Nonglycosylated Protein Research Articles

Biochemical and Biophysical Characterization of the Mg2+-induced 90-kDa Heat Shock Protein Oligomers

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051 T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051 T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.

Genetically engineered mannosylated-human serum albumin as a versatile carrier for liver-selective therapeutics

Human serum albumin (HSA), a non-glycosylated protein, is widely employed as carrier for drug delivery systems. A series of recombinant, mannosylated-HSA mutants (Man-rHSAs: D63N, A320T and D494N) and their triple mutant (TM-rHSA: D63N/A320T/D494N) were prepared, that can be selectively delivered to the liver via mannose receptor (MR) on the liver non-parenchymal cells. A pharmacokinetic analysis of 111In-Man-rHSAs in mice showed that they were rapidly cleared from the blood circulation, and were largely taken up by the liver rapidly in the order: TM-rHSA > D494N ≫ A320T = D63N, consistent with their degree of mannosylation. In vivo competition experiments with an excess amount of chemically modified Man-BSA or mannan suggested that the hepatic uptake of TM-rHSA was selectively mediated by MR on Kupffer cells. Lastly, a TM-rHSA–NO conjugate, S-nitrosylated TM-rHSA, effectively delivered NO to the liver and then exhibited a significant inhibitory effect against hepatic ischemia/reperfusion injury model rats, accompanied by the induction of heme oxygenase-1.

Filgrastim biosimilar: desde el desarrollo al registro

Ratiograstim ® is the first filgrastim biosimilar approved by EMEA. The reference medicinal product is Neupogen ®. The active substance is filgrastim (the manufacturing laboratory, ratiopharm, is using the name XM02). Filgrastim is a non glycosylated recombinant methionyl human granulocyte colony stimulating factor expressed in E. coli. Differences with human granulocyte-colony stimulating factor (G-CSF) are a N-terminal methionyl extension and be a non-glycosylated protein. Ratiograstim ® has the same indications as the reference product: – Cytotoxic chemotherapy induced neutropenia. – Neutropenia caused by myeloablative therapy followed by BMT. – Mobilisation of peripheral blood progenitor cells (PBPC). – Congenital, cyclic or idiopathic neutropenia. – Persistant neutropenia in HIV-patients. Authorisation according to the EMEA guidelines for biosimilars by centralized approval procedure, to demonstrate quality, efficacy and safety compared to the reference product Neupogen ®. Comprehensive phase I and phase III clinical studies involving 880 subjects and patients have been completed (table 1).

Role of the UL45 protein in herpes simplex virus entry via low pH-dependent endocytosis and its relationship to the conformation and function of glycoprotein B

Herpesviruses commandeer distinct cellular pathways to enter target cells. The mechanism by which herpes simplex virus (HSV) selects a pH-dependent, endocytic route or a pH-independent route remains to be elucidated. We investigated the role of the non-glycosylated viral envelope protein UL45 in HSV entry via endocytosis. UL45 plays a role in mediating cell–cell fusion and has been proposed to functionally interact with gB to regulate membrane fusion. Thus, we also probed the impact of UL45 on the structure and function of gB present in virions. A UL45 deletion virus successfully entered cells via low pH, endocytic pathway with wild type kinetics. In the absence or presence of UL45, the antigenic conformation of virion gB appeared unaltered. Antibodies to gB neutralized infection of the UL45-deletion virus and wild type virus to a similar extent, regardless of whether the target cells supported low pH endocytic or non-endocytic entry routes. Lastly, HSV virions were inactivated by low pH regardless of the presence of UL45. The results, together with previous studies, suggest that UL45 plays distinct roles in cell–cell fusion and virus–cell fusion during acid-dependent entry.

Molecular Cloning and Expression of Canine Prolactin Gene

We isolated canine prolactin cDNA from a dog pituitary cDNA library. The 930-bp nucleotide sequence covered the entire open reading frame encoding the putative 229 amino acids. It was located in chromosome 35, and had five exons. The amino acid sequence was highly homologous to the feline and porcine sequences. To generate recombinant canine prolactin, plasmids for full-length canine prolactin were constructed and transfected into the mammalian HEK293 cell line. The recombinant prolactin was secreted into the medium as an N-linked glycosylated (31 kDa) or non-glycosylated (27 kDa) protein. Western blotting revealed both of these bands were canine pituitary protein extracts.

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.

Immunocytochemical characterization of the cytopathic effect induced by bovine respiratory syncytial virus strain RC 98 on Hep-2 cells

family (Meyer et al., 2008). Its genome has approximately 15,140 nucleotides and encodes 10 different mRNA molecules (Pastey and Samal, 1995; Buchholz et al., 1999; Arns et al., 2003). The genome of BRSV encodes 11 proteins, three of them are transmembrane surface proteins: the G, F, and SH and three other proteins are nonglycosylated virion matrix proteins: M, M2-

Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2

Chicken pancreatic phospholipase A(2) (ChPLA(2)) was purified from delipidated pancreases using ammonium sulfate and ethanol precipitation, followed by sequential column chromatography steps on MonoQ Sepharose and size exclusion HPLC columns. ChPLA(2) was found to be a nonglycosylated monomeric protein with a molecular mass of 14 kDa and a specific activity of 400 U x mg(-1) in the presence of 1 mM sodium taurodeoxycholate and 4 mM CaCl(2) with phosphatidylcholine as substrate. The N-terminal sequence of the first 15 amino acids of ChPLA(2) was determined, and showed a high degree of homology with known mammal pancreatic phospholipases A(2). The gene encoding the mature ChPLA(2) was cloned and sequenced. The deduced amino acid sequence of the mature ChPLA(2) confirmed the high level of identity with mammal pancreatic PLA(2). To investigate the structure-activity relationships, a 3D model of group IB ChPLA(2) was built using the porcine pancreatic phospholipase A(2) structure as template.

