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First published online 20 July 2004
doi: 10.1242/jcs.01261

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Diglons are heterodimeric proteins composed of IgLON subunits, and Diglon-CO inhibits neurite outgrowth from cerebellar granule cells

¹ Department of Human Anatomy and Cell Biology, Liverpool University, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
² Department of Medicine, Liverpool University, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK

View larger version (46K): [in a new window]   Fig. 1. CGC express LAMP, OBCAM and CEPU-1 on their surface. (A) RT-PCR was carried out on cDNA prepared from E15 cerebellum. Primers were designed to amplify a central region of LAMP and OBCAM occurring in all known isoforms, or to detect CEPU-1 and CEPU-Se, specifically. All three members of the family and both GPI-anchored and secreted forms of CEPU-1 are expressed. (B) Immunofluorescence microscopy of live-stained CGC revealed that LAMP, OBCAM and CEPU-1 are expressed on the cell surface. In all cases, specific punctate staining could be observed. Bar, 25 µm.

View larger version (29K): [in a new window]   Fig. 2. Recombinant IgLON-Fc proteins show differential interactions with CGCs. CGCs adhere preferentially to CEPU-1-Fc and fail to adhere to LAMP-Fc. Dissociated CGCs were incubated on coverslips sequentially coated with protein A and recombinant IgLON-Fc for (A) 4 hours (n=2) or (B) 18 hours (n=3), and adhering cells were counted. After 4 hours, the number of cells adhering to CEPU-1-Fc was approximately 60% of that adhering to poly-L-lysine and was significantly above background (P<0.0001), whereas adhesion to LAMP and OBCAM was similar to background. After 18 hours, adhesion to CEPU-1-Fc had increased to 80% of the poly-L-lysine control and adhesion to OBCAM-Fc was now 50% of the poly-L-lysine control and significantly above background (P<0.0001). Adhesion to LAMP-Fc remained similar to background. (C) Live staining of CGC with IgLON-Fc revealed a punctate stain with both CEPU-1-Fc and OBCAM-Fc, but LAMP-Fc staining was similar to the secondary antibody alone. In keeping with the adhesion results shown above, the CEPU-1-Fc staining was more intense than that observed with OBCAM-Fc. Bar, 25 µm.

View larger version (14K): [in a new window]   Fig. 3. IgLON homodimers have different binding affinities. Fluorescent beads coupled to protein G coated with recombinant LAMP-Fc, OBCAM-Fc or CEPU-1-Fc were allowed to aggregate for 15 minutes and analysed by flow cytometry. (A) CEPU-1-Fc-coated beads showed the best aggregation, with only 22% of beads remaining single, compared with 30% for OBCAM-Fc and 45% for LAMP-Fc. An unrelated lymphocyte protein Ox40-HuIg Fc gave 68%, and protein-G-coupled beads gave 80% (n=4). CEPU-1 and OBCAM were significantly different from LAMP (P<0.004 and P<0.04, respectively). (B) CEPU-1-Fc-coated beads formed larger aggregates than LAMP-Fc-coated beads. The number of single CEPU-1-Fc beads was small compared with single LAMP beads, and the number of CEPU-1 aggregates with three or more beads was much larger.

View larger version (17K): [in a new window]   Fig. 4. LAMP binds with highest affinity to OBCAM and CEPU-1. CHO cell lines were grown in 96-well plates and incubated with varying concentrations of chimaeric proteins. (A) For LAMP-CHO, strong binding was observed with OBCAM-Fc and CEPU-1-Fc, both of which displayed almost identical absorbance for each concentration tested. However, with LAMP-Fc, the absorbance remained at background levels, even at 10 µg ml–1. (B) For OBCAM-CHO, highest absorbance values were observed with LAMP-Fc at 10 µg ml–1 (0.75), as predicted by the previous result. The next highest was CEPU-1-Fc at 10 µg ml–1 (0.35) and the lowest was OBCAM-Fc at 10 µg ml–1 (0.2). (C) For CEPU-CHO, highest absorbance was observed with LAMP-Fc at 10 µg ml–1 (0.8), followed by CEPU-1-Fc at 10 µg ml–1 (0.7) and the lowest was OBCAM-Fc at 10 µg ml–1 (0.35), as expected. Error bars lie within the symbol for some points. (D) Taking together the relative affinities of the homophilic interactions from Fig. 3 and the comparative affinity of the homo- and heterophilic interactions, we propose this hierarchy for the relative affinity of IgLON interactions in trans.

