CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRPα). Interactions between CD47 and SIRPα regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRPα. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRPα binding in a dose-dependent fashion. Protein binding assays using SIRPα domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRPα, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRPα interactions in regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents.
During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IB␣/ NF-B signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor ␣-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.The epithelial cells lining the gastrointestinal tract form a highly specialized barrier that separates two very distinct environments, thus maintaining the delicate balance between the gut lumen and the underlying tissue (1, 2). As part of its barrier function, the intestinal epithelium is able to detect surface attached enteric pathogens like Salmonella enterica, serovar Typhimurium (S. typhimurium), and orchestrate a proinflammatory response. This multifaceted response involves the rapid secretion of chemoattractants, the development of a chemotactic gradient in the surrounding subepithelial matrix (3-5), and the migration of neutrophils to the site of infection, ultimately triggering secretory diarrhea (6 -11). The concurrent apical release of a soluble factor, designated PEEC (for pathogenelicited epithelial chemoattractant), aids in directing the final polymorphonuclear leukocyte (PMN) movement across the epithelial tight junctions into the gut lumen (12). In addition to IL-8, 1 a variety of other chemokines are secreted in response to S. typhimurium attachment (13-15). Salmonella-induced secretion of IL-8 is known to require the activation of the nuclear transcription factor 17). In quiescent cells, the NF-B heterodimer (p50/p65) is held in a stable, inactive, cytoplasmic complex with IB␣ molecules until activated by an as yet undefined Salmonella...
The widely distributed protein-L-isoaspartyl, D-aspartyl carboxylmethyltransferase (EC 2.1.1.77) is hypothesized to play a role in the repair or metabolism of deamidated and isomerized proteins that are spontaneously generated during the aging of proteins in cells. The yeast two-hybrid system was used to identify proteins that potentially interact with the methyltransferase in a cellular processing pathway. Two cDNAs, both encoding calmodulin, were isolated from a human fetal brain cDNA library using the human methyltransferase as the bait. Enzymatic assays with purified components revealed a complex set of interactions between the methyltransferase and calmodulin. Calmodulin weakly stimulated protein carboxylmethyltransferase activity in vitro at concentrations of the two proteins reflecting their representation in mammalian brain. Calmodulin stimulation of methyltransferase was observed in both the presence and absence of calcium, although the effect was greater in the presence of calcium. Native calmodulin was not a substrate for the carboxylmethyltransferase, but deamidated variants of calmodulin act as substrates for the methyltransferase, with calculated K m values of 3.6 and 8.6 M for calcium-liganded and unliganded calmodulin, respectively. Both the effector and substrate interactions of calmodulin with the protein isoaspartyl methyltransferase likely contributed to the positive results obtained with the two-hybrid system. Protein-L-isoaspartyl, D-aspartyl carboxyl methyltransferase (PCMT)1 (E.C. 2.1.1.77), a well conserved enzyme detected in a broad spectrum of organisms from Escherichia coli to humans (1-6), specifically modifies L-isoaspartyl and D-aspartyl residues that arise in proteins due to spontaneous deamidation and racemization reactions involving asparaginyl and aspartyl residues (7,8). Because the formation of L-isoaspartate can adversely affect the stability and biological potency of many proteins (9 -13), it has been proposed that methylation of the damaged protein initiates either the repair or degradation of the substrate. Experimental support for a structural repair function for PCMT has been obtained in purified systems using isoaspartyl peptides as substrates for the PCMT (14 -16). The esterified isoaspartyl peptides formed by PCMT hydrolyze into both aspartyl peptides and isoaspartyl peptides, the latter of which can be remethylated by the PCMT. The nearly quantitative conversion of isoaspartyl to aspartyl forms of the peptides has been interpreted as evidence for a repair function, although the process lacks the efficiency characteristic of enzymatic processes, and no isoaspartyl residues are converted into asparaginyl residues, the likely origin of most isoaspartyl sites in cellular proteins (2).Because of the structural complexity of protein substrates for the PCMT, very little information is available regarding the fate of isoaspartyl residues in protein substrates. Deamidation of some proteins, including calmodulin (11) and the bacterial HPr phosphocarrier protein (17), generate...
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