α-mannosidase I-dependent N-linked glycosylation affects hERG channel functional availability during reactivation

O-mannosylation and furin processing are critical maturation events for the KIAA1549 protein implicated in pediatric pilocytic astrocytomas.

The gene fusion KIAA1549::BRAF is a common oncogenic driver in pediatric pilocytic astrocytoma. The chimeric fusion protein that comprises most of the type 1 transmembrane KIAA1549 protein fused in its C-terminal cytosolic domain to the truncated BRAF oncogene without its N-terminal autoinhibitory domains. KIAA1549 has a large N-terminal ectodomain that undergoes extensive O-mannosylation directed by the POMT1/2 protein O-mannosyltransferase complex localized in the ER. Here, we investigated how posttranslational modifications contribute to KIAA1549 protein maturation in the secretory pathway. Using a panel of KIAA1549 expression constructs and glycoengineered HEK293 cells, we provide evidence that O-mannosylation of the KIAA1549 ectodomain is required for ER exit, acquisition of complex N-glycans, and subsequent furin-like cleavage in the Golgi. In the absence of POMT1/2 O-mannosylation KIAA1549 is retained in ER and fails to undergo proprotein processing. Our findings confirm and extend our previous studies with the KIAA1549::BRAF fusion protein, where oncogenic transformation was dependent on POMT1/2 O-mannosylation and affected on furin processing. Thus, these intrinsic posttranslational modifications of the KIAA1549 ectodomain are critical determinants of the cellular trafficking that lead to activation of the BRAF oncogene, potentially involving a cascade of cleavage events in the Golgi that ultimately release a truncated but fully active BRAF kinase domain into the cytosol.

Impacts of N-glycanase1 (NGLY1) Down Regulation on the Function of Mitochondria.

Structural and molecular basis of O-GlcNAcylation-phosphorylation crosstalk in protein Kinase-B (Akt2).

Post Translational Modifications significantly influence conformational preferences and activity of proteins. In Protein Kinase-B (Akt2), numerous residues undergo phosphorylation and O-GlcNAcylation, in same region. This results in a crosstalk mechanism modulating Akt2 activity, and is dysregulated in metabolic diseases such as type-II diabetes. However, atomic level details of how such modifications influence Akt2 dynamics and activity is poorly understood. In this work, we employed in silico approaches to explore the conformational preferences and functional consequences of Akt2 in O-GlcNAcylated, phosphorylated and unmodified states. Multi-microsecond simulations, resulting in total sampling of 7.6μs were performed on Akt2 in all the above states, and the structural effects of such modifications were analyzed. We found that such diverse modifications result in considerable difference in Akt2 dynamics. In phosphorylated state, the activation loop of Akt2 is highly flexible and has high solvent accessibility, in contrast with the buried nature of O-GlcNAcylated Akt2. This involved contacts between the O-GlcNAc residues and the pleckstrein homology domain, resulting in its burial. In the phosphorylated state, an elaborate hydrogen bonded network was observed, where phosphorylated threonine residues and active site residues formed hydrogen bonds with the phosphatidylinositol (3,4,5)-trisphosphate (PIP3) binding residues, forming the structural basis of autoinhibition and phosphatase shielding by Akt2. We further sampled for first time, the conformational changes associated with the PIP3 induced Akt2 activation from the Akt2 simulations in presence of PIP3, ATP and Mg2+ cofactor. This work demonstrated the structural effects of varied post-translational modifications, which form a crosstalk mechanism regulating Akt2 activity.

The protein sequence modulates terminal GalNAc incorporation during N-glycan biosynthesis.

N-linked glycosylation of the EP3α receptor is essential for the regulation of platelet homeostasis

Platelet activation and aggregation represent pivotal events in hemostasis and thrombosis. Prostaglandin E2 (PGE 2 ) receptor subtype 3 (EP3), the most abundantly expressed PGE 2 receptor in platelets, regulates platelet activation. Yet, the underlying mechanisms remain incompletely defined. Glycosylation, one of the most common post-translational modifications, plays a crucial role in maintaining platelet function. However, the specific contribution of EP3 glycosylation in platelets has not yet been investigated. We identified N16 and N193 of the EP3α isoform as sites for N-linked glycosylation and demonstrated that EP3α glycosylation stabilizes its capacity for G i coupling and sustains G i -mediated cAMP and Ca 2+ flux, both key modulators of platelet function. In contrast, EP3α mutants impaired U46619-, ADP- and thrombin- induced platelet aggregation and thrombin-induced clot retraction. Notably, both EP3α mutants and EP3 deficiency attenuated FeCl 3 -induced arterial thrombosis. Mechanistically, EP3α glycosylation enhanced its binding to PGE 2 and supports efficient G i coupling. The study concludes that N-linked glycosylation of EP3α is critical for platelet regulation through modulation of PGE 2 /EP3α/G i -mediated cAMP and Ca 2+ signaling.

Cell-surface N-linked glycans manipulation of K562 cells for augmented susceptibility to natural killer cell killing

Glycosylation of proteins is arguably the most diverse post-translational modification that is altered in almost all cancer types, which has been demonstrated to play a crucial role in creating an immunosuppressive microenvironment that promotes immune tolerance and evasion. However, the biosynthesis of N-linked glycan is mediated by a series of enzymatic reactions catalysed by glycosyltransferases and glycosidases in a template-independent manner, hindering our understanding of specific structure-function relationships and roles of specific glycans on specific proteins. Here we use HLA class I-negative cell line K562, a known reference target for NK-mediated cytolysis, to establish a model investigating how cell surface glycan dynamics influence its susceptibility to cytolysis mediated by NK-92 cells. Treatment of K562 cells with kifunensine, swainsonine, 2F-peracetyl-fucose, or 3Fax-peracetyl Neu5Ac, inhibitors of N-linked glycan processing, resulted in drastic alterations in cell surface carbohydrate phenotype, as could be shown by flow cytometric analysis of the lectiNbinding properties of the cells. Despite these clear changes in carbohydrate phenotype, only K562 cells treated with either kifunensine or 3Fax-peracetyl Neu5Ac exhibited higher susceptibility to the cytolysis medidated by NK-92 cells accompanied with an increased CD107a expression by NK-92 cells. Although K562 cells overexpressing gene MGAT3 exhibited a decreased NK-susceptibility, we further found that this decrease was not exclusively determined by the overexpression of gene MGAT3 product bisecting β1,4-GlcNAc, because the treatment of 3Fax-peracetyl Neu5Ac reversed the resistance of K562 cell against NK-92 cell in despite of expressing higher levels of bisecting β1,4-GlcNAc. Expressing HLA-G on cell surface as extravillous trophoblast did not change the NK-susceptibility of K562 cells, despite evidence that HLA-G molecules expressed by K562 cells can bind to inhibitory receptor ILT2 expressed on NK-92 cell surface. These findings suggest that the level of terminal sialylation, outweighing other components in N-linked glycan, determines the NK-susceptibility of K562 cell, offering a new strategy to weaken the resistance of cancer cells so that the immune system can maximise the elimination.