N-linked Carbohydrate Chains Research Articles

Reduced Anticancer Potency of Carboplatin in the Presence of Sialic Acid in SH-SY5Y Cells

Background: Neuroblastoma (NB) is the most common solid childhood tumor with aggressive behavior and a high mortality rate. Multiple therapeutic approaches have been developed and applied to treat neuroblastoma, but resistance to therapies is inevitable, leading to treatment failure and cancer relapse. Therefore, perceiving the mechanisms of reduced drug sensitivity seems appropriate and promising in NB treatment to determine synergistic effects strategies like combination therapy. Objectives: The present study aimed to investigate the modulation effect of the therapeutic efficacy of carboplatin (a chemotherapeutic agent) by co-administration of sialic acid (an alpha-keto acid sugar with a 9-carbon backbone located at the outermost end of N-linked and O-linked carbohydrate chains and in lipid-associated glycoconjugates showing aberrant expression in tumor cells caused tumor-genesis) in NB cells. Methods: In the present study, the effects of sialic acid, carboplatin, and Sia-Carbo (sialic acid in combination with carboplatin) were evaluated on viability and apoptosis using the determination of EC50 and IC50. Also, the capability of metastasis and apoptosis were assessed using real-time PCR. Moreover, the expression of MRP-1 encoded by ABCC1 (as a target for therapeutic suppression in high-risk neuroblastoma) was studied in different treatment groups. Conclusions: Regarding IC50 increasing carboplatin in the presence of sialic acid, the results showed that sialic acid treatment significantly modulated carboplatin's effect on cell apoptosis and induced ABCC1 expression. These findings show that sialic acid metabolic engineering can be a good approach to neuroblastoma therapy. It was suggested that targeting aberrant sialylation in combination with carboplatin exerts more profound apoptotic and anticancer effects on the neuroblastoma SH-SY5Y cell line than carboplatin monotherapy.

Development of expression systems for the production of recombinant human Fas ligand extracellular domain derivatives using <em>Pichia pastoris</em> and preparation of the conjugates by site-specific chemical modifications: A review

Human Fas ligand extracellular domain (hFasLECD) is a biomedically important glycoprotein with three potential N-linked carbohydrate-chain attachment sites. hFasLECD can induce an apoptotic cell-death in many malignant cells. Hence, the creation of novel molecular tools exhibiting useful biological activities, based on the exploitation of this protein domain as their components, opens up a great possibility of the advancements in future medical applications. This review mainly focuses on the development of expression systems for obtaining various derivatives of recombinant hFasLECD using Pichia pastoris and the preparation of the conjugates by site-specific chemical modifications of the expressed products. Firstly, a brief introduction of human Fas ligand protein and an overview of the previous works, on the heterologous expression systems for recombinant hFasLECD as well as the associated derivatives aimed at medical applications, were described. Then, the experimental results, obtained during our investigations into the development of the expression systems for the recombinant hFasLECD derivatives using chemically synthesized artificial genes in Pichia pastoris, were summarized. After that, the current state of the methodology for preparation of the hFasLECD conjugates by site-specific chemical modifications, and the functional characterizations of the prepared conjugates, were presented. Finally, conclusions, including a relevant discussion and future perspectives, are provided.

EXPRESSION OF MODIFIED RECOMBINANT HUMAN ERYTHROPOIETIN IN CHO-K1 CELLS AND ITS IN VITRO PROLIFERATION ASSAY IN TF-1 CELLS

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIV-AIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional N-linked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 N-linked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation. Key words: CHO-K1, erythropoietin, glycosylation, MTT assay and TF-1 cell line.

Desialylation of N-Linked Carbohydrate Chain of Fibrinogen

Acceleration of fibrin polymerization occurs by the cleavage of sialic acids at the nonreducing terminal ends of N-linked carbohydrate chains as well as the cleavage of the entity of carbohydrate chains. In order to characterize and clarify the role of terminal sialic acid in the fibrin polymerization, mixing effects of desialylated fibrinogen with the intact one on the polymerization behavior were investigated by turbidity measurements in the course of polymerization. Marked accelerated fibrin polymerization was observed for the mixing of even a little amount of desialylated fibrinogen. Cleavage of the terminal sialic acid resulted in almost the equivalent accelerating effect to those of the deglycosylated fibrinogen, in which the entity of N-linked carbohydrate chain was cleaved. These results suggest that the terminal sialic acids regulate the fibrin polymerization in an inhibitory manner, and the cleavage of them induces the switchover from the protofibril growth to the lateral aggregation of fibrin polymerization process, resulting in the preferential fibrin polymerization.

