Caroline H. Damsky

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.

Cleavage of a 135 kD cell surface glycoprotein correlates with loss of fibroblast adhesion to fibronectin

We have previously described a group of three plasma membrane glycoproteins that are recognized by an adhesion-disrupting antiserum and that are involved in fibronectin-mediated BHK cell adhesion. A peculiar property of these molecules is their resistance to tryptic digestion. We have now extended this study in the attempt to identify the active component within this group of molecules. SR/BALB mouse fibroblasts, used in this work, expose at their surface only two trypsin-resistant glycoproteins, gp1 (150 K) and gp2 (135 K), that are recognized by the adhesion-disrupting anti-BHK serum. Controlled proteolysis of the cell surface in the presence of a reducing agent results in the loss of cell adhesion to fibronectin-coated substratum. gp2 is selectively cleaved under these conditions. Moreover, cells treated with trypsin and reducing agent can no longer adsorb the adhesion-relevant antibodies from the anti-BHK serum. These data indicate that gp2 plays a critical role in the adhesion of SR/BALB fibroblasts to fibronectin-coated substratum, and that disulfide bonds are important in the conformation and function of this molecule.

Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development

Early mouse embryos undergo several morphogenetic processes, such as compaction, trophoblast attachment, and endoderm formation that can be studied in vitro. Several polyspecific and monospecific antisera have been used to perturb these processes in a nontoxic, reversible fashion. One of the antibody-defined molecules, cell CAM 120/80, promotes epithelial cell adhesion, embryo compaction, and endoderm formation. The results suggest the presence of another such molecule(s) involved in these same processes. Evidence is also presented that another set of antibody-defined molecules, GP 140, involved in attachment of somatic cells to the substrate, mediates trophoblast attachment of the mouse blastocyst. The possible role of these molecules in governing the processes leading to cell lineages in the mouse embryo is discussed.

Integral Membrane Glycoproteins in Cell-Cell and Cell-Substratum Adhesion

The focus of this chapter is on the integral membrane glycoproteins of the animal cell involved in cell-cell and cell-substratum adhesion. The approach to the subject, both intellectually and experimentally, reflects not only the prejudices of the authors but also the ideas and concepts gleaned from the literature and from conversations with colleagues. For the free interchange of ideas, we are grateful; for misinterpretations or oversights, we apologize. We have focused attention on that body of experimental data that has used both biochemical and biological approaches in an attempt to understand the adhesion process. Immunology, with its potential for specificity, has played a particularly crucial role in the discovery of adhesion-related glycoproteins and, with the increased use of monoclonal antibody technology, will play an increasingly important role in exploring this area. It is a tool that must be used with caution, however, as will be noted throughout the chapter.

Effect of tunicamycin on the synthesis, intracellular transport and shedding of membrane glycoproteins in BHK cells☆☆☆

Abstract Tunicamycin (TM) has been shown in many systems to prevent glycosylation of glycoproteins but not to inhibit synthesis of the polypeptide portions of glycoproteins. We report that TM causes selective inhibition of protein glycosylation in BHK fibroblasts as well. Data are presented which indicate that, during the first 12 h of exposure to TM, cells continue to shed proteins and glycoproteins made before the addition of TM and are able to transfer non-glycosylated polypeptides from their site of synthesis in the endoplasmic reticulum to the cell surface. These data suggest: 1. 1. that cells can shed glycosylated proteins even though the supply of fully glycosylated replacement molecules is severely restricted 2. 2. that protein glycosylation and removal of cell membrane glycoproteins from the surface are not coupled 3. 3. that the oligosaccharide moities linked N -glycosidically to many membrane glycoproteins are not required for their intracellular transport.

Recovery of concentrated protein samples from sodium dodecyl sulfate-polyacrylamide gels

Abstract Procedures are described for the isolation, concentration, and further analysis of proteins that have been subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After analysis by SDS-PAGE, particular proteins are eluted electrophoretically from fixed and stained or from unstained gels into SDS-containing or nonionic detergent-containing buffers in a specifically designed apparatus consisting of gel particle and protein elution chambers, Eluted proteins can be further concentrated if necessary into volumes suitable for reanalysis on SDS or urea gels by precipitation with trichloroacetic acid, followed by filtration onto polycarbonate filters and reextraction into SDS or nonionic detergent buffers. Protein recoveries are 65–90% at each step.

Integral Membrane Adhesion Glycoproteins: What is their Fate During Metastasis?

Data from a large number of laboratories over several decades have shown that adhesive interactions among cells and between cells and extracellular matrix (ECM) are essential to early cell lineage segregation, cell recognition, cell migration and maintenance of normal tissue architecture in the adult. Adhesive interactions are also a fundamental feature of the complex metastatic process. Part of the job of understanding both normal and aberrant adhesive behavior is very basic: identification of the molecules involved at adhesion sites. This job is becoming increasingly complicated for several reasons. We have learned that any one cell type has the ability to adhere to more than one type of extracellular matrix ligand (1, 2) and that cells can adhere to one another not by one, but by several kinds of adhesive complexes (reviewed in 3). Thus, the cells of mature tissues appear to express a repertoire of adhesive molecules. In several well-documented situations, it is clear that the repertoires of different tissue types are distinct. For example, nervous tissue expresses calcium dependent and independent cell-cell adhesion molecules that are distinguishable from analagous molecules expressed by epithelial or connective tissue cells (4, 5, 6, 7).