key: cord-0009827-lt9yzs99 authors: Skubitz, K.M.; Campbell, K.D.; Skubitz, A. P. N. title: Synthetic peptides from the N‐domains of CEACAMs activate neutrophils date: 2008-12-08 journal: J Pept Res DOI: 10.1034/j.1399-3011.2001.00931.x sha: 7ac32d1a5bf74ab9e422875203de65b7729ae6e5 doc_id: 9827 cord_uid: lt9yzs99 Abstract: Four members of the carcinoembryonic antigen family, CEACAM1, CEACAM8, CEACAM6 and CEACAM3, recognized by CD66a, CD66b, CD66c and CD66d monoclonal antibodies (mAb), respectively, are expressed on human neutrophils. CD66a, CD66b, CD66c and CD66d mAb binding to neutrophils triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to human umbilical vein endothelial cells. Molecular modeling of CEACAM1 using IgG and CD4 as models has been performed, and three peptides from the N‐terminal domain were found to increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. The peptides were 14 amino acids in length and were predicted to be present at loops and turns between β‐sheets. To better understand the amino acid sequences critical for this biological activity, in the present study we examined the other neutrophil CEACAMs and the highly homologous CEACAM, CEA. Molecular modeling of the N‐terminal domains of human CEACAM8, ‐6, ‐3 and CEA was performed. Twenty peptides, each 14 amino acids in length, that were homologous to the previously reported peptides from the N‐domains of CEACAM1, were synthesized and tested for their ability to alter neutrophil adhesion. Only one new peptide, from the N‐domain of CEA, was found to increase neutrophil adhesion, and this peptide differed from the corresponding CEACAM1 peptide by only a single conservative amino acid substitution. Importantly, minor amino acid differences between active and inactive homologous peptides suggest regions of these peptides that are critical for biological activity. The data suggest that the regions SMPF of peptide CD66a‐1, QLFG of peptide CD66a‐2 and NRQIV of peptide CD66a‐3 are critical for the activities of these peptides, and for the native CEACAMs. The mechanism(s) by which CEACAMs transmit signals (e.g. activation in neutrophils, or growth regulating signals in epithelial cells and carcinomas) are unclear. However, CEACAM1 is phosphorylated on its cytoplasmic domain, largely on tyrosine with a lower level of phosphoserine, in neutrophils and colon cancer cells (12,62±66). In addition, associated protein tyrosine kinase and phosphatase activities may be involved in CEACAM signaling (62, 64, 65) . Because of the adhesive and signaling properties of Normal peripheral blood neutrophils were prepared by a modi®cation of the method described in Ref. 68 Each experiment was performed at least three times using different HUVEC subcultures. Effects of peptides on neutrophil adhesion to HUVECs was analyzed by the Student's t-test when appropriate. Analyses of CD11b and CD62L expression were performed as previously described (67) . Synthetic peptides of human CEACAMs as described in the text. Amino acids underlined and shown in bold represent amino acid residues that differ from the homologous CEACAM1 peptide. cells were then incubated, washed and analyzed by¯ow cytometry as above. As CEACAMs are members of the Ig superfamily, we modeled CEACAM8, CEACAM6, CEACAM3 and CEA using the known crystallographically determined structure of the IgV and Ig C2-like domains of IgG and CD4. The amino acid sequences were also analyzed by a hydropathy plot using Kyte±Doolittle and Chou±Fassman analyses, and sequences predicted to be exposed on the surface of the molecules based on hydrophilicity were identi®ed. There was good agreement in general between the peptides selected using these two methods. A series of 32 peptides of 14 amino acids in length was then identi®ed that were predicted to contain turns and loops between b-sheets ( CD66e-6 and also CD66a-7, which is identical to CD66c-7, CD66d-7 and CD66e-7, was not successful. Peptide CD66a-6, which is identical to CD66c-6, was not soluble in our assay conditions and was therefore not studied further. The CEACAM1 peptides CD66a-1, CD66a-2 and CD66a-3 were previously reported to activate neutrophils and to increase neutrophil adhesion to HUVECs (67 CD66c-6, CD66c-7 and CD66c-8 were not tested as they are identical to the CD66a-6 (insoluble), CD66a-7 (unsuccessful synthesis) and CD66a-8 (inactive in this assay) peptides, respectively, previously reported (67) . Peptide CD66d-7 was not tested as it is identical to the CD66a-7 peptide. We were not able to synthesize peptide CD66d-6 in three separate attempts. Six peptides from CEA (CD66e-1 to CD66e-5 and CD66e-8) were also tested for their ability to alter neutrophil adhesion to HUVECs (Fig. 1) . Peptide CD66e-3, but none of the other CEA peptides, altered neutrophil adhesion to HUVECs. Peptide CD66e-7 was not tested as it is identical to CD66a-7. We were not able to synthesize peptide CD66e-6 in three separate attempts. The CEA peptide CD66e-3 was further tested for its effects on the adhesion of neutrophils