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Annexin 6 is a putative cell surface receptor for chondroitin sulfate chains

¹ Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
² Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan

View larger version (48K): [in a new window]   Fig. 1. A schematic representation of the cell attachment assay (A) and the attachment of cells to CSPE-coated substratum in this assay (B). When cells were plated onto adhesive- or 5 µg/ml CSPE-coated wells, they remained attached to the entire bottom of the well after the centrifugation. When cells were plated onto non-adhesive protein, BSA (10 mg/ml), the centrifugation formed a small pellet of cells at the bottom of the well.

View larger version (22K): [in a new window]   Fig. 6. Expression of the deletion mutants of annexin 6 and the effect on attachment to the CSPE substratum. (A) Schematic representation of the deletion mutant structures of human annexin 6. The abbreviations of the mutants used in the figure were indicated in the text. All constructs were designed with a hemagglutinin A tag at the C-terminus. The numbers indicate human annexin 6 amino-acid residues encoded by each construct. Western blotting of transfectants with those deletion mutants was performed using 50 µg of protein from cell lysates. The proteins were detected using anti-hemagglutinin A antibodies as described in the Materials and Methods. (B) The attachment abilities of those transfectants to CSPE substratum were examined using the cell attachment assay. The results are expressed as mean values and standard deviations of determinations from at least three independent experiments.

View larger version (15K): [in a new window]   Fig. 2. (A) Effect of the simultaneous presence of CS chains in the substratum on cell attachment to FN or ConA substratum. The attachment of human IMR90 cells was assessed by the cell attachment assay as described in the Materials and Methods. The wells were coated with 5 µg/ml FN, 8 µg/ml ConA and 5 µg/ml CSPE. +RGD, the addition of 2 mM GRGDSP peptide to medium; +RGE, the addition of 2 mM GRGESP peptide to the medium. (B) Attachment of cells to CSPE substratum via a specific interaction with CS chains. Compared with the attachment to FN, human IMR90 cells weakly but significantly attached to CSPE substratum. +CSase, the pretreatment of the wells with chondroitinase ABC; +CS, the addition of 1 mg/ml CS (shark cartilage) to the medium; +HA, the addition of 1 mg/ml HA to the medium. The addition of CS (whale cartilage) and dermatan sulfate at the same concentration as above had the same inhibitory effect, and chondroitin had no significant effect (data not shown). (C) Dependency of the cell attachment to CSPE substratum on Ca2+. IMR 90 cells were plated onto CSPE-coated wells containing the media with variable concentrations of EGTA and Ca2+. The results are expressed as mean values and standard deviations of determinations from at least three independent experiments.

View larger version (66K): [in a new window]   Fig. 3. Purification and characterization of a putative receptor for CS chains. (A) Human fibroblast WI38 extracts (lanes 1-4) and chick embryonic fibroblasts (lanes 5 and 6) were subjected to CS affinity chromatography in the presence of Ca2+. Cell membrane extracts (lane 1), the flow-through fraction from the CS column (lane 2), the elution fraction with EDTA buffer (lanes 3 and 5) and the subsequent fraction obtained by further elution with 4M urea (lanes 4 and 6) were subjected to SDS-PAGE (10%) and visualized by Coomassie brilliant blue staining. (B) Comparison of the amino-acid sequence of the 68 kDa protein with annexin 6. (C) Proteins in the elution fraction from human WI38 fibroblasts extracts were subjected to SDS-PAGE and western blot analysis. Lane 1, Coomassie brilliant blue staining of SDS-PAGE (10%). Lane 2, identification of immunoreactive human annexin 6 in the elution fraction by western blot analysis with rabbit anti-annexin 6 antibodies. An identical blotting pattern was obtained by analysis using mouse monoclonal antibody to human annexin 6.

View larger version (39K): [in a new window]   Fig. 4. The inability of A431 cell to attach to CSPE substratum is due to lack of annexin 6 expression. (A) The ability of various human cultured cell lines, IMR90, MG63, MRC5, WI38 and A431 cells to attach to CSPE substratum was examined using the cell attachment assay as described in the Materials and Methods. The results are expressed as the means and ranges of determinations from at least three experiments. (B) 50 µg of protein from cell lysates were analysed by western blotting of annexin 6 and annexin 4 with mouse monoclonal antibodies to human annexin 6 and human annexin 4, respectively.

View larger version (34K): [in a new window]   Fig. 5. The relationship between the expression of annexin 6 and the cell attachment ability of A431 cells. (A) The expression of annexin 6 was examined by western blotting of two stable transfectants with annexin 6 cDNA (Anx#1 and Anx#2) and one with mock vector (Mock#1), together with IMR90 and the parent A431 cells. 50 µg of protein of cell lysates were used for western blotting. Human annexin 6 was detected using mouse monoclonal anti-annexin 6 antibody. (B) Attachment of the transfectants to the CSPE substratum was assayed. The results are expressed as mean values and standard deviations of determinations from at least three independent experiments.

View larger version (35K): [in a new window]   Fig. 7. Flow cytometric analysis of the annexin-6-expressing transfectant. (A) One million stable transfectants with the annexin 6 cDNA (Anx#1) or mock vector (Mock#1) were labeled with various antibodies. Anti-FAK antibody (1:200 dilution) was used as a negative control. Anti-annexin 6 and anti-ß1-integrin polyclonal antibodies were used at the final dilutions of 1:500 and 1:200, respectively. (B) Anx#1 was also labeled with two different monoclonal (ZYMED and Transduction Laboratories) and one polyclonal antibodies (Santa Cruz) to annexin 4 at the final dilutions of 1:200. The source of the anti-annexin 4 antibody is indicated in brackets. Propidium iodide was used for the detection of any damaged or leaky cells. Gates for propidium iodide staining and FITC intensity were placed on the basis of values of negative cells. Numbers in quadrants represent the percentage of gated cells.