Avian IgY is selectively incorporated into the egg yolks of oocytes by discriminating Fc amino acid residues located on the Cυ3/Cυ4 interface

In avian species, maternal IgY is selectively incorporated into the egg yolks of maturing oocytes, but the relevance of receptor-mediated uptake is unclear. Here we investigated the critical amino acid residues of IgY required for egg yolk transport by conducting mutational analyses of selected residues located along the Cυ3 and Cυ4 domains of chicken IgY. Recombinant wild-type IgY-Fc (WT) and its mutants were synthesized, and their uptakes into the egg yolks of quail were determined. Among the 17 amino acid residues located on the Cυ3/Cυ4 interface, the substitution of Y363 at the Cυ3 domain to alanine abolished the IgY-Fc uptake into egg yolks. The comprehensive substitution of Y363 with other amino acids revealed that the residue at 363 needs to be allocated with aromatic amino acids to maintain the high transport ability. The deglycosylation of the N-linked carbohydrate chain by substituting N407 at the Cυ3 domain with alanine also caused a marked reduction of IgY-Fc uptake. The microscopic detection of the injected WT and Y363A mutant in ovarian follicles showed that the WT was concentrically accumulated in yolk granules, whereas the Y363A mutant was hardly accumulated in yolk granules, but it had infiltrated into the granulosa cell layer, suggesting that a major hurdle disturbing the infiltration of the Y363A mutant lies on the inside of the granulosa cell layer. The identification of important amino acid residues required for efficient IgY transport enhances our understanding of the molecular mechanisms underlying IgY transport through a specific IgY receptor in ovarian follicles.

The carbohydrate ligands on the host embryo mediate intercellular migration of the parasitic wasp embryo

Invasive stage embryos of the parasitic wasp Copidosoma floridanum transmigrate through the epithelium of phylogenetically distant host embryos in a manner that is similar to mammalian leukocyte infiltration. Host embryonic cells appear to recognize the invading wasp embryo by components on the cell surface. We developed an in vitro wasp entry inhibition assay and found that C-type lectins of the wasp embryo bound to N-linked carbohydrate chains with fucose residues on the surface of host embryo. This is the first report showing a receptor–ligand interaction between heterologous multicellular organisms.

Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2 kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.

A computational investigation on the role of glycosylation in the binding of alpha1 nicotinic acetylcholine receptor with two alpha‐neurotoxins

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism.

ChemInform Abstract: Xylose-Containing Carbohydrate Chains Derived from N-Glycoproteins

Xylose-containing N-linked carbohydrate chains are integral parts of certain plant and animal glycoproteins. In all of the known structures, p-D-xylose is 1-2-linked to p-D- mannose of the trimannosyl-N,N'-diacetylchitobiose unit. If a-L-fucose is present at the asparagine-linked N-acetyl-Pglucosamine, then there are differences in the sites of its attachment, namely, al-3- or al-6-linked, depending on the biological origin. The a+ mannose residues can be substituted with additional monosaccharides or 3-0-methylated. The state of the art with respect to structural analysis, organic synthesis, conformational analysis, biosynthesis, lectin binding, and immunological aspects is reviewed.

Maintenance of cell surface glycan density by lectin-glycan interactions: A homeostatic and innate immune regulatory mechanism

