Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.

Phtsmin mai,dy cle;tved the ArlI~,$eP bond o1' ArlpVaI.Leu.Pro.Arll.inletleukin.8 (AVLPR-IL-a} produced by human derm.I Itbroblast=t, which r¢=ulted in the conversion orAVLPR.IL.8 to IL.~t and the in,clive pentapeptide, though, minor cleavall~ of AVLPR-IL.It by phtsmin at Ly~':,Ght'* bond occurred. MATERIALS AND METHODS Purtfication of FDNCFHuman dermal fibroblasts (SF-TY cell line) were maintained in Dulbecco's modified Eagle's medium supplemented with 5°7o fetal calf sert~,m, 25 mM Hepes, penicillin (0, I mg/ml) and streptomycin (O. 1 mg/ml) until they reached confluence. The cells were cultured in serum-free culture medium supplemented with tO "m M recombinant human IL-I# and 0.1o70 bovine serum albumin. After culture for 2 days, cell.free conditioned medium was obtained by centrifugation at 1600× g for 20 rain. The chemoattractant in the conditioned medium Abbreviations; AVLPR-IL-8, Ala-Val-Leu-Pro-Arg-interleukin-8; FDNCF fibroblast-derived neutrophil chemot~ctic factor; RP. HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodccyl sulfate polyacrylamide gel electrophoresJs.Correspondence address: H. Nakagawa, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyarna 930-01, Japan, was purified essentially ns described previously [11]; the sample was chromatoliraphedsequential/y on CM-Seph~dex, Sephadex O-?$ and RP-HPLC, Tree/men/of FDNCF =+'ith plasminFDNCF (40 j+g) in a final volume of 0,2 ml ¢0f 50 mM Tris.HCI buffer (pH 8.0) was incubated with 0,gpg of plasmin (purified from human plasma; Sigma, MO. USA) at 37*C for 30 rain, and the rent. lion was stopped by the addition of leupeptin (final 1 raM), The reae. lion mixture was concentrated by a Speed-Vac centrifuge, dissolved in 6 M 8uanidine solution and loaded onto a C.18 reverse-phase col. umn (0.45 x l$cm; ODS.120T, Tosoh Co, Tokyo, Japan).Chemotaetic factors were ehtted with a linear co~centration gradient of aeetonitrile from 0% to 50.4% in 0.05% trifluoroacetic acid at z~ flow rate of 0.8 ml/min, Neutrophil chemotaxls of plasmin.treated FDNCF was performed in vitro by using rat and human neutrophils as described previously [121, As an index of ehemotaxis, the number of neutrophils migrated into the lower chamber was expressed as percentage (migration rate) of that of neutrophils applied in the upper chamber, $DS-PAGE was carried out on a slab gel of a discontinuous 9,6%/16,507o acrylamide in tricine buffer system [13]. NH2.terminal atnino acid sequencing analysisNH,-terminal amino acid sequence determination was performed by automated Edman degradation on a gas-plmse protein sequencer (model 470A, Applied Biosystems, CA, USA) e¢~uipped with a PTH analyzer (model 120A HPLC system). RESULTS AND DISCUSSIONHuman dermal fibroblasts stimulated with 10 -t° M IL-1 mainly produced FDNCF, which is the NH2-termina] extended 77-residue variant of IL-8 (AVLPR-IL-8). The homogeneity of the purified FDNCF was confirmed by SDS-PAGE, its NH2-terminal amino acid sequence and its COOHterminal amino...

