Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced

Direct Determination of Multiple Ligand Interactions with the Extracellular Domain of the Calcium Sensing Receptor

Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca2+ homeostasis by sensing Ca2+ and L-Phe. However, direct interaction of Ca2+ and Phe with the receptor’s ECD and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of CaSR (ECD) (residues 20~612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ~50% -helix, ~20% -sheet content and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca2+ with a Kd of 3.0~5.0 mM. The local conformational changes of the proteins induced by their interactions with Ca2+ were visualized by NMR with specific 15N Phe-labeled forms of the ECD. Saturation transfer difference (STD) NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and L-Phe. We further demonstrated that L-Phe increases the binding affinity of the CaSR ECD for Ca2+. Our findings provide new insights into the mechanisms by which Ca2+ and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily

Recombinant Sialyltransferase Infusion Mitigates Infection-Driven Acute Lung Inflammation

Inappropriate inflammation exacerbates a vast array of chronic and acute conditions with severe health risks. In certain situations, such as acute sepsis, traditional therapies may be inadequate in preventing severe organ damage or death. We have previously shown cell surface glycan modification by the circulating sialyltransferase ST6Gal-1 regulates de novo inflammatory cell production via a novel extrinsic glycosylation pathway. Here, we show that therapeutic administration of recombinant, bioactive ST6Gal-1 (rST6G) mitigates acute inflammation in a murine model mimicking acute exacerbations experienced by patients with chronic obstructive pulmonary disease (COPD). In addition to suppressing proximal neutrophil recruitment at onset of infection-mediated inflammation, rST6G also muted local cytokine production. Histologically, exposure with NTHI, a bacterium associated with COPD exacerbations, in rST6G-treated animals revealed consistent and pronounced reduction of pulmonary inflammation, characterized by smaller inflammatory cuffs around bronchovascular bundles, and fewer inflammatory cells within alveolar walls, alveolar spaces, and on pleural surfaces. Taken together, the data advance the idea that manipulating circulatory ST6Gal-1 levels has potential in managing inflammatory conditions by leveraging the combined approaches of controlling new inflammatory cell production and dampening the inflammation mediator cascade

Biochemical characterization of functional domains of the chaperone Cosmc

<div><p>Cosmc is an endoplasmic reticulum chaperone necessary for normal protein O-GalNAc glycosylation through regulation of T-synthase, its single client. Loss-of-function of Cosmc results in expression of the Tn antigen, which is associated with multiple human diseases including cancer. Despite intense interest in dysregulated expression of the Tn antigen, little is known about the structure and function of Cosmc, including domain organization, secondary structure, oligomerization, and co-factors. Limited proteolysis experiments show that Cosmc contains a structured N-terminal domain (CosmcΔ256), and biochemical characterization of CosmcΔ256 reveals wild type chaperone activity. Interestingly, CosmcE152K, which shows loss of function <i>in vivo</i>, exhibits wild type-like activity <i>in vitro</i>. Cosmc and CosmcE152K heterogeneously oligomerize and form monomeric, dimeric, trimeric, and tetrameric species, while CosmcΔ256 is predominantly monomeric as characterized by chemical crosslinking and blue native page electrophoresis. Additionally, Cosmc selectively binds divalent cations in thermal shift assays and metal binding is abrogated by the CosmcΔ256 truncation, and perturbed by the E152K mutation. Therefore, the N-terminal domain of Cosmc mediates T-synthase binding and chaperone function, whereas the C-terminal domain is necessary for oligomerization and metal binding. Our results provide new structure-function insight to Cosmc, indicate that Cosmc behaves as a modular protein and suggests points of modulation or regulation of <i>in vivo</i> chaperone function.</p></div

Modulation of the NOTCH1 Pathway by LUNATIC FRINGE Is Dominant over That of MANIC or RADICAL FRINGE

Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG

SEC of Cosmc, CosmcE152K, and CosmcΔ256, and EDTA treated proteins.

<p>Cosmc was injected at 1.5 mg/ml onto a Sephacryl S300 size exclusion column (SEC) with phosphate buffered saline as the running buffer. SEC profiles are shown for untreated and EDTA treated <b>(A)</b> Cosmc WT; <b>(B)</b> CosmcE152K; <b>(C)</b> CosmcΔ256.</p

Oligomeric behavior of WT, CosmcE152K, and CosmcΔ256.

<p><b>(A)</b> Chemical crosslinking followed by SDS PAGE shows products corresponding to the mass of Cosmc monomer (1), dimer (2), trimer (3), and tetramer (4) for Cosmc and CosmcE152K, while CosmcΔ256 yields only monomer and small amounts of dimer species. Cosmc was treated with either the crosslinker DSG (+), or mock treated (-), along with negative and positive controls, BSA and IgG respectively, for protein oligomerization. <b>(B</b>) BN-PAGE analysis of Cosmc along with monomeric and oligomeric control proteins, BSA and apoferritin, respectively. WT and CosmcE152K show heterogeneous oligomers, while CosmcΔ256 shows a single predominant species. <b>(C)</b> Chemical crosslinking with DSG of full length Cosmc from crude HEK293SC lysates shows monomeric, dimeric, and trimeric species. Cosmc from cell lysates was detected by western blotting.</p