View larger version (37K): [in a new window]   Fig. 5. Do IgLONs form heterodimers in cis? We propose that IgLONs preferentially form heterodimers on the surface of neurons. The homodimeric IgLON-Fc proteins might not bind to the heterodimers because dimerization induces a conformational change that interferes with binding, because of steric hindrance or because the affinity of one head to one of the heterodimeric subunits is too low to stabilize the complex.

View larger version (90K): [in a new window]   Fig. 6. Heterodimeric proteins form on the surface of CHO cells expressing both LAMP and OBCAM. CHO cells stably expressing LAMP or CEPU-1 were transiently transfected with pOIG. Cells were stained with LAMP-Fc, OBCAM-Fc or CEPU-1-Fc. (A) LAMP-CHO cells bound OBCAM-Fc with high affinity but, when OBCAM was expressed, staining was specifically reduced (arrow). Non-transiently transfected cells have either LAMP monomers or dimers on their cell surface but, after expression of OBCAM, LAMP is sequestered by OBCAM in heterodimers. OBCAM:LAMP heterodimers no longer stabilize OBCAM-Fc binding. If OBCAM expression exceeds LAMP expression then OBCAM monomers or dimers will also be available, but these will similarly fail to stabilize OBCAM-Fc binding. (B) LAMP-CHO cells bound CEPU-1-Fc with high affinity but, when OBCAM was expressed, staining was specifically reduced (arrow). CEPU-1-Fc binds well to cells expressing only LAMP, but binding is destabilized by the introduction of OBCAM and the subsequent incorporation of LAMP into LAMP:OBCAM dimers. (C) LAMP-CHO cells bound LAMP-Fc very weakly, but expression of OBCAM increased LAMP-Fc binding owing to greater retention of LAMP-Fc by the LAMP:OBCAM dimers (compared with LAMP homodimers) and perhaps the presence of free OBCAM, which would bind LAMP-Fc with relatively high affinity. Introduction of OBCAM into CEPU-1-CHO cells resulted in little change in fluorescence intensity. (D) LAMP-Fc bound with similar (high) affinity to CEPU-1-CHO and to the OBCAM:OBCAM dimers that might form. However, it is unclear from this experiment whether CEPU-1:OBCAM dimers also form and whether LAMP-Fc can interact with them. Intense fluorescence staining of all cells was observed. (E) OBCAM-Fc and (F) CEPU-1-Fc stained CEPU-1-CHO cells less intensely than LAMP-Fc but, again, OBCAM expression did not change the intensity of fluorescence staining. It is important that, in these last three experiments, transient transfection of OBCAM cDNA was insufficient on its own to change the ability of the chimaeric proteins to bind. For all cases, expression of EGFP alone did not alter IgLON binding. Bar, 25 µm. The fluorescence intensity of untransfected and pOIG-transfected LAMP-CHO cells was quantified. Transfection of OBCAM significantly reduced the binding of (G) OBCAM-Fc or (H) CEPU-1-Fc as judged by the reduction in fluorescence intensity. (I) The fluorescence intensity of untransfected and pOIG-transfected CEPU-1-CHO cells was quantified. Transfection of OBCAM did not significantly alter the binding of LAMP-Fc as judged by fluorescence intensity. (J) The expression of OBCAM on pLIG-transfected CHO cells was unchanged as judged by immunofluorescence staining with antisera to OBCAM.