Cadherin is a cell adhesion molecule crucial for epithelial and endothelial cell monolayer integrity. The previously solved x-ray crystallographic structure of the E-CAD12 cis-dimer displayed an unpaired Cys 9 , which protruded away from the Cys 9 on the other protomer. To investigate the possible biological function of Cys 9 within the first repeat (the E-cadherin-derived Nterminal repeat), E-CAD1 was overexpressed and secreted into the periplasmic space of Escherichia coli cells. Recombinant E-CAD1 produced a mixed monomer and dimer in an equilibrium fashion. The dimer was linked by a disulfide through Cys 9 pairing. Analysis by high pressure liquid chromatography and electron microscopy suggested the existence of oligomeric complexes. Mutation at Trp 2 appears to indicate that these oligomeric complexes trans-dimerize. Interestingly, mutation of Cys 9 affected not only the cis-dimerization, but also the trans-oligomerization of E-CAD1. Accordingly, it is plausible that, under oxidative stress, the homophilic interactions of E-cadherin through E-CAD1 may be promoted and stabilized by this disulfide bond.E-cadherin, a Ca 2ϩ -dependent member of the cadherin family of cell adhesion molecules, is crucial in providing cell polarity, tightness, and integrity of the intercellular junctions (1, 2). In most cancerous tissues, nonfunctional E-cadherin leads to the disturbance of the integrity of the intercellular junctions and consequentially promotes higher mobility and invasiveness of the cancer cells (3-5).In recent years, considerable information has been gathered on the adhesion mechanism of classical cadherins from both structural and functional studies. The modular architecture of classical cadherins is characterized by the five repeats in the extracellular domain (6). The N-terminal of the extracellular domain is believed to be critical for homophilic cadherin interactions. The structure of the N-terminal fragment derived from the first repeat of the neuronal N-cadherin (N-CAD1) (7) and the epithelial E-cadherin (E-CAD1) (8) displays a resemblance to the structural fold of immunoglobulin.Biochemical analysis of the first extracellular repeat E-CAD1 revealed only the presence of monomers (8). The solution structure of this monomer form was indeed determined by nuclear magnetic resonance (9, 10). This is in contrast with the crystal lattice structure of N-CAD1 (7), which contains a unique mixture of two different populations. One population consists of monomers interacting closely to form cis-dimers (parallel), which are stabilized by the exchange between the N-terminal ␤A-strand and the intercalation of Trp 2 into the partnering hydrophobic core. The other population reveals the pairing between antiparallel-oriented cisdimers, designated as trans-dimers to reflect the possible head-to-head contacts between two cadherin molecules of the apposing cells. Alternating the cis-and trans-interactions forms a zipper-like structure (7). Crystallographic analysis of E-CAD12 also showed cis-dimers linked together by calciu...

No abstract

Transient modulation of E-cadherin-mediated cell-cell adhesion may improve paracellular drug delivery through biological barriers. Therefore, there is a need to develop an efficient method to evaluate cadherin peptides that can modulate the intercellular junctions. The objective of this study was to establish a novel assay to evaluate peptide activity in modulating E-cadherin-mediated homophilic interactions, based on the homotypic adhesion of Caco-2 cells. Fluorescence-labeled Caco-2 single cells were incubated with Caco-2 monolayers that were treated beforehand with Ca 2ϩ -free medium. The homotypic adhesion in the presence or absence of peptide and antibody was determined fluorometrically. The Ca 2ϩ -deficient pretreatment dramatically increased the number of single cells bound to the monolayers. Immunofluorescence staining showed that some of E-cadherins became accessible without surfactant-induced permeabilization of Caco-2 cell monolayers after the Ca 2ϩ -deficient pretreatment. The homotypic adhesion was largely dependent on extracellular Ca 2ϩ concentrations and significantly inhibited by the presence of anti-E-cadherin monoclonal antibody DECMA-1. In contrast, DECMA-1 did not inhibit E-cadherin-independent adhesion, such as the homotypic adhesion of Caco-2 cells in the absence of Ca 2ϩ or the heterotypic adhesion of Molt-3 T cells to Caco-2 monolayers. These results indicate the predominant involvement of E-cadherin-mediated cell-cell adhesion in this assay. E-cadherinderived peptides, which had been shown in our previous studies to inhibit E-cadherin-mediated cell-cell adhesion, significantly inhibited homotypic adhesion in a dose-dependent manner. These results, taken together, suggest that the present assay can be used for evaluation of peptide, protein, or antibody activity in modulating the E-cadherin-mediated homophilic interactions in the context of whole live cells.