Externalization of tropomyosin isoform 5 in colon epithelial cells

Ulcerative colitis (UC) is associated with autoantibody response to a cytos keletal protein, human tropomyosin (hTM) isoform-5 (hTM5). Because hTM5 is an intracellular protein, it may remain inaccessible to the autoantibodies. Therefore, we have investigated the possibility of externalization of hTM5 in colon epithelial cells. Freshly isolated colonic and small intestinal e pithelial cells and LS-180 colon cancer cell line were examined for surface expression of hTM5 by flow cytometric analysis using hTM isoform-specific MoAbs. The extracellular release of hTM5 was determined by Western blot and radioimmunoprecipitation analyses. Physical association of hTM5 with a mem brane-associated colon epithelial protein (CEP) was examined by co-immunopr ecipitation of hTM5 with anti-CEP MoAb, and CEP with anti-hTM5 MoAb. Cell s urface expression of hTM5 was observed in colonic epithelial and LS-180 cel ls but not in small intestinal epithelial cells. LS-180 cells spontaneously released hTM5 as well as CEP into the culture medium that was significantl y stimulated by a calcium ionophore, A23187, but inhibited by phorbol-12-my ristate-13-acetate, monensin and methylamine. Co-immunoprecipitation experi ments revealed that hTM5 forms a complex with CEP. We conclude that hTM5 is externalized in colon but not in small intestinal epithelial cells. The ph ysical association of hTM5 with CEP suggests a possible chaperone function of CEP in the transport of hTM5, a putative target autoantigen in UC.