A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

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. We report a novel effect of IBV infection on cellular endomembranes, namely, the compaction of the pericentrosomal endocytic recycling compartment (ERC) defined by the GTPase Rab11, which coincides with the previously described Golgi fragmentation, as well as virus release. Despite Golgi disassembly, the IC elements containing the major IBV membrane protein (M)-which mostly associates with newly formed virus particles-maintain their close spatial connection with the Rab11-positive endocytic recycling system. Moreover, partial colocalization of the M protein with Rab11 was observed, whereas M displayed negligible overlap with LAMP-1, indicating that IBV egress does not occur via late endosomes or lysosomes. Synchronization of virus release using temperature-shift protocols was accompanied by increased colocalization of M and Rab11 in vesicular and vacuolar structures in the pericentrosomal region and at the cell periphery, most likely representing IBV-containing transport carriers. In conclusion, these results add CoVs to the growing list of viruses exploiting the endocytic recycling apparatus defined by Rab11 for their assembly and/or release.

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. The viral nucleocapsid, consisting of the genomic RNA complexed with nucleocapsid proteins obtains a membrane envelope during virus budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. From here, several alternative routes for CoV extracellular release have been proposed. Strikingly, recent studies have shown that CoV infection leads to the disassembly of the Golgi ribbon and the mobilization of host cell compartments and protein machineries that are known to promote Golgi-independent trafficking to the cell surface. Here, we discuss the life cycle of CoVs with a special focus on different possible pathways for virus egress.

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. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.

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. We here show that glycosaminoglycan (GAG) modification of PGs is significantly reduced, regardless which COG subunit that is missing in HEK293T cells. Least reduction was observed for cells lacking COG1 and COG8 subunits, that bridge the A and B lobes of the complex. Lack of these subunits did not reduce GAG chain lengths of secreted PGs, which was reduced in cells lacking any other subunit (COG2-7). COG3 knock out (KO) cells had particularly reduced ability to polymerize GAG chains. For cell-associated GAGs, the mutant cell lines, except COG4 and COG7 KO, displayed longer GAG chains than wild-type cells, indicating that COG subunits play a role in cellular turnover of PGs. In light of the important roles PGs play in animal development, the effects KO of individual COG subunits have on GAG synthesis could explain the variable severity of COG associated CDGs.

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. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.

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. Challenging a commonly held view that ribbon architecture involves the formation of homotypic tubular bridges between the Golgi stacks, we present an alternative model, based on direct interaction between the biosynthetic (pre-Golgi) and endocytic (post-Golgi) membrane networks and their connection with the centrosome. We propose that the central domains of these permanent pre- and post-Golgi networks function together in the biogenesis and maintenance of the more transient Golgi stacks, and thereby establish "linker compartments" that dynamically join the stacks together. This model provides insight into the reversible fragmentation of the Golgi ribbon that takes place in dividing and migrating cells and its regulation along a cell surface - Golgi - centrosome axis. Moreover, it helps to understand transport pathways that either traverse or bypass the Golgi stacks and the positioning of the Golgi apparatus in differentiated neuronal, epithelial, and muscle cells.

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. The tissue distribution of NDRG1 has been described in humans and rodents, but this has not been studied in the dog. RESULTS: By immunolabeling and Western blotting, we present a detailed mapping of NDRG1 in dog tissues and primary canine Schwann cell cultures, with particular emphasis on peripheral nerves. High levels of phosphorylated NDRG1 appear in distinct subcellular localizations of the Schwann cells, suggesting signaling-driven rerouting of the protein. In a nerve from an Alaskan malamute homozygous for the disease-causing Gly98Val mutation in NDRG1, this signal was absent. Furthermore, NDRG1 is present in canine epithelial cells, predominantly in the cytosolic compartment, often with basolateral localization. Constitutive expression also occurs in mesenchymal cells, including developing spermatids that are transiently positive for NDRG1. In some cells, NDRG1 localize to centrosomes. CONCLUSIONS: Overall, canine NDRG1 shows a cell and context-dependent localization. Our data from peripheral nerves and primary Schwann cell cultures suggest that the subcellular localization of NDRG1 in Schwann cells is dynamically influenced by signaling events leading to reversible phosphorylation of the protein. We propose that disease-causing mutations in NDRG1 can disrupt signaling in myelinating Schwann cells, causing disturbance in myelin homeostasis and axonal-glial cross talk, thereby precipitating polyneuropathy.

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. Ephrin-B3 binding to HEK-293T cells is blocked by a secretory variant of CD44 (v3-v10), while over-expression of membrane associated CD44 increased ephrin-B3 binding. In addition, ephrin-B3 precipitated CD44 expressed by the oral squamous carcinoma cell line H376. Moreover, ephrin-B3 binding affinities to heparin and CD44 in solution was strong. In conclusion, we have identified secretory and cell associated PGs with high ability to bind ephrin-B3 and suggest that ephrin-B3 can bind to a protein complex organized by a membrane associated PG.

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. Furthermore, both processed ESM powder and the ESM-derived carbohydrate fraction had immunomodulation properties in monocytes and macrophage-like cells. Under inflammatory conditions induced by lipopolysaccharide, the ESM powder and the isolated carbohydrate fraction reduced the activity of the transcription factor nuclear factor-κB. The expression of the immune regulating receptors toll-like receptor 4 and ICAM-1, as well as the cell surface glycoprotein CD44, all important during inflammation response, were down-regulated by these fractions. Interestingly, our experiments show that the two fractions regulated cytokine secretion differently: ESM depressed inflammation by increased secretion of the anti-inflammatory cytokine IL-10 while the carbohydrate fraction reduced secretions of the pro inflammatory cytokines IL-1ß and IL-6. Also, the phosphorylation of p65 and p50 subunits of nuclear factor-κB, as well as nuclear localization, differed between processed ESM powder and carbohydrate fraction, suggesting different down-stream regulation during inflammation. In conclusion, processed ESM powder and its soluble carbohydrate components possess anti-inflammatory properties, demonstrating the potential of ESM as a novel biological wound dressing for treatment of chronic inflammatory wounds.

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. Using simple instrumentation, the analysis time per sample was below 10min and the method was characterized by easy sample preparation. The method was used to monitor decreasing levels of PAPS as function of sodium chlorate treatment (an inhibitor of PAPS synthesis) in whole-cell lysates as well as Golgi-fractions. The method allowed PAPS to be chromatographically separated from ADP and ATP, which can interfere with measurements if a less resolving LC-MS method is used.

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. Which type of GAG chain becomes attached to the linker tetrasaccharide is influenced by the structure of the protein core, modifications occurring to the linker tetrasaccharide itself, and the biochemical environment of the Golgi apparatus, where GAG polymerization and modification by sulfation and epimerization take place. The same cell type may produce different GAG chains that vary, depending on the extent of epimerization and sulfation. However, it is not known to what extent these differences are caused by compartmental segregation of protein cores en route through the secretory pathway or by differential recruitment of modifying enzymes during synthesis of different PGs. The topic of this review is how different aspects of protein structure, cellular biochemistry, and compartmentalization may influence GAG synthesis.

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. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.

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. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

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. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.

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. With time, however, more physiological mechanisms and roles have been proposed for an increasing number of secretory processes that bypass the Golgi apparatus.

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. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.