IMMUNOCYTOCHEMICAL DETECTION OF EXTRACELLULAR ANNEXIN-II IN CULTURED HUMAN SKIN KERATINOCYTES AND ISOLATION OF ANNEXIN-II ISOFORMS ENRICHEDIN THE EXTRACELLULAR POOL

Monoclonal antibodies were raised against trypsinized human skin epide rmal cells and selected for their staining of the epidermal cells in a cell periphery pattern. One antibody, CP-1, immunoprecipitated a 36 k Da protein that was identified as annexin II, heavy chain by microsequ encing of a CNBr-generated peptide fragment from the antigen and by cr oss-identification with another anti-annexin II antibody. In addition to staining a broad cell periphery band in keratinocytes, CP-1 also de tected annexin II outside and in between the top layer cells before ce ll permeabilization. Double-labeling of annexin II and F-actin reveale d a distinct topographical relationship between the two, with intercel lular annexin II flanked by the submembranously located actin of the j uxtapositioned cells. Annexin II was isolated from cultured keratinocy tes via immunoaffinity column chromatography in one step, using the sa me monoclonal antibody CP-1 and was found to be resolved into multiple isoforms when analyzed by two-dimensional gel electrophoresis. The pr edominant components of annexin II were basic, with pI of 6.5-8.5, and some of them formed disulfide-linked monomeric multimers under non-re ducing conditions. Acidic annexin II isoforms with pI 5.4-5.8 were bar ely detectable among the total annexin II isolated but were selectivel y enriched in an extracellular pool created by 0.05% ethylenediaminete traacetic acid (EDTA) dispersion of the cultured cells into single cel l suspensions. Furthermore, they can be separated from the rest of ann exin II by using a different elution condition. A 46 kDa protein, the identity of which is unclear, co-eluted with the acidic isoforms in th e EDTA washes. These acidic isoforms, which co-eluted with the 46 kDa protein, are suspected of corresponding to the extracellular annexin I I detected immunocytochemically.