The data are derived from a single representative experiment of three independent experiments

The data are derived from a single representative experiment of three independent experiments. with Forssman glycosphingolipid. The oligosaccharide moiety of Forssman glycosphingolipid, Forssman pentasaccharide, KPT-9274 exhibits exceptionally high affinity for the N-terminal CRD of Gal-9 (4). In contrast to Forssman glycosphingolipid, the structures and glycosylation site(s) of KPT-9274 glycoprotein glycans through which Gal-9 exerts its effect are generally difficult to determine unequivocally. Currently, the glycans responsible for the conversation with Gal-9 have not been identified in most glycoprotein receptors except for GLUT2. Vertebrate GLUT2 has a single IgE with an oxidized oligosaccharide moiety). These results indicate that this conversation between Gal-9 and IgE glycan(s) is usually indispensable for the suppressive effect of Gal-9. In the present study, we aimed at identification of glycosylation site(s) and structural Mouse monoclonal to CD4.CD4, also known as T4, is a 55 kD single chain transmembrane glycoprotein and belongs to immunoglobulin superfamily. CD4 is found on most thymocytes, a subset of T cells and at low level on monocytes/macrophages analysis of IgE glycans responsible for the Gal-9-induced suppression of RBL-2H3 cell degranulation. Because most studies, including our previous ones, concerning the function and therapeutic potential of Gal-9 were performed with a mouse model (11,C17), mouse monoclonal IgE was used as a target molecule for both human and mouse Gal-9. Experimental Procedures Cloning and Sequence Analysis of TIB-141 cDNA An anti-2,4,6-trinitrophenyl (TNP) mouse monoclonal IgE-producing hybridoma cell line (IGELb4) was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). The term TIB-141 will be used as the name of the monoclonal antibody in this paper, although it is the ATCC catalogue number for the hybridoma cell line. The cDNA sequences of both the L and H chains of TIB-141 were determined to confirm previous reports by other investigators. Total RNA was extracted from IGELb4 hybridoma cells by the method of Chomczynski and Sacchi (18). About 2 g of total RNA was reverse transcribed using the GeneAmp RNA PCR kit with oligo(dT) primers (PerkinElmer Life Sciences). cDNAs for the light chain (L chain) and heavy chain (H chain) of TIB-141 were amplified using gene-specific primer KPT-9274 pairs of mIGLK-1/-2 and mIGH-1/-2, respectively (19,C21): mIGLK-1, 5-ATGAAGTTGCCTGTTAGGCTGTTG-3; mIGLK-2, 5-CTAACACTCATTCCTGTTGAAGCT-3; mIGH-1, 5-ATGGAATTGATCTGGGTCTTTCTC-3; mIGH-2, 5-CTAGGAGGGACGGAGGGAGGTGTT-3. The amplified cDNAs were cloned into the pGEM-T Easy vector using a TA cloning system (Promega Corp., Madison, WI). The DNA sequence was determined by using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3130 genetic analyzer (Applied Biosystems). The sequences (supplemental Fig. S1) were deposited in the DDBJ database under the accession numbers LC031494 (H chain) and LC031495 (L chain). The sequence of the L chain completely coincided with those reported previously, whereas there were differences in the sequence of the H chain between the present data and the data reported by Liu (20). In the DNA database, however, we found DNA sequences identical to that of the H chain shown in supplemental Fig. S1. Currently, it is not clear whether the differences are due to sequencing error, PCR artifacts, or genetic variation of the hybridoma cell line. The sequences decided in the present study were used to interpret the results described below. Expression and Purification of Recombinant Gal-9 Expression of tag-free recombinant proteins, human stable form Gal-9 (hG9Null), and mouse stable form Gal-9 (mG9Null), in BL21(DE3) was carried out as described previously (22). Recombinant proteins were purified by affinity chromatography on a lactose-agarose column (Seikagaku Corp., Tokyo, Japan). The purified proteins were dialyzed against PBS and then sterilized KPT-9274 by filtration. All of the preparations were found to be >95% pure, as judged on SDS-PAGE. The protein concentrations were decided using BCA protein assay reagent (Pierce) and BSA as a standard. The concentrations of peptides purified by HPLC were determined from the UV absorbance at 215 and 225 nm using the formula, concentration (mg/ml) = ((25) (consecutive glycosidase digestion and RP-HPLC analysis). Results Effects of hG9Null and mG9Null on Degranulation of RBL-2H3 Induced by TIB-141 and TNP-BSA TIB-141 in combination with the antigen (TNP-BSA) induced degranulation of RBL-2H3 cells in a similar manner as reported previously, where mouse monoclonal IgE (Spe7) and dinitrophenyl-human serum albumin were used instead (10). hG9Null almost completely depressed degranulation at concentrations higher than 0.3 m in a lactose-sensitive manner (Fig. 2represents the mean value for a control sample (without TIB-141 and TNP-BSA). Lactose was used at a concentration of 20 mm. The data shown in are derived from a single representative experiment of two impartial experiments. The results are presented as means S.D. (= 3). and in Fig. 3and and = 3). Identification of Gal-9-interacting Peptides To identify the substances present in the peak fractions, especially P22 and P23, the P4CP23 preparations with and without treatment with PNGase F were subjected to SDS-PAGE, N-terminal amino acid sequence analysis, and MALDI-TOF MS analysis. The P23 preparation contained a major component with an apparent molecular weight of 20,000C23,000 (P23C), as revealed by SDS-PAGE (Fig. 5, and = 9684.3, which is closely similar to the mass ([M + H]+) of.