Nonglycosylated Protein Research Articles

Cocaine Sensitization Increases I h Current Channel Subunit 2 (HCN2) Protein Expression in Structures of the Mesocorticolimbic System

Alteration of the biological activity among neuronal components of the mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The hyperpolarization-activated cyclic nucleotide current, I h, is a prominent mixed cation current present in neurons. The biophysical properties of the I h and its potential modulatory role in cell excitability depend on the expression profile of the hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN₂ channel's subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens, and hippocampus were analyzed using Western blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN₂ subunit in both its glycosylated and non-glycosylated protein isoforms in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated with alterations in the subunit composition of their HCN channels.

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.

An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi‐lectin affinity chromatography

Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.

Probing the stability of nonglycosylated wild-type erythropoietin protein via reiterative alanine ligations

Nonglycosylated erythropoietin bearing acetamidomethyl protecting groups at the cysteine residues has been synthesized via chemical methods. Alanine ligation was used to assemble four peptide fragments, themselves prepared by solid phase peptide synthesis. This work outlines a route for the synthesis of homogeneous glycosylated erythropoietin.

Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30

Previously, we reported the ice-binding protein (LeIBP) from the Arctic yeast Leucosporidium sp. AY30. In this study we provide physicochemical characterization of this IBP, which belongs to a class of IBPs that exhibited no significant similarity in primary structure to other known antifreeze proteins (AFPs). We compared native, glycosylated and non-glycosylated recombinant LeIBPs. Interestingly, size-exclusion chromatography and analytical ultracentrifugation revealed that LeIBP self-associates with a reversible dimer with Kd values in the range 3.45–7.24×10−6M. Circular dichroism (CD) spectra showed that LeIBP, glycosylated or non-glycosylated, is predominantly composed of β-strand secondary structural elements (54.6%), similar to other β-helical antifreeze proteins (AFPs). In thermal hysteresis (TH) activity measurements, native LeIBP was twice more active (0.87°C at 15mg/mL) than that of the recombinant IBPs (0.43–0.42°C at 10.8mg/mL). This discrepancy is probably due to uncharacterized enhancing factors carried over during ice affinity purification, because glycosylated and non-glycosylated recombinant proteins displayed similarly low activity. Ice recrystallization inhibition (RI) activities of the native and recombinant LeIBPs were comparable. Measurements of CD, TH activity, and RI showed that glycosylation does not cause structural changes and is not required for function. An ice-etching experiment using green fluorescent protein-tagged IBP revealed that LeIBP binds, just as hyperactive AFPs, to both basal and pyramidal prism planes of the ice crystal. Taken together, our results indicate that LeIBP, structurally similar to hyperactive AFPs, is moderately active and that a reversible dimer has no effect on its activity.

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.

P38α limits the contribution of MAP17 to cancer progression in breast tumors

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.

Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.

Purification and characterization of endoxylanase Xln-2 from Aspergillus niger B03

An extracellular multiple form of endoxylanase was isolated from the xylanolytic complex of Aspergillus niger B03. The enzyme was purified to a homogenous form using ultrafiltration, anion exchange chromatography, and gel filtration. It was a nonglycosylated protein with a molecular weight of 20,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 21,000 Da as determined by gel filtration. The optimal pH for the enzyme action was 5.0 and the optimal temperature was 55 °C. Endoxylanase stability was significantly improved in the presence of glycerol and sorbitol. The enzyme activity was activated by Mn^{2+} and Co^{2+}, and it was inhibited by Ag^+, Cu^{2+}, Fe^{3+}, Fe^{2+}, and Pb^{2+}. The substrate specificity and the product profile of the enzyme suggested that it was an endoxylanase. The enzyme showed a synergism with another endoxylanase from Aspergillus niger B03 in xylan hydrolysis.

MAP17, a ROS-dependent oncogene

MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas. Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition, decreased apoptotic sensitivity, and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis. MAP17 is overexpressed in a great variety of human carcinomas, including breast tumors. Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression. Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and, especially, for malignant progression.

Secretory Expression and Purification of the Recombinant Duck Interleukin-2 in Pichia pastoris

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal OD600 of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.

Extremely high salivary β2-microglobulin and Na+ levels in a Sjögren syndrome patient

Extremely high salivary β2-microglobulin and Na+ levels in a Sjögren syndrome patient

Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.

Glycosylation Increases the Thermostability of Human Aquaporin 10 Protein

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.

Yos9, a control protein for misfolded glycosylated and non-glycosylated proteins in ERAD

The endoplasmic reticulum (ER) is responsible for folding and delivery of secretory proteins to their site of action. One major modification proteins undergo in this organelle is N-glycosylation. Proteins that cannot fold properly will be directed to a process known as endoplasmic reticulum associated degradation (ERAD). Processing of N-glycans generates a signal for ERAD. The lectin Yos9 recognizes the N-glycan signal of misfolded proteins and acts as a gatekeeper for the delivery of these substrates to the cytoplasm for degradation. Presence of Yos9 accelerates degradation of the glycosylated model ERAD substrate CPY∗. Here we show that Yos9 has also a control function in degradation of the unglycosylated ERAD substrate CPY∗0000. It decelerates its degradation rate.

Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.

Occluding the Mannose Moieties on Human Immunodeficiency Virus Type 1 gp120 with Griffithsin Improves the Antibody Responses to Both Proteins in Mice

To assess the influence of mannosylated glycans on the immunogenicity of human immunodeficiency virus type 1 (HIV-1) Env proteins, we immunized mice with monomeric gp120 in the presence and absence of the mannose-binding protein, griffithsin (GRFT). For comparison, other groups of mice received the nonglycosylated HIV-1 Gag protein, with and without GRFT. Coimmunization with GRFT increased the anti-gp120 IgG reactivity significantly, but had no effect on the anti-Gag response. We also investigated the IgG response to GRFT and found that gp120, but not Gag, enhanced its immunogenicity. For both proteins, IgG1 antibodies dominated the IgG response, with IgG2b as the next most prevalent subclass. We conclude that gp120-GRFT complexes are more immunogenic than the free proteins, for both components, and that occluding the mannose moieties on monomeric gp120 can improve the humoral immune response to this protein.