View larger version (67K): [in a new window]   Fig. 7. Heterodimeric proteins form on the surface of CHO cells expressing LAMP and either OBCAM or CEPU-1. (A) Expression of LAMP in OBCAM-CHO cells destabilized the surface binding of LAMP-Fc. The arrowed cell, identified by GFP fluorescence, showed essentially no staining for LAMP-Fc despite its rounded, phase-bright morphology. Notice cells indicated with arrowheads that were also phase bright and stained intensely with LAMP-Fc. Expression of LAMP on the cell surface results in sequestration of free OBCAM into LAMP:OBCAM dimers, leaving no detectable OBCAM monomers or homodimers. (B) Expression of LAMP in OBCAM-CHO cells produced a small increase in fluorescence intensity when stained with OBCAM-Fc, possibly owing to increased affinity for LAMP:OBCAM dimers or overexpression of LAMP. Similar results were observed for CEPU-1-Fc (data not shown). (C) Expression of LAMP in CEPU-1-CHO cells also resulted in loss of LAMP-Fc binding (arrow). This is explained by the formation of LAMP:CEPU-1 dimers in cis, which destabilizes the binding of LAMP-Fc. (D) OBCAM-Fc binding increased as a result of LAMP expression, again because of the greater affinity of OBCAM-Fc for the heterodimer and/or the overexpression of LAMP. Similar results were observed for CEPU-1-Fc (data not shown). Bar, 25 µm.

View larger version (26K): [in a new window]   Fig. 8. CEPU-1/OBCAM-expressing CHO cells inhibit neurite outgrowth from CGCs. CGCs were seeded at 1x105 cells per well onto confluent CHO cell monolayers growing on coverslips and incubated at 37°C in a 5% CO2 atmosphere for 28-31 hours. Cells were fixed, permeabilized and stained with chicken-GAP-43 antibody to identify the neurons. (A) At least 100 individual neurons were counted per coverslip, comparing the number with and without neurites. CO-CHO cells inhibited the number of neurons that had extended neurites by 40% (with respect to wild-type CHO cells) and 45% (with respect to the average of the CEPU-1- and OBCAM-expressing cell lines) (n=5). *P<0.001 compared with CEPU-1- or OBCAM-expressing CHO cells and P<0.02 compared with wild-type CHO cells. (B) At least 220 random neurites greater than 10 µm long were measured per CHO cell line using Metamorph. Neurites from CGCs seeded on OBCAM-CHO were slightly longer and those on CEPU-1- and CO-CHO slightly shorter, respectively, than those from CGCs seeded on wild-type CHO cells (n=5). (C) CHO cell lines were grown in a 96-well plate and incubated with 1 µg ml–1 LAMP-Fc. LAMP-Fc bound well to CEPU-1- and OBCAM-expressing CHO cells, but its affinity for CO-CHO cells was comparable to that of the LAMP-CHO cells.

View larger version (73K): [in a new window]   Fig. 9. Proposed IgLON and Diglon CAMs. We have shown that LAMP associates in cis with either OBCAM or CEPU-1, and that CEPU-1 and OBCAM also associate in cis; we also propose that the same combinations might be possible for Kilon. (A) The proposed dimeric IgLONs (Diglons). (B) Diglon-LO might, in principle, bind to one or more of the other Diglons. (C) It is unclear whether IgLONs will also function as independent CAMs; this will be the case if cells express only one member of the family or unequal amounts of two or more IgLONs. Based on the results presented here, the most likely combinations are shown. LAMP and OBCAM are unlikely to function as homophilic CAMs, but a cell expressing only LAMP will adhere effectively to an adjacent cell expressing only OBCAM or CEPU-1. It remains to be seen whether CEPU-1 is unique in acting as a homophilic CAM or whether this is an activity restricted to the minor isoforms of CEPU-1 (and LAMP) that have a ß exon.

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