It has been recently proposed that lectins such as galectins, C-type lectins and siglecs in innate immunity bind to foreign pathogens by density-dependent recognition of surface glycans (Dam and Brewer 2010). In many cases, foreign pathogens including viruses and bacteria possess glycan epitopes such as Man and LacNAc residues that are also found on host cells (Vasta 2009). Host lectins appear to bind to the pathogens due to their high density and number of glycan epitopes relative to those on host cells (Dam and Brewer 2010). Unique “weak” glycan epitopes on foreign pathogens appear to be strong epitopes when presented in high-density presentations such as polysaccharides and lipopolysaccharides (Dam and Brewer 2010). Thus, the concept of lectins as pattern recognition receptors in innate immunity (Medzhitov and Janeway 2000; Vasta 2009) has been replaced with lectins as density-dependent glycan binding receptors. However, in order for host lectins to bind to density-dependent expression of glycans on foreign pathogens, the glycan density of host cells must be established and controlled. Evidence that metazoans do regulate their surface glycan density comes from a new study on the effects of mutations in the pathway for the formation of a specific branch chain in Nlinked glycoproteins in mouse cells that results in apparent global compensation of glycan epitope number (density) in the remaining branch chains of the mutant’s N-linked carbohydrates (Takamatsu et al. 2010). N-acetylglucosaminyltransferase-IV (GnT-IV) exists as two isoenzymes, a and b, that initiate the synthesis of the GlcNAcβ1-4 branch on the core Manα1-3 arm of N-glycans. One effect of the GlcNAcβ1-4 branch chain is to increase the glycan epitope density per N-linked carbohydrate chain. Takamatsu et al. (2010) engineered and characterized GnT-IVb-deficient mice and double-IVa/IVbdeficient mice. Wild-type mice have GnT-IVa expression restricted to gastrointestinal tissue, while GnT-IVb is broadly expressed among organs. GnT-IVb-deficient mice show aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern, and hence, the GnT-IVb-deficient mice show mild phenotypic alterations in hematopoietic cells and hemostasis. Importantly, GnT-IVa/IVb double-deficient mice have completely abolished GnT-IV activity, and thus there is a complete disappearance of the GlcNAcβ1-4 branch on the Manα1-3 arm of the N-linked glycans that was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and gas chromatography-MS analyses. The absence of the GlcNAcβ1-4 branch, however, was shown by MS analyses as additional glycan epitope extensions on the remaining branches in the tissues of the N-linked glycans of the GnT-IVa/IVb double-deficient mice. For example, the Le epitope is found in greater number in the N-linked glycans from mouse kidney cells, and biosynthetic compensation is observed in biand triantennary N-glycans of the double mutant relative to the tetra-antennary N-glycans of the wild-type cells (Figure 1) (Takamatsu et al. 2010). Similar biosynthetic compensation was observed for other glycan epitopes such as polylactosamine in the pancreas. Analysis shows that these additional glycan epitopes in the GnT-IVa/IVb double-deficient mice are due to elevated expression of glycosyltransferases that are normally involved in their biosynthesis. Remarkably, the phenotype of the GnT-IVa/IVb double-deficient mice was similar to that of the GnT-IVa single-deficient mice, which is relatively mild compared to the wild-type mice. The ability of the GnT-IVa/IVb double-deficient mice to effectively restore glycan epitope density in the remaining N-linked glycans of the different organs of the mice suggests that maintenance of this density is critical to the homeostatic function of the cells and organs of the mouse. The authors describe these findings as the first example of induced glycomic compensation of glycosyltranserase activities of cells from different organs of the mice (Takamatsu et al. 2010). They also suggest that maintenance of the overall expression of glycan ligands for endogenous lectins in the double-mutant mice appears to prevent cellular dysfunctions, but no mechanism was suggested for these effects. We suggest that a possible mechanism for regulating cell surface glycan density in both the wild-type and GnT-IVa/

A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression

Glutathione-S-transferases (GSTs), a major superfamily of antioxidative enzymes, play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The delta-class GST cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the Chinese mitten crab, Eriocheir sinensis. The 938 bp E. sinensis GST gene, encoding a polypeptide of 216 amino acids, showed significant similarity to homologous genes in insects. The deduced amino acid sequence of the crab GST contains conserved features of this gene family including the G-site (1–82 aa, tripeptide glutathione binding site) in the N-terminal region and an H-site (88–213 aa, substrate binding site) in the C-terminus. Additionally, a kinase C phosphorylation site (ITI) and one putative N-linked glycosylation site (DNIT) for N-linked carbohydrate chains were also identified. Quantitative real-time RT-PCR (qRT-PCR) was employed to investigate the distribution of GST mRNA in different tissues and its temporal expression in haemocytes of crabs challenged with Aeromonas hydrophila. Different levels of GST mRNA expression were detected in haemocytes, hepatopancreas and muscle, while it was not detected in the gills, intestines and stomach. GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge ( P < 0.05) and dropped to basal levels at 12 h, presumably down-regulated by increased bacteremia by that time. These findings suggest that GST could play a critical role in immunity, and this positive feedback mechanism can allow for efficient activation of the early innate immune response to infection.

