Targeting immune complex-mediated hypersensitivity with recombinant soluble human FcgammaRIA (CD64A)

Binding of Ag-Ab immune complexes to cellular FcgammaR promotes cell activation, release of inflammatory mediators, and tissue destruction characteristic of autoimmune disease. To evaluate whether a soluble FcgammaR could block the proinflammatory effects of immune complexes, recombinant human (rh) versions of FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA were prepared. Binding of rh-FcgammaRIA to IgG was of high affinity (KD=1.7x10(-10) M), whereas rh-FcgammaRIIA and rh-FcgammaRIIIA bound with low affinity (KD=0.6-1.9x10(-6) M). All rh-FcgammaR reduced immune complex precipitation, blocked complement-mediated lysis of Ab-sensitized RBC, and inhibited immune complex-mediated production of IL-6, IL-13, MCP-1, and TNF-alpha by cultured mast cells. Local or systemic delivery only of rh-FcgammaRIA, however, reduced edema and neutrophil infiltration in the cutaneous Arthus reaction in mice. 125I-labeled rh-FcgammaRIA was cleared from mouse blood with a rapid distribution phase followed by a slow elimination phase with a t1/2gamma of approximately 130 h. The highest percentage of injected radioactivity accumulated in blood approximately liver approximately carcass>kidney. s.c. dosing of rh-FcgammaRIA resulted in lower serum levels of inflammatory cytokines and prevented paw swelling and joint damage in a murine model of collagen Ab-induced arthritis. These data demonstrate that rh-FcgammaRIA is an effective inhibitor of type III hypersensitivity.     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells

Infection of T-lymphocytes and macrophages by human immunodeficiency virus (HIV) is mediated by the binding of the HIV envelope glycoprotein to the cell-surface receptor glycoprotein CD4. A soluble, recombinant CD4 molecule (rCD4), produced by expression of a truncated CD4 gene in Chinese hamster ovary (CHO) cells [Smith et al. (1987) Science 238, 1704-1707], is in clinical trials as a potential therapeutic agent in the treatment of acquired immunodeficiency syndrome (AIDS). In the present study, the structures of the Asn-linked oligosaccharides of soluble rCD4 have been elucidated. The rCD4 molecule has two potential sites for N-glycosylation, Asn-271 and Asn-300. Tryptic glycopeptides containing either of the sites were purified by reversed-phase HPLC, and their oligosaccharides were released enzymatically. The structures of the oligosaccharides were determined by methylation analysis, high-pH anion-exchange chromatography, fast-atom bombardment mass spectrometry, and 1H NMR spectroscopy at 500 MHz. Asn-271 was found to carry diantennary N-acetyllactosamine-type ("complex") oligosaccharides, of which 8% were asialo, 55% were monosialyl, and 37% were disialyl. Approximately 18% of these structures contained fucose alpha(1-->6) linked to the reducing GlcNAc residue. Two different hybrid structures were found to account for 34% of the oligosaccharides attached to Asn-300. The remainder of the oligosaccharides attached to Asn-300 were diantennary N-acetyllactosamine-type, of which 10% were asialo, 61% were monosialyl, and 29% were disialyl. Approximately 9% of the hybrid structures and 40% of the N-acetyllactosamine structures at Asn-300 were found to contain fucose alpha(1-->6) linked to the innermost GlcNAc residue.     

Purification of recombinant human tissue factor

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.     

Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.     

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.The authors contributed equally to this work.     

Purification and properties of human lymphocyte activating factor (LAF).

Lymphocyte-activating factor (LAF) has been shown to be produced by LPS-stimulated human adherent cells (monocytes) and peripheral leukocytes, but many non-macrophage cell lines failed to produce LAF. Other macrophage activators including latex microspheres, antigen-antibody complexes, and barium sulfate induce the production of LAF. There is a delay of 6 hr before significant amounts of LAF activity appear in the supernatant medium and maximum activity is found after 12 to 24 hr. Chromatography of concentrated crude supernatant fractions containing LAF activity on Sephadex G-100 gave two peaks of activity (approximately 85,000 and 13,000 daltons). The latter constitutes the major activity and has been purified at least 500-fold with Sephadex G-100, anion exchange, and adsorption chromatography. Optimal stimulation with LAF induces mitosis in 10% of murine thymocytes. The purified activity is sensitive to chymotrypsin and is not affected by treatment with sodium periodate, sulfhydryl reagents, and phenylmethanesulfonylfluoride. The response of thymocytes to LAF decreases with age after 10 weeks and thymocytes obtained from animals injected with cortisone or tumor-bearing animals have an increased responsiveness to LAF.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.     