to resting and TNF stimulated HUVECs in the presence of FMLP. HUVECs at concentrations as low as 75 mg/mL (< 50 mm) ( Fig. 2) . The effects of the CEA peptide CD66e-3 on surface expression of CD11b on neutrophils was next examined. Twenty peptides were synthesized from regions of the N-domains of human CEACAM8, CEACAM6, CEACAM3 and CEA, that we predict form loops and turns between regions of b-sheets and may be exposed on the surface of the molecule. These peptides are homologous to a series of peptides from CEACAM1 that we described previously (67) that included three peptides that had activity in an assay examining stimulated neutrophil adhesion to HUVECs. Although 19 of the 20 peptides tested in this study were not active in this assay, it is possible that they might have functional activity in a different assay. We chose to focus our study on the N-domains of Comparison of active synthetic peptides of human CEACAM1 (CD66a-1 to CD66a-3), with homologous peptides of CEACAM8, CEACAM6, CEACAM3, and CEA as described in the text. Amino acids underlined and shown in bold represent amino acid residues that differ from the homologous CEACAM1 peptide. Activity in the neutrophil stimulation assay as described in the text is shown in the right column. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues CEA adhesion molecules ± multifunctional proteins with signal-regulatory properties Carcinoembryonic antigen gene family: molecular biology and clinical perspectives Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus in¯uenzae The structural basis of CEACAM-receptor targeting by neisserial opa proteins: response Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV Binding of the coronavirus mouse hepatitis virus A59 to its receptor expressed from a recombinant vaccinia virus depends on posttranslational processing of the receptor glycoprotein Receptor for mouse hepatitis virus is a member of the carcijojembryonic antigen family of glycoproteins Mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors Coronavirus receptor speci®city CD66 family members are associated with tyrosine kinase activity in human neutrophils Anti-serum to carcinoembryonic antigen recognizes a phosphotyrosine-containing protein in human colon cancer cell lines Association of p60 c±src with biliary glycoprotein (CD66a), an adhesion molecule of the carcinoembryonic antigen family downregulated in colorectal carcinomas Synthetic peptides of CEACAMs activate neutrophils Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells Tyrosine phosphorylation of biliary glycoprotein, a cell adhesion molecule related to carcinoembryonic antigen Synthetic peptides of CD66a stimulate neutrophil adhesion to endothelial cells Monoclonal antibodies that recognize lacto-N-fucopenatose III (CD15) react with adhesion-promoting glycoprotein family (LFA-1/HMAC-1/GP 150,95) and CR1 on human neutrophils Rapid analysis of leukocyteendothelial adhesion CD63 associates with tyrosine kinase activity and CD11/CD18, and transmits an activation signal in neutrophils CD50 monoclonal antibodies inhibit neutrophil activation Intercellular adhesion molecule-1 gene expression in human endothelial cells Traf®c signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes Leukocyteendothelial adhesion molecules Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve ®tting procedure: implications for cellular adhesion Extracellular N-domain alone can mediate speci®c heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8 The Identi®cation of Critical Adhesiotopes on the N-Domain of Human CEACAM1 Required for Homophilic Interactions Self recognition in the Ig superfamily. Identi®cation of precise subdomains in carcinoembryonic antigen required for intercellular adhesion Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59 Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen A CD66a-speci®c, activation-dependent epitope detected by recombinant human signal chain fragments (scFvs) on CHO transfectants and activated granulocytes Evidence for regulated dimerization of cell±cell adhesion molecule (C-CAM) in epithelial cells The dimeric structure of carcinoembryonic antigen (CEA) Calmodulin binds to speci®c sequences in the cytoplasmic domain of C-CAM and down-regulates C-CAM self-association Biliary glycoprotein (BGP) expression on T cells and on a natural-killer-cell sub-population Susceptibility of colorectal carcinoma cells to natural-killer-mediated lysis: relationship to CEA expression and degree of differentiation CEA expression of colorectal adenocarcinomas is correlated with their resistance against LAK-cell lysis Binding of Escherichia coli and Salmonella strains to members of the carcinoembryonic antigen family: differential binding inhibition by aromatic a-glycosides of mannose Identi®cation of the speci®c oligosaccharide sites recognized by type 1 ®mbriae from Escherichia coli on nonspeci®c cross-reacting antigen, a CD66 cluster granulocyte glycoprotein We thank Dr W. Gleason for a critical review of the manuscript.