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

Current and Upcoming Erythropoiesis-Stimulating Agents, Iron Products, and Other Novel Anemia Medications

Treatment for anemia has come a long way in the last 20 years since the first recombinant human erythropoietins were licensed for the management of anemia in chronic kidney disease. The first-generation epoetins were succeeded by the development and production of a longer-acting erythropoietin (EPO) analog, darbepoetin alpha, which allowed less frequent dosing, usually once weekly or once every 2 weeks. More recently, another EPO-related molecule has been manufactured called Continuous Erythropoietin Receptor Activator with an even longer half-life, and although for patent reasons this is not available in the United States, it is licensed and is already being used in Europe. Other molecules are in development or are becoming licensed in Europe, including biosimilar epoetin products/follow-on biologics, and elsewhere in the world there are cheaper-production "copy" epoetins. Indeed, it is estimated that up to 80 such products may be sold in countries with less stringent regulatory control of pharmaceutical products. Two different biosimilar epoetins have already been licensed in Europe, one under 2 different brand names and one under 3 different brand names, and others may follow. Hematide is a synthetic peptide-based EPO receptor agonist that, interestingly, has no structural homology with EPO, and yet is still able to activate the EPO receptor and stimulate erythropoiesis. This agent is currently in phase III clinical trials. Research continues for orally active antianemic therapies, and several strategies are being investigated, although none is imminently available. Two new intravenous iron preparations have recently been developed, one in the United States (Ferumoxytol; AMAG Pharmaceuticals, Inc., Cambridge, MA) and one recently licensed in Europe (ferric carboxymaltose [Ferinject; Vifor Pharma, Zurich, Switzerland]). In conclusion, the development of effective therapies for the treatment of anemia has been a highly active field, both scientifically and economically, over the last two decades.

Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown. Mesangial IgA is of the IgA1 subclass, and since no consistent antigenic target for the IgA1 has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or IgA2. Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgA1, and the O-linked sugars of IgA1 and C1 inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.

Improved secretion of human Fas ligand extracellular domain by N-terminal part truncation in Pichia pastoris and preparation of the N-linked carbohydrate chain trimmed derivative

Human Fas ligand is a medically important transmembrane glycoprotein directing the induction of apoptosis. The influence of N-terminal part (Q103-P138) truncation of human Fas ligand extracellular domain (hFasLECD) on the expression of N-terminal FLAG-(Gly) 5-tagged hFasLECD (NFG5-hFasLECD) with partial N-glycosylation-sites deletion in Pichia pastoris was investigated. The N-terminal part truncation significantly improved the secretion level of both singly (N184Q) and doubly (N184Q, N250Q) N-glycosylation-sites deleted NFG5-hFasLECD. The highly purified N-terminal truncated NFG5-hFasLECD with the double N-glycosylation-sites deletion mutation was obtained using single-step cation-exchange chromatography. The isolation yield was about 24 mg from one liter culture supernatant, which amounted to approximately five times higher than that of the previously reported non-truncated NFG5-hFasLECD with N184Q mutation. The remaining N-linked carbohydrate chain in the purified product was digested with a high-mannose type glycochain specific endoglycosidase, Endo Hf, under non-denatured condition. The N-linked carbohydrate chain trimmed product was purified through Con A-agarose column fractionation and another cation-exchange chromatography from the reaction mixture. The final product showed the molecular weight exact to that of NFG5-hFasLECD-[Δ(103–138), N250Q] mutant with single N-acetylglucosamine residue in MALDI-TOF mass-spectrometric analysis, and existed as a trimer in solution. The N-terminal truncated product either with or without N-linked carbohydrate chain exhibited the specific binding activity toward soluble human Fas receptor extracellular domain—human IgG 1-Fc domain fusion protein, which revealed that the presence of N-linked carbohydrate chain was not essential for the functional activity of hFasLECD. The sample preparation system developed here may be applicable to the structural analysis of hFasLECD.

