In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Evidence for the role of Rab11-positive recycling endosomes as intermediates in coronavirus egress from epithelial cells.

After their assembly by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface, coronaviruses (CoVs) are released from their host cells following a pathway that remains poorly understood. The traditional view that CoV exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves unconventional secretion. Here, using the avian infectious bronchitis virus (IBV) as a well-established model virus, we have applied confocal microscopy to investigate the pathway of CoV egress from epithelial Vero cells.

The life cycle and enigmatic egress of coronaviruses.

There has been considerable recent interest in the life cycle of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of the Covid-19 pandemic. Practically every step in CoV replication-from cell attachment and uptake via genome replication and expression to virion assembly has been considered as a specific event that potentially could be targeted by existing or novel drugs. Interference with cellular egress of progeny viruses could also be adopted as a possible therapeutic strategy; however, the situation is complicated by the fact that there is no broad consensus on how CoVs find their way out of their host cells.

Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and Sorting in Filter-Grown and Polarized Epithelial Cells.

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades.

Proteoglycan synthesis in conserved oligomeric Golgi subunit deficient HEK293T cells is affected differently, depending on the lacking subunit.

The Conserved Oligomeric Golgi (COG) complex is an eight subunit protein complex associated with Golgi membranes. Genetic defects affecting individual COG subunits cause congenital disorders of glycosylation (CDGs), due to mislocalization of Golgi proteins involved in glycosylation mechanisms. While the resulting defects in N-and O-glycosylation have been extensively studied, no corresponding study of proteoglycan (PG) synthesis has been undertaken.

Assembly and Cellular Exit of Coronaviruses: Hijacking an Unconventional Secretory Pathway from the Pre-Golgi Intermediate Compartment via the Golgi Ribbon to the Extracellular Space.

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC.

A New Look at the Functional Organization of the Golgi Ribbon.

A characteristic feature of vertebrate cells is a Golgi ribbon consisting of multiple cisternal stacks connected into a single-copy organelle next to the centrosome. Despite numerous studies, the mechanisms that link the stacks together and the functional significance of ribbon formation remain poorly understood. Nevertheless, these questions are of considerable interest, since there is increasing evidence that Golgi fragmentation - the unlinking of the stacks in the ribbon - is intimately connected not only to normal physiological processes, such as cell division and migration, but also to pathological states, including neurodegeneration and cancer.

Cell and context-dependent sorting of neuropathy-associated protein NDRG1 - insights from canine tissues and primary Schwann cell cultures.

Background: Mutations in the N-myc downstream-regulated gene 1 (NDRG1) can cause degenerative polyneuropathy in humans, dogs, and rodents. In humans, this motor and sensory neuropathy is known as Charcot-Marie-Tooth disease type 4D, and it is assumed that analogous canine diseases can be used as models for this disease. NDRG1 is also regarded as a metastasis-suppressor in several malignancies.

Ephrin-B3 binds both cell-associated and secreted proteoglycans.

The ephrin family of membrane proteins binds Eph tyrosine kinase receptors. We have previously shown that ephrin-B3 also binds to heparan sulfate proteoglycans (HSPGs). We now show that ephrin-B3 can bind both secretory and cell associated PGs, such as agrin, collagen XVIII, Perlecan, and CD44, and indicate that such interaction with cell associated PGs involves a complex including 20 and 45 kDa proteins.

The extracellular matrix of eggshell displays anti-inflammatory activities through NF-κB in LPS-triggered human immune cells.

Avian eggshell membrane (ESM) is a natural biomaterial that has been used as an alternative natural bandage on burned and cut skin injuries for >400 years in Asian countries, and is available in large quantities from egg industries. Our aim was to characterize ESM that was separated and processed from egg waste, and to study whether this material possesses anti-inflammatory properties, making it suitable as an ingredient in industrial production of low cost wound healing products. Our results show that the processed ESM particles retain a fibrous structure similar to that observed for the native membrane, and contain collagen, and carbohydrate components such as hyaluronic acid and sulfated glycosaminoglycans, as well as N-glycans, mostly with uncharged structures.

Determination of 3'-phosphoadenosine-5'-phosphosulfate in cells and Golgi fractions using hydrophilic interaction liquid chromatography-mass spectrometry.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a key player in the sulfation of biomolecules, but methods for selective measurements are lacking. A liquid chromatography-mass spectrometry (LC-MS) approach for measuring PAPS was developed. A central feature of the method was employing hydrophilic interaction liquid chromatography (HILIC), which is highly suited for separating very polar/charged compounds, and is compatible with electrospray MS.

Determinants of Glycosaminoglycan (GAG) Structure.

Proteoglycans (PGs) are glycosylated proteins of biological importance at cell surfaces, in the extracellular matrix, and in the circulation. PGs are produced and modified by glycosaminoglycan (GAG) chains in the secretory pathway of animal cells. The most common GAG attachment site is a serine residue followed by a glycine (-ser-gly-), from which a linker tetrasaccharide extends and may continue as a heparan sulfate, a heparin, a chondroitin sulfate, or a dermatan sulfate GAG chain.

PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.

Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles.

Serglycin is implicated in the promotion of aggressive phenotype of breast cancer cells.

Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material.

Reduced sulfation of chondroitin sulfate but not heparan sulfate in kidneys of diabetic db/db mice.

Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides.

Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus.

Arrivals and departures at the plasma membrane: direct and indirect transport routes.

Studies carried out during the last 2 decades have dramatically increased our knowledge of the pathways and mechanisms of intracellular membrane traffic, most recently due to the developments in light microscopy and in vivo imaging of fluorescent fusion proteins. These studies have also revealed that certain molecules do not behave according to the classical transportation rules first documented in cell biology textbooks in the 1980s and 1990s. Initially, unconventional mechanisms of secretion that do not involve passage of cargo through the stacked Golgi cisternae were thought to confer on cells the ability to discard excess amounts of protein products.

N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain.

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005.

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment.

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA).

Ephrin-B3 binds to a sulfated cell-surface receptor.

The ephrins are a family of proteins known to bind the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinase family. In the present paper, we provide data showing that ephrin-B3 binds a sulfated cell-surface protein on HEK-293T (human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40) and HeLa cells, a binding that is nearly completely blocked by treatment of these cell lines with chlorate or heparinase, or by addition of the heavily sulfated glycosaminoglycan heparin. This indicates that heparan sulfate on these cells is essential for cell-surface binding of ephrin-B3.

Epigenetics: methylation-associated repression of heparan sulfate 3-O-sulfotransferase gene expression contributes to the invasive phenotype of H-EMC-SS chondrosarcoma cells.

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown.

A secretory Golgi bypass route to the apical surface domain of epithelial MDCK cells.

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae. We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER.