Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

The fungal pathogen Magnaporthe oryzae can cause rice blast and wheat blast diseases, which threatens worldwide food production. During infection, M. oryzae follows a sequence of distinct developmental stages adapted to survival and invasion of the host environment. M. oryzae attaches onto the host by the conidium, and then develops an appressorium to breach the host cuticle. After penetrating, it forms invasive hyphae to quickly spread in the host cells. Numerous genetic studies have focused on the mechanisms underlying each step in the infection process, but systemic approaches are needed for a broader, integrated understanding of regulatory events during M. oryzae pathogenesis. Many infection-related signaling events are regulated through post-translational protein modifications within the pathogen. N-linked glycosylation, in which a glycan moiety is added to the amide group of an asparagine residue, is an abundant modification known to be essential for M. oryzae infection. In this study, we employed a quantitative proteomics analysis to unravel the overall regulatory mechanisms of N-glycosylation at different developmental stages of M. oryzae. We detected changes in N-glycosylation levels at 559 glycosylated residues (N-glycosites) in 355 proteins during different stages, and determined that the ER quality control system is elaborately regulated by N-glycosylation. The insights gained will help us to better understand the regulatory mechanisms of infection in pathogenic fungi. These findings may be also important for developing novel strategies for fungal disease control. Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

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