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Characterization of GPR56 protein and its suppressed expression in human pancreatic cancer cells

GPR56 is an atypical G protein-coupled receptor (GPCR) with an unusually large N-terminal extracellular region, which contains a long Ser/Thr-rich region forming a mucin-like stalk and due to this feature, GPR56 is thought to be an adhesion GPCR. Recent studies demonstrate that GPR56 plays a role in brain development and tumorigenesis. Here, we report that human GPR56 undergoes GPS (GPCR proteolytic site)-mediated protein cleavage to generate its extracellular domain as an N-terminal fragment (GPR56-N). We also show that GPR56-N is highly glycosylated with N-linked carbohydrate chains. Mouse Gpr56 is ubiquitously expressed in various tissues, with high levels in kidney and pancreas. GPR56 mRNA is detected in diverse human cancer cells including pancreatic cancer cells PANC-1, Capan-1, and MiaCaPa-2. Interestingly, GPR56 protein is either negligible or undetectable in these pancreatic cancer cells, despite the fact that high levels of GPR56 mRNA are observed. Moreover, we have found that protein levels of GPR56 in pancreatic cancer cells were not affected when cells were treated with a proteasome inhibitor MG132. Taken together, these results define the biochemical properties of GPR56 protein, and suggest that the expression of GPR56 protein is suppressed in human pancreatic cancer cells.

Crystal structure of the extracellular domain of nAChR α1 bound to α-bungarotoxin at 1.94 Å resolution

We determined the crystal structure of the extracellular domain of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to alpha-bungarotoxin at 1.94 A resolution. This structure is the first atomic-resolution view of a nAChR subunit extracellular domain, revealing receptor-specific features such as the main immunogenic region (MIR), the signature Cys-loop and the N-linked carbohydrate chain. The toxin binds to the receptor through extensive protein-protein and protein-sugar interactions. To our surprise, the structure showed a well-ordered water molecule and two hydrophilic residues deep in the core of the alpha1 subunit. The two hydrophilic core residues are highly conserved in nAChRs, but correspond to hydrophobic residues in the nonchannel homolog acetylcholine-binding proteins. We carried out site-directed mutagenesis and electrophysiology analyses to assess the functional role of the glycosylation and the hydrophilic core residues. Our structural and functional studies show essential features of the nAChR and provide new insights into the gating mechanism.

Nanomedicines in the treatment of anemia in renal disease: focus on CERA (Continuous Erythropoietin Receptor Activator)

Anemia is a common complication of chronic kidney disease (CKD), with erythropoietin deficiency being the major contributing factor. The availability of erythropoiesis-stimulating agents (ESAs) has been a seminal advance in the treatment of anemia related to chronic kidney disease. Over the course of the last decade and a half, newer generations of ESAs have become available. The first-generation ESAs or epoetins have a relatively shorter half-life and have traditionally been administered up to 3 times per week intravenously or subcutaneously to maintain adequate hemoglobin (Hb) levels. At the turn of the century, darbepoetin alfa, a hyperglycosylated form, became available for clinical use. It conferred greater metabolic stability in vivo owing to two additional N-linked carbohydrate chains attached to the protein backbone and has a half-life 3 times longer than that of epoetin. Recently developed and undergoing phase III clinical trials is the third-generation ESA, Continuous Erythropoiesis Receptor Activator (CERA), which has a methoxy-polyethylene glycol polymer chain integrated and has a longer elimination half-life than the first- and second-generation ESAs. Its receptor binding characteristics also differ from those of previous ESAs. Its major advantage is that extended dosing intervals are possible in the management of anemia related to erythropoietin deficiency.

The glycoprotein hGC-1 binds to cadherin and lectins

Human granulocyte colony stimulating factor stimulated clone-1 (hGC-1, also known as GW112, OLM4, and hOlfD) is an olfactomedin-related glycoprotein of unknown function. We performed a series of biochemical studies to characterize its function. Using hGC-1 purified from baculovirus Sf9 cells we demonstrated that hGC-1 is a secreted glycoprotein containing N-linked carbohydrate chains and forms disulfide-bonded multimers. It binds to cell surfaces and to the locutions ricinus communis agglutinin I, concanavalin A and wheat germ agglutinin. Purified hGC-1 enhanced NIH3T3 and 293T/17 cell spreading and attachment, as did hGC-1-enriched culture supernatants of 293T/17 cells transfected with an hGC-1 expression vector. Coimmunoprecipitation studies demonstrated that hGC-1 interacts with cadherin in 293T/17 cells. This interaction depends on the C-terminal olfactomedin domain, but does not require the five well-conserved cysteine residues. However, cysteine residues at 83, 85, 246 and 437 are essential for secretion, and cysteine 226 is critical for hGC-1 multimer formation. Our studies demonstrated that hGC-1, an extracellular matrix glycoprotein, facilitates cell adhesion. Its potential interaction with endogenous cell surface lectins and cadherin may mediate this function.