Utility of cystatin C to monitor renal function in duchenne muscular dystrophy

Creatinine as a marker of renal function has limited value in Duchenne muscular dystrophy (DMD) because of reduced muscle mass. Alternative methods of assessing renal function are sorely needed. Cystatin C, a nonglycosylated protein unaffected by muscle mass, is potentially an ideal biomarker of nephrotoxicity for this population but requires validation. In all, 75 subjects were recruited: 35 DMD (mean age 10.8 +/- 5.4 years, corticosteroids n = 19, ambulatory n = 26), 29 healthy controls, 10 with renal disease, and one DMD with renal failure. Cystatin C levels in DMD were normal irrespective of age, ambulation, or corticosteroid treatment. Serum cystatin C was 0.67 +/- 0.11 mg/l compared to normal controls 0.69 +/- 0.09. mg/l. In these same individuals serum creatinine was severely reduced (0.27 +/- 0.12 mg/dl) versus normals (0.75 +/- 0.15 mg/dl, P < 0.01). In one DMD subject in renal failure, cystatin C was elevated. This study demonstrates the potential value of cystatin C as a biomarker for monitoring renal function in DMD. Its applicability extends to other neuromuscular diseases.

Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris

The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7 kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c- myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.

Latherin: A Surfactant Protein of Horse Sweat and Saliva

Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg ml−1), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 Å thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material.

Functional Expression and Characterization of Keratinase from Pseudomonas aeruginosa in Pichia pastoris

Recombinant keratinase (rK) from Pseudomonas aeruginosa was secreted by Pichia pastoris SMD1168H with a final yield of 580 mg/L (1.03 kU/mL) after 72 h of induction. The rK was purified after nickel affinity chromatography and was stable at pH 6.0-9.0 and 10-60 degrees C. It was nonglycosylated protein with a molecular mass of 33 kDa and had an optimal pH and temperature at 8.0 and 60 degrees C, respectively. Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylene diamine tetraacetic acid, and p-chloromercuribenzoate inhibited its activity. rK could hydrolyze broad substrates and cleave hydrophobic and aromatic amino acids at P(1) position, behaving as those from the wild type strain and E. coli transformant.

Synthesis and application of novel phenylboronate affinity materials based on organic polymer particles for selective trapping of glycoproteins

We report on synthesis concepts for the fabrication of various novel phenylboronate affinity materials based on polymethacrylate epoxy beads (Fractogel EMD Epoxy (M) 40-90 microm) and the testing of these functionalized polymer particles for selective trapping of a glycoprotein from a standard mixture containing a glycosylated and a nonglycosylated protein. Two inherently different approaches for the functionalization of the bare beads with boronate groups have been elucidated. In the first, the epoxy residues of the polymer particles were converted into reactive thiol groups which were subsequently used as anchor moieties for the immobilization of 4-vinylphenylboronic acid by radical addition or radical polymerization reaction. Three different ways for the generation of sulfhydryl groups have been examined leading to materials with distinct linker chemistries. In the second and more straightforward approach, the epoxy groups were reacted with 4-mercaptophenylboronic acid. The novel materials were thoroughly characterized by (i) quantitation of the sulfur content by elemental analysis, (ii) reactive sulfhydryls were determined in a photospectrometric assay, (iii) boron content was measured by inductively coupled plasma-atomic emission spectrometry, and (iv) the amount of reactive boronate groups was evaluated in a fast binding assay employing adenosine as test compound. A maximum concentration of 1.2 mmol boronate groups per gram dry beads could be achieved by the presented synthesis routes. Employing the novel phenylboronate affinity materials in capture and release experiments in the batch mode, a standard glycoprotein, viz. transferrin (Tf) from human serum was separated from a nonglycosylated protein, BSA. A commercial boronate affinity material based on 3-aminophenylboronic acid modified agarose gel was employed as reference material and was found to perform significantly worse compared to the herein presented novel polymethacrylate particles.

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the beta-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycan's first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc(2), improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.

Stability of dimeric interface in banana lectin: Insight from molecular dynamics simulations

Banana lectin (Banlec) is a homodimeric non-glycosylated protein. It exhibits the beta-prism I structure. High-temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study elucidates the behavior of the dimeric protein at four different temperatures and compares the structural and conformational changes to that of the minimized crystal structure. The process of unfolding was monitored by following the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, and the stability is mostly contributed by interfacial interactions. It maintains its overall conformation during high-temperature (400-500 K) simulations, with only the unstructured loop regions acquiring greater momentum under such condition. Nevertheless, at still higher temperatures (600 K) the tertiary structure is gradually lost which later extends to loss of secondary structural elements. The pattern of hydrogen bonding within the subunit and at the interface across different stages has been analyzed and has provided rationale for its intrinsic high stability.

Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea

A novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed.

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post-translational processing of human alpha(1)-antichymotrypsin (AACT) in Bright Yellow-2 (BY-2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse-chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non-glycosylated form, in contrast with secreted variants undergoing multiple post-translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N-glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY-2 tobacco cells led to the production of two protein products: (i) a stable, non-glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post-translational N-glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process . Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.