γ-glutamyltransferase in human diploid fibroblasts and other mammalian cells

Summary γ-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S₃ and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that γ-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme. Preliminary reports of some of this work were presented at meetings of The American Society of Biological Chemists in Minneapolis (Abstracts Fed. Proc. 33: 957, 1974) and at Atlantic City (Abstracts Fed. Proc. 34: 2243, 1975). This work was supported by Grant NIH 1 P01 HD 07173. The WI-38 starter cultures and cell pack used in these studies were obtained through Contract M01 HD 42828 to Stanford University from the National Institute of Aging.     

Mouse Fcγ RI: identification and functional characterization of five new alleles

 The mouse Fcgr1 gene encoding the high-affinity IgG receptor (FcγRI) exists as two known alleles, FcγRI-BALB and FcγRI-NOD, and these alleles exhibit functional differences. To determine whether other alleles exist in mouse strains, Fcgr1 coding regions from 35 strains of mice were sequenced and a further five alleles were identified. The FcγRI-BALB and NOD alleles are now designated the "a" and "d" alleles, respectively. Analysis of the five new alleles revealed that although no polymorphisms were observed in the two leader exons, nucleotide and subsequent amino acid changes were observed in the exons encoding the extracellular domains, and transmembrane and cytoplasmic tail. The cDNA of the seven alleles (a–g) were isolated and transiently transfected into COS cells, and IgG-binding studies were performed. Receptors encoded by four of the five new alleles (b, c, f, g) bound IgG2a with high affinity, displaying IgG binding characteristics similar to the a allele (previously FcγRI-BALB). The d allele (previously FcγRI-NOD) and the e allele [derived from Mus spretus (SPRET/Ei)] encoded receptors which showed broader specificity by binding monomeric IgG2a, IgG2b, and IgG3. Received: 26 May 1999 / Revised: 25 October 1999     

Engineering, expression, and purification of “soluble” human cytochrome P45017α and its functional characterization

To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a "soluble" form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered "soluble" form of human CYP17 is used as a subject for crystallization of the hemeprotein.     

A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC₅₀ was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.

     

Selection and characterization of FcεRI phospho-ITAM specific antibodies

ABSTRACT

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y₂₁₈ or Y₂₂₄; 2) phosphorylation of the Y₂₂₈ tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP₂₄-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y₆₅) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.     

Molecular cloning and expression of the porcine high-affinity immunoglobulin G Fc receptor (FcγRI)

Receptors for the Fc region (FcγRs) of immunoglobulin G (IgG) play a crucial role in the immune system and host protection against infection. In this study, we describe the cloning, sequencing, and expression of the high-affinity IgG receptor from pig. By screening a translated Expressed Sequence Tags database with the human FcγRI (CD64) protein sequence, we identified a putative porcine homologue. Subsequent polymerase chain reaction amplification confirmed that the identified full-length cDNA was expressed in porcine cells. Rosetting analysis shows that COS-7 cells transfected with a plasmid containing the cloned cDNA were able to bind chicken erythrocytes sensitized with porcine IgG. Scatchard analysis indicated that monomeric IgG bound to transiently transfected cells with an affinity of approximately 4×10⁷ M⁻¹. The porcine FcγRI cDNA is 1,038 nucleotides long and is predicted to encode a 346-amino-acid transmembrane glycoprotein composed of three Ig-like domains, a transmembrane region, and a short cytoplasmic tail. The overall identity of the porcine FcγRI to its human and mouse counterparts at the level of the amino acid sequence was 75% and 57%, respectively. Identification of porcine FcγRI will aid in the understanding of the molecular basis of the porcine immune system and further studies of the receptor function.Gaiping Zhang and Songlin Qiao contributed equally to this study.The GenBank accession number of the nucleotide sequence reported here is DQ026063.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for 2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat 2,6-ST was expressed in a recombinant CHO cell line making interferon-, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon- being linked in the 2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of 2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN- productivity or other aspects of IFN- glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.Abbreviations AT-III antithrombin-III - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - EPO erythropoietin - IFN- human interferon- - NEO neomycin - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - ST sialyltransferase - tPA tissue plasminogen activator     

High-level soluble expression, purification, and functional characterization of the recombinant human leukemia inhibitory factor: a potential general strategy for the recombinant expression of cytokines consisting of four α-helices in a bundle

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95? purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four α-helices in a bundle for basic research, as well as clinical applications.     

Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.     

Increase in histamine content and enhancement of high affinity IgE receptor FcεRI expression in the human leukemia KU812 cells upon treatment with hydrocortisone

Hydrocortisone was investigated for its ability todifferentiate human leukemia KU812 cells into maturehematopoietic cells including basophils. Hydrocortisonetreatment increased the amount of intracellular histamine byup-regulation of L-histidine decarboxylase (HDC) mRNA andenhanced cell surface expression of the high affinity IgEreceptor FcRI. Histamine is catalyzed from L-histidine byHDC, which in blood cell types is only expressed in basophilsand mast cells. Cells, on which the FcRI expression wasenhanced by hydrocortisone, were shown to release histaminewhen stimulated with anti-IgE antibody after sensitizationwith myeloma IgE, implying that the induced FcRI moleculeswere able to transduce a signal for degranulation. Theseresults suggest that hydrocortisone promotes differentiationof KU812 cells into functionally mature basophilic cells.     

