Junctional adhesion molecules (JAMs) are a family of immunoglobulin‐like single‐span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 Å resolution. rsJAM consists of two immunoglobulin‐like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U‐shaped dimer with highly complementary interactions between the N‐terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U‐shaped JAM dimers are oriented in cis on the cell surface and form a two‐dimensional network by trans‐interactions of their N‐terminal domains with JAM dimers from an opposite cell surface.

Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. Mutant presenilin 1 and 2 (PS1, PS2)1 genes are linked to early-onset familial Alzheimer's disease (1-5). The PS gene products are integral membrane proteins localized in Golgi and ER compartments (6 -8). They share ϳ65% sequence identity and similar structural features, i.e. 6 -8 transmembrane domains, hydrophilic regions at their N and C termini, and a large hydrophilic loop C-terminal to transmembrane domain 6 (6, 9, 10). The loop and the N terminus are the regions of significant sequence divergence between PS1 and PS2. More than 30 mutations in PS1 and 2 mutations in PS2 have been genetically linked to early-onset FAD (3, 4). The biological functions of PS currently remain unknown. Studies on the structural homologue in Caenorhabditis elegans, SEL-12, suggest a role in protein trafficking and in the Notch signaling pathway (11). Human carriers of PS FAD mutations show a small but significant increase (approximately 1.5-3-fold) in plasma levels of A␤ 1-42 compared with normal controls, whereas levels of A␤ 1-40 are unchanged (12-14). A specific effect on ␥-secretase processing of ␤-amyloid precursor protein has been proposed, and this enhanced misprocessing of ␤-amyloid precursor protein is thought to be crucial for FAD pathogenesis. Recent experiments have indicated a regulatory role for PS2 in cell apoptosis. C-terminal fragment of mouse PS2 reportedly protected T-cells or PC12 cells from different apoptosis-inducing challenges, whereas the susceptibility to the same challenges was enhanced when full-length PS2 was overexpressed (15,16).Extensive proteolytic cleavage of PS1 resulting in a 27-28-kDa N-terminal fragment and a 16 -17-kDa C-terminal fragment was reported for PS1-transfected COS-1 cells, mice transgenic for human PS1, and endogenous PS1 in human and murine tissues (12,(17)(18)(19). Proteolytic processing has also been reported for human PS2 resulting in a 35-kDa N-terminal fragment and a 20-kDa C-terminal peptide (3,10,13). In the present study we have identified proteolytic processing sites in PS1 and PS2 and demonstrate that processing occurs through caspase-type protease(s). We also observed additional, caspaseindependent cleavage products suggesting alternative pathways of presenilin processing. EXPERIMENTAL PROCEDURESAntibodies-Amino acids 1-80 of human PS2 (N terminus) and 270 -387 of murine PS2 (large hydrophilic loop between transmembrane domains 6 and 7) were expressed in Escherichia coli as (NANP) 19 -fusion proteins and used for immunization of mice. PST20 is a monoclonal antibody specific for the hydrophilic N-termi...

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

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