Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B heterohexamer and B pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B pentamer showed an unexpectedly specific localization in the medial/trans-Golgi.

Functional magnetic nanoparticles for protein delivery applications: understanding protein-nanoparticle interactions.

Iron oxide nanoparticles (IONPs) surface functionalised with thermo-responsive polymers can encapsulate therapeutic proteins and release them upon heating with an alternating magnetic field above the lower critical solution temperature (LCST). In order to make this delivery system clinically-relevant, we prepared IONPs coated with poly--isopropylmethacrylamide (PNIPMAM), a polymer with LCST above human body temperature. The optimal polymer chain length and nanoparticle size to achieve LCST of 45 °C were 19 kDa PNIPMAM and 16 nm IONPs.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors.

Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady-State Organization of Golgi-Resident Proteins.

The organization of Golgi-resident proteins is crucial for sorting molecules within the secretory pathway and regulating posttranslational modifications. However, evaluating changes to Golgi organization can be challenging, often requiring extensive experimental investigations. Here, we propose a systems biology approach in which changes to Golgi-resident protein sorting and localization can be deduced using cellular N-glycan profiles as the only experimental input.

Computational Modeling of Glycan Processing in the Golgi for Investigating Changes in the Arrangements of Biosynthetic Enzymes.

Modeling glycan biosynthesis is becoming increasingly important due to the far-reaching implications that glycosylation can exhibit, from pathologies to biopharmaceutical manufacturing. Here we describe a stochastic simulation approach, to overcome the deterministic nature of previous models, that aims to simulate the action of glycan modifying enzymes to produce a glycan profile. This is then coupled with an approximate Bayesian computation methodology to systematically fit to empirical data in order to determine which set of parameters adequately describes the organization of enzymes within the Golgi.

Leveraging glycomics data in glycoprotein 3D structure validation with Privateer.

The heterogeneity, mobility and complexity of glycans in glycoproteins have been, and currently remain, significant challenges in structural biology. These aspects present unique problems to the two most prolific techniques: X-ray crystallography and cryo-electron microscopy. At the same time, advances in mass spectrometry have made it possible to get deeper insights on precisely the information that is most difficult to recover by structure solution methods: the full-length glycan composition, including linkage details for the glycosidic bonds.

Magnetically Triggered Release of Entrapped Bioactive Proteins from Thermally Responsive Polymer-Coated Iron Oxide Nanoparticles for Stem-Cell Proliferation.

Nanoparticles could conceal bioactive proteins during therapeutic delivery, avoiding side effects. Superparamagnetic iron oxide nanoparticles (SPIONs) coated with a temperature-sensitive polymer were tested for protein release. We show that coated SPIONs can entrap test proteins and release them in a temperature-controlled manner in a biological system.

The -Glycosylation Processing Potential of the Mammalian Golgi Apparatus.

Heterogeneity is an inherent feature of the glycosylation process. Mammalian cells often produce a variety of glycan structures on separate molecules of the same protein, known as glycoforms. This heterogeneity is not random but is controlled by the organization of the glycosylation machinery in the Golgi cisternae.

Modeling Glycan Processing Reveals Golgi-Enzyme Homeostasis upon Trafficking Defects and Cellular Differentiation.

The decoration of proteins by carbohydrates is essential for eukaryotic life yet heterogeneous due to a lack of biosynthetic templates. This complex carbohydrate mixture-the glycan profile-is generated in the compartmentalized Golgi, in which level and localization of glycosylation enzymes are key determinants. Here, we develop and validate a computational model for glycan biosynthesis to probe how the biosynthetic machinery creates different glycan profiles.

Glycans modify mesenchymal stem cell differentiation to impact on the function of resulting osteoblasts.

Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both - and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later.

Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay.

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the -Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP.

One Filter, One Sample, and the N- and O-Glyco(proteo)me: Toward a System to Study Disorders of Protein Glycosylation.

A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NHOH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate.

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11.

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking.

The role of acroblast formation during Drosophila spermatogenesis.

Protein recycling is important for maintaining homeostasis of the Golgi and its cisternae. The Vps54 (Scat) protein, a subunit of the GARP tethering complex, is a central factor in retrograde transport to the trans-Golgi. We found the scat(1) mutant to be male sterile in Drosophila with individualization problems occurring during spermatogenesis.

Molecular architecture of the complete COG tethering complex.

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.

Glycan Profiling Shows Unvaried N-Glycomes in MSC Clones with Distinct Differentiation Potentials.

Different cell types have different N-glycomes in mammals. This means that cellular differentiation is accompanied by changes in the N-glycan profile. Yet when the N-glycomes of cell types with differing fates diverge is unclear.

COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits.

The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches.

Bridging the Gap between Glycosylation and Vesicle Traffic.

Glycosylation is recognized as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae.

Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.

We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis.

Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus.

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions.

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport.

COG complexes form spatial landmarks for distinct SNARE complexes.

Vesicular tethers and SNAREs (soluble N-ethylmalemide-sensitive fusion attachment protein receptors) are two key protein components of the intracellular membrane-trafficking machinery. The conserved oligomeric Golgi (COG) complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Here, using yeast two-hybrid and co-immunoprecipitation approaches, we show that three COG subunits, namely COG4, 6 and 8, are capable of interacting with defined Golgi SNAREs, namely STX5, STX6, STX16, GS27 and SNAP29.