Heterologous expression and purification of biologically active domains 3 and 4 of human polymeric immunoglobulin receptor and its interaction with choline binding protein A of Streptococcus pneumoniae.

Streptococcus pneumoniae, one of the common causes of pneumonia, colonises the epithelium via the interaction between a choline binding protein of S. pneumoniae and the human polymeric immunoglobulin receptor (pIgR). One of the functions of pIgR is to mediate the transcytosis of polymeric immunoglobulins from the basolateral to the apical surface of epithelial cells. S. pneumoniae invades human epithelial cells by exploiting the transcytosis machinery. Due to an increase in the prevalence of antibiotic resistant strains of S. pneumoniae, and the limitations and expense of the vaccines available, extensive research may provide insights into the potential of new therapeutic regimes. This study investigated the potential of pIgR domains as an alternative non-antibiotic immune therapy for treating pneumonia. The aim was to determine the binding affinity of recombinant D3D4 protein, the domains of pIgR responsible for binding S. pneumoniae, to recombinant R1R2 repeat domains of choline binding protein A of S. pneumoniae. Biologically active recombinant D3D4 was produced in Escherichia coli using a gel filtration chromatography refolding method, a novel approach for the refolding of pIgR domains, after the purification of inclusion bodies using nickel affinity chromatography. Surface Plasmon resonance (SPR) spectroscopy showed that purified recombinant D3D4 binds recombinant R1R2 with an equilibrium dissociation constant (KD) of 3.36×10(-7)M.

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation.

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 "stalk" domain, exCD23>sCD23>derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.

An acidic loop within the human soluble CD23 protein may direct the interaction between sCD23 and the αXß2 integrin.

CD23 is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor - the ß2 integrin subfamily. It has been shown that CD23 is also capable of interacting with the ß3 and ß5 integrin ß-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. In this study the interaction was probed for whether or not the RKC motif governs the interaction between CD23 and the αXß2 integrin as well. This was done by performing bioinformatic docking predictions between CD23 and αXß2 integrin αI domain and SPR spectroscopy analysis of the interaction. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, in the presence of divalent metal cations the interaction showed the involvement of a novel acidic motif within the CD23 protein. This same pattern of interaction was seen in docking predictions between CD23 and the ß3I-like domain. This study thus presents an alternative site as a possible contributor to the CD23-integrin interaction exhibiting cation-dependence.

Differential effects of detergents, fatty acids, cations and heating on ostrich skeletal muscle 20S proteasome.

The 20S proteasome, the catalytic core of the 26S proteasome, has previously been isolated, purified and partially characterised from ostrich skeletal muscle (Thomas, A.R., Oosthuizen, V., Naude, R.J., Muramoto, K. 2002. Biol. Chem. 383, 1267-1270). Due to the apparent latency of the 20S proteasome purified from various sources, this study focuses on further characterising the ostrich enzyme in terms of the effects of selected detergents, fatty acids and cations, as well as heating at 60 degrees C, on four of its activities. Results showed that ostrich skeletal muscle 20S proteasome was affected in a non-concentration-dependent manner by the selected detergents and fatty acids. Monounsaturated fatty acids, unlike unsaturated fatty acids, showed no major effects on the activities of the ostrich enzyme. The enzyme did not show sensitivity towards monovalent cations and the only divalent cations that showed a relevant effect were Ca2+ and Mg2+. Heating at 60 degrees C for 1-2 min had a substantial activating effect only on the peptidylglutamylpeptide-hydrolase (PGPH) and caseinolytic activities. In conclusion, many of the effects by the abovementioned reagents and conditions were noticeably different to those shown on different sources of the enzyme, further demonstrating the unique kinetic characteristics of the ostrich skeletal muscle 20S proteasome.

The structure of Fc receptor/Ig complexes: considerations on stoichiometry and potential inhibitors.

Recently, Fc receptors for immunoglobulins moved further into the focus of pure and applied research as a role in the induction and development of autoimmune diseases and allergies is becoming more probable. Indeed by connecting the humoral with the cellular immune response FcRs possess an important role in the immune system in which the initial, crucial step is the binding of an immunoglobulin to the cell bound receptor. Thereafter a variety of effector functions depending on the cell and the receptor is triggered. This key event could recently be visualised with the solution of the crystal structure of a human Fc receptor in complex with its native ligand the Fc fragment of IgG1. In this paper the reasons for a 1:1 stoichiometry between Fc receptor and Fc Fragment and the medical applications of potential inhibitors of complex formation are highlighted.

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation.

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Biological inferences from IgM binding characteristics of recombinant human secretory component mutants.

The polymeric immunoglobulin receptor (pIgR) or membrane secretory component (SC) selectively transports polymeric IgA and IgM across secretory epithelial cells to mucosal surfaces. The ligand binding ectodomain consists of five homologous Ig-like domains with domain I being an absolute requirement for binding. The role of DII to V in IgM binding remains unknown. Here, using in vitro refolded non-glycosylated recombinant domain deletion mutants of human SC, we show by biological and biophysical binding assays that DII to V are required for high affinity binding to IgM. Competitive binding analysis, by whole cell ELISA, showed that DII-V significantly increase the affinity of recombinant SC for IgM (K(i)=2.42 nM) as opposed to recombinant DI only (K(i)=44.8 nM). Lastly, we provide qualitative data highlighting the complexity of measuring the IgM/SC interaction using surface plasmon resonance spectroscopy.

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis.

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.

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.

Purification and characterization of the 20S proteasome from ostrich skeletal muscle.

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700,000, pI 6.67 and a 'ladder' of 22.2-33.5 kDa bands on SDS-PAGE. The amino acid composition and amino-terminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0-11.0 and 40-70 degrees C, and stabilities between 5-12 and up to 40-60 degrees C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.