Increased expression of FcγRI/CD64 on circulating monocytes parallels ongoing inflammation and nephritis in lupus

Introduction  

The high-affinity receptor for IgG Fcγ/CD64 is critical for the development of lupus nephritis (LN). Cross-linking Fc receptor on recruited monocytes by IgG-containing immune complexes is a key step in immune-complex-mediated nephritis in systemic lupus erythematosus (SLE). The goal of this study was to determine whether expression of Fc receptor (FcγR) I on circulating monocytes is associated with systemic inflammation and renal disease in SLE patients.     

Production and characterization of mice transgenic for the A and B isoforms of human FcγRIII

Fcγ receptor (FcγR) engagement is pivotal for many effector functions of macrophages, polymorphonuclear neutrophils (PMN), and natural killer (NK) cells. Mice transgenic for the A and B isoforms of human (h) FcγRIII on macrophages, PMN, and NK cells were constructed to permit the study of mechanisms and potential in vivo strategies to utilize the cytotoxic effector and antigen-presenting functions of cells expressing the hFcγR. The present report characterizes the phenotypic and functional expression of hFcγRIII in transgenic mice derived by crossing hFcγRIIIA and hFcγRIIIB transgenic mice. Interleukin-2 (IL-2) induces hFcγRIII expression by myeloid cells and their precursors, and these transgenic receptors promote in vitro cytotoxicity and anti-hFcγRIII antibody internalization. Splenocytes from untreated and IL-2-treated hFcγRIIIA, hFcγRIIIB, and hFcγRIIIA/B mice exhibited enhanced in vitro cytotoxicity toward HER-2/neu-overexpressing SK-OV-3 human ovarian carcinoma cells when incubated with the murine bispecific mAb 2B1, which has specificity for HER-2/neu and hFcγRIII. These results indicate that hFcγRIII transgenes are expressed on relevant murine cellular subsets, exhibit inducible up-regulation patterns similar to those seen in humans, and code for functional proteins. hFcγRIII transgenic mice exhibiting specific cellular subset expression will permit the examination of strategies designed to enhance hFcγRIII-dependent immunological effector functions and will provide a model system in which to evaluate preclinically potential candidate molecules that recognize hFcγRIII for the immunotherapy of cancer. Received: 5 December 1998 / Accepted: 14 July 1999     

Pentagalloylglucose down-regulates mast cell surface FcεRI expression in vitro and in vivo

Mast cell activation by immunoglobulin E (IgE)-mediated stimuli is a central event in the pathogenesis of allergic disorders. The present report shows that treatment with pentagalloylglucose (PGG) resulted in a down-regulation of FcεRI surface expression on mucosal-type murine bone marrow-derived mast cells (mBMMCs), which correlated with a reduction in IgE-mediated activation of mBMMCs. Furthermore, PGG prevented development of allergic diarrhea in a food-allergy mouse model and suppressed the up-regulated FcεRI surface expression on mast cells derived from the food-allergy mouse colon. These findings on PGG suggest its therapeutic potential for allergic diseases through suppressing the FcεRI surface expression.     

Helicobacter pylori pediatric infection changes FcεRI expression in dendritic cells and Treg profile in vivo and in vitro

H. pylori infection shows an inverse relationship with allergies. Dendritic cells regulate mucosal immune responses including the induction of T regulatory cells which are fundamental in Helicobacter pylori-induced dampening of allergies. In this respect expression of high-affinity IgE receptor (FcεRI) has been associated with a regulatory dendritic cell profile. Therefore we aimed to evaluate possible mechanisms by which H. pylori infection might modify atopy in pediatric patients. Here we show that H. pylori-infected children exhibited both increased expression of FcεRI on peripheral myeloid and plasmacytoid dendritic cells and higher levels of Foxp3 and Latency Associated Peptide on T regulatory cells. Moreover, exposure to H. pylori drove increased FcεRI expression and IL-10 secretion by both pediatric H. pylori-exposed monocyte derived dendritic cells and T cells. Finally, we show a positive correlation between expression of FcεRI in circulating myeloid DCs and total Treg cells, suggesting that in children, H. pylori infection may have a modulating role in atopy, mediated by both altered surface expression of FcεRI on children's DC and an increased T regulatory cell profile.     

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6?mM isopropyl β-D-1-thiogalactopyranoside for 9?h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ～10?mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu²⁺, Mn²⁺, Zn²⁺, and Al³⁺ ions. Furthermore, the K_M and k_cat values for purified Aaβ-agarase were ～0.02?mM and ～45/s, respectively. These kinetic values were up to approximately 15–100-fold lower than the K_M values reported for other agarases and approximately 7–30-fold higher than the k_cat/K_M values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.     

Cytoplasmic expression of a soluble synthetic mammalian metallothionein-α domain in Escherichia coli

Bacteria are commonly used for bioremediation of heavy metal pollution and strategies to improve their performance in this respect are desirable. In this study, an Escherichia coli strain was engineered to express a common metallothionein-α domain. The metallothionein-α domain was over-expressed in the cytoplasm of E. coli as a fusion to the carboxyl terminal of maltose binding protein. The fusion protein was highly soluble in the cytoplasm of E. coli. When grown in the presence of cadmium, cells expressing the metallothionein-α fusion protein showed increased viability compared with control cells. Cells expressing the metallothionein-α also demonstrated increased accumulation of cadmium.     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.