title: Immunocytochemical detection of extracellular annexin II in cultured human skin keratinocytes and isolation of annexin II isoforms enriched in the extracellular pool.
abstract: Monoclonal antibodies were raised against trypsinized human skin epidermal cells and selected for their staining of the epidermal cells in a cell periphery pattern. One antibody, CP-1, immunoprecipitated a 36 kDa protein that was identified as annexin II heavy chain by microsequencing of a CNBr-generated peptide fragment from the antigen and by cross-identification with another anti-annexin II antibody. In addition to staining a broad cell periphery band in keratinocytes, CP-1 also detected annexin II outside and in between the top layer cells before cell permeabilization. Double-labeling of annexin II and F-actin revealed a distinct topographical relationship between the two, with intercellular annexin II flanked by the submembranously located actin of the juxta-positioned cells. Annexin II was isolated from cultured keratinocytes via immunoaffinity column chromatography in one step, using the same monoclonal antibody CP-1 and was found to be resolved into multiple isoforms when analyzed by two-dimensional gel electrophoresis. The predominant components of annexin II were basic, with pI of 6.5-8.5, and some of them formed disulfide-linked monomeric multimers under non-reducing conditions. Acidic annexin II isoforms with pI 5.4-5.8 were barely detectable among the total annexin II isolated but were selectively enriched in an extracellular pool created by 0.05% ethylenediaminetetraacetic acid (EDTA) dispersion of the cultured cells into single cell suspensions. Furthermore, they can be separated from the rest of annexin II by using a different elution condition. A 46 kDa protein, the identity of which is unclear, co-eluted with the acidic isoforms in the EDTA washes. These acidic isoforms, which co-eluted with the 46 kDa protein, are suspected of corresponding to the extracellular annexin II detected immunocytochemically.
authors.affiliation: Department of Medicine, University of Chicago, Illinois 60637.
authors.forename: A S
authors.initials: AS
authors.lastname: Ma
authors.forename: D J
authors.initials: DJ
authors.lastname: Bell
authors.forename: A A
authors.initials: AA
authors.lastname: Mittal
authors.forename: H H
authors.initials: HH
authors.lastname: Harrison
mesh_terms: D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology* / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012867:Skin / Q000166:cytology*; D012879:Skin Physiological Phenomena
chemical_list: D017306:Annexin A2; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments