Biochemical characterization of CD39L4

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Chemical and biological conjugation strategies for the development of multivalent protein vaccine nanoparticles

The development of subunit vaccine platforms has been of considerable interest due to their good safety profile and ability to be adapted to new antigens, compared to other vaccine typess. Nevertheless, subunit vaccines often lack sufficient immunogenicity to fully protect against infectious diseases. A wide variety of subunit vaccines have been developed to enhance antigen immunogenicity by increasing antigen multivalency, as well as stability and delivery properties, via presentation of antigens on protein nanoparticles. Increasing multivalency can be an effective approach to provide a potent humoral immune response by more strongly engaging and clustering B cell receptors (BCRs) to induce activation, as well as increased uptake by antigen presenting cells and their subsequent T cell activation. Proper orientation of antigen on protein nanoparticles is also considered a crucial factor for enhanced BCR engagement and subsequent immune responses. Therefore, various strategies have been reported to decorate highly repetitive surfaces of protein nanoparticle scaffolds with multiple copies of antigens, arrange antigens in proper orientation, or combinations thereof. In this review, we describe different chemical bioconjugation methods, approaches for genetic fusion of recombinant antigens, biological affinity tags, and enzymatic conjugation methods to effectively present antigens on the surface of protein nanoparticle vaccine scaffolds.     

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG₁ molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.     

Biochemical and biological characterization of a neuroendocrine-associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.     

Killing kinetics of simian immunodeficiency virus-specific CD8+ T cells: implications for HIV vaccine strategies

Both the magnitude and function of vaccine-induced HIV-specific CD8+ CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-gamma were simultaneously studied in SIV Gag(164-172) KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-gamma and with limited (<5%) degranulation and granzyme B release. Vaccination with live-attenuated SIV resulted in a rapid cytolytic profile of SIV-specific CTLs with rapid (<0.5 h) and robust (>50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIV(mac251) challenge of prime-boost-vaccinated macaques, albeit with less IFN-gamma expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure.     

Rapid viral quasispecies evolution: implications for vaccine and drug strategies

High mutation rates occurring during replication allow RNA viruses to evolve rapidly and adapt continuously to new environments. This poses an enormous challenge to vaccine and drug development which, to be effective, must consider RNA virus variability and follow approaches that minimize the probability of escape or resistant mutants arising.     

Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells

The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.     

Chemical and/or biological therapeutic strategies to ameliorate protein misfolding diseases

Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation.     

Biochemical characterization of a SET and transposase fusion protein, Metnase: its DNA binding and DNA cleavage activity

Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.     

Biochemical and functional characterization of the Ebola virus VP24 protein: implications for a role in virus assembly and budding

The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding.     

Review: TTR amyloidosis-structural features leading to protein aggregation and their implications on therapeutic strategies

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.     

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.     

Biochemical and biological characterization of a novel anti-aromatase coumarin derivative

Estrogen stimulates the proliferation of estrogen receptor (ER)-positive breast cancer cells. Aromatase is the enzyme responsible for the conversion of androgens into estrogens, and synthetic aromatase inhibitors such as letrozole, anastrozole, and exemestane have proven to be effective endocrine regimens for ER-positive breast cancer. In a recent study, we have found that 4-benzyl-3-(4'-chlorophenyl)-7-methoxycoumarin is a potent competitive inhibitor of aromatase with respect to the androgen substrate. Its K(i) value was determined to be 84 nm, significantly more potent than several known aromatase inhibitors. The specific interaction of this compound with aromatase was further demonstrated by the reduction of its binding by several mutations at the active site region of aromatase and evaluated by computer modeling analysis. The structure-activity studies have revealed that three functional groups (i.e. 3-(4'-chlorophenyl), 4-benzyl, and 7-methoxyl) of this coumarin are important in its inhibition of aromatase. In addition, through a matrigel thread three-dimensional cell culture, this compound was shown to behave like known aromatase inhibitors that suppress the proliferation of aromatase and estrogen receptor positive MCF-7aro breast cancer cells. This coumarin has been shown not to be cytotoxic at up to 40 mum. It was found not to be an inhibitor of steroid 5alpha-reductase that also utilizes androgen as the substrate and not to be a ligand of ERalpha, ERbeta, estrogen-related receptors, or androgen receptor. These results demonstrate that coumarins (a common type of phytochemical) or their derivatives can be potent inhibitors of aromatase and may be useful in suppressing aromataseand ER-positive breast tumors.     

Biochemical characterization and evaluation of a Brugia malayi small heat shock protein as a vaccine against lymphatic filariasis

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential.     

CD38 and CD157: biological observations to clinical therapeutic targets

The application of molecular knowledge for developing new medical technologies is the goal of molecular medicine. Success in this area is highly dependent on the interaction of investigators from fields as diverse as biochemistry, cell biology, immunology, physiology, epidemiology, and physics, with an eye toward applying their insights and discoveries to improving human health. Such interdisciplinary approaches rarely find the common ground and language necessary to achieve this goal. Recently, a meeting of researchers studying the ectoenzymes CD38 and CD157 brought together insights into the regulation of calcium signaling, the metabolism of pyridine nucleotides by CD38 and CD157, and subsequent effects on immune function. Together, these discoveries were being applied to the development of novel therapeutics and diagnostics for myeloma and chronic lymphocytic leukemia. This issue of Molecular Medicine, featuring several short reviews based on a conference held in Turin, Italy, 10-12 June 2006, showcases the current state of this field and highlights some recent progress in molecular medicine.