.

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity.

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.

Proteomics Reveals Profound Metabolic Changes in the Alcohol Use Disorder Brain.

Changes in brain metabolism are a hallmark of alcohol use disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in prefrontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.