Purification of transgenic tobacco-derived recombinant human monoclonal antibody

Purification of recombinant monoclonal antibody from transgenic plant extract is technically challenging as it involves the processing of large volume of material, containing low titre of antibody, present along with large quantities of native proteins and other impurities. The conventional approach of capturing antibody from a clarified extract using packed-bed chromatography is therefore not particularly suitable. This study evaluates the suitability of using a combination of ultrafiltration and chromatography for purifying transgenic tobacco-derived human monoclonal antibody. A two-stage cascade ultrafiltration process removed about 97% impurities while ensuring almost complete recovery of antibody, providing 32-fold antibody enrichment in the process. The primary objective of the ultrafiltration step was to reduce the burden on the subsequent chromatographic steps. A two-step chromatographic process was then used to eliminate remaining impurities. Using this approach, recombinant human antibody expressed in tobacco could be purified to greater than 95% purity with 50% overall recovery (ca. 12.5 mg antibody/kg tobacco tissues).     

Purification and characterization of recombinant human prostacyclin synthase

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.     

可溶性CD14基因克隆及序列分析

针对人可溶性CD14(sCD14）的cDNA序列设计引物,通过反转录PCR技术,从U937细胞中,扩增出编码人可溶性CD14的基因序列,并将其克隆至质粒pGEM－T中,通过PCR、酶切及序列测定鉴定重组载体。结果表明获得了人可溶性CD14基因片段,为下一步进行CD14表达及生物学活性研究奠定了基础。     

N-glycosylation profile of recombinant human soluble Fcgamma receptor III

N-glycans of human Fcgamma receptor III (FcgammaR III) are believed to be involved in the interaction with complement receptor type 3 (CR3) (Sehgal et al. [1993] J. Immunol., 150, 4571-4580). Recombinant human soluble FcgammaRIII (rhsFcgammaRIII), which is produced in baby hamster kidney (BHK) cells, has been shown to interact with CR3 in a manner similar to native FcgammaRIII. We elucidated the N-glycosylation profiles of rhsFcgammaRIII by the 3D high-performance liquid chromatography mapping technique. It was revealed that the N-glycans of rhsFcgammaRIII are much more divergent (consisting of 20 neutral, 7 monosialyl, 4 disialyl, 5 trisialyl, and 1 tetrasialyl oligosaccharides) than those previously determined for BHK-expressed mouse sFcgammaRII, notwithstanding close structural similarity of polypeptide chains between the two sFcgammaRs. Particularly, high-mannose type oligosaccharides are specifically expressed on rhsFcgammaRIII.     

Purification and biochemical characterization of human lymphocyte mitogenic factor (LMF).

Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.     

Purification of soluble guanylate cyclase enzyme from human platelets

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.     

Production of biologically active recombinant human soluble CD23 and its effect on PBMCs isolated from hyper-IgE blood

A recombinant form of human soluble CD23 (sCD23), the low affinity receptor for IgE (FcepsilonRII), was produced by PCR cloning the lectin-binding domain sequence into a bacterial expression vector. After renaturation and purification, the sCD23 bound IgE and divalent metal ions, indicating its activity. The recombinant human sCD23 exhibited similar proinflammatory properties as the native protein. Although interleukin-1beta, tumour necrosis factor-alpha, and nuclear factor-kappaB appeared not to be enhanced significantly in unstimulated RPMI 8866 B-lymphoblastoid and U937 promonocytic cell lines with 24 h incubation of recombinant sCD23, they were produced in both healthy and hyper-IgE-derived peripheral blood mononuclear cells, especially tumour necrosis factor-alpha. This study concludes that while recombinant and chimeric sCD23 may be useful in blocking IgE binding to immune cells and decreasing IgE synthesis by B-lymphocytes, the production of proinflammatory cytokines, particularly tumour necrosis factor-alpha will enhance immune responses in cases of asthma, allergy, and hyper-IgE syndrome.