Identification of the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R) using synthetic peptides

To identify the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R), peptides derived from the membrane-distal extracellular domain (EC1) of boFcgamma2R corresponding to the homologous region of human FcalphaRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F-G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2-binding. Such functional epitope peptide should be very useful for understanding the IgG-Fcgamma interaction and development of FcR-targeting drugs.     

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.     

A Preliminary Study on Proteome Variations Associated with Gall Formation in <Emphasis Type="Italic">Zizania latifolia</Emphasis> Trucs

Zizania latifolia Trucs is a uniquely flavored aquatic vegetable found in southern and eastern Asia. Several physiology and genetic approaches have been employed to increase our knowledge about the physiological basis of gall formation; however, as yet, data at the proteomic level are not available. Protein yield and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to determine the most appropriate protein extraction methods for use in this study. Total proteins were extracted from the culm tissue at three relevant developmental stages and separated by two-dimensional gel electrophoresis. The number and abundance of spots varied among the two-dimensional gel electrophoresis gels at the three stages. Proteins with more than 1.7-fold abundance between the different stages were monitored. We identified 10 well-resolved spots by matrix-assisted laser-desorption ionization/time-of-flight peptide mass fingerprinting and tandem mass spectrometry. Some spots linked to signal transduction of the phytohormone could be identified. The expression volume of these spots transiently increased during the expansion phase. In contrast, the spots linked to phytoene dehydrogenase and methionine synthase or pyrophosphate-dependent phosphofructokinase were more abundant during gall formation, showing an increase in spot intensity during development and maximal abundance in mature gall. Higher intensity was also found in the spots linked to stress response. We discuss protein variations, considering their potential role during gall formation and comparing our results with established variations at metabolite-profiling levels.