scholarly journals The Specificity for the Differentiation Blocking Activity of Carcinoembryonic Antigen Resides in Its Glycophosphatidyl-Inositol Anchor

2000 ◽  
Vol 150 (3) ◽  
pp. 613-626 ◽  
Author(s):  
Robert A. Screaton ◽  
Luisa DeMarte ◽  
Petr Dráber ◽  
Clifford P. Stanners
Keyword(s):  
Carcinoembryonic Antigen ◽  
Adhesion Molecule ◽  
Signal Sequence ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Gpi Anchor ◽  
Ig Superfamily ◽  
Blocking Activity ◽  
Differentiation Block ◽  
Glycophosphatidyl Inositol

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.

scholarly journals Human carcinoembryonic antigen, an intercellular adhesion molecule, blocks fusion and differentiation of rat myoblasts.

1993 ◽  
Vol 123 (2) ◽  
pp. 467-475 ◽  
Author(s):  
F J Eidelman ◽  
A Fuks ◽  
L DeMarte ◽  
M Taheri ◽  
C P Stanners
Keyword(s):  
Carcinoembryonic Antigen ◽  
Cell Line ◽  
Adhesion Molecule ◽  
Early Stage ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Parental Cell Line ◽  
Proliferative Potential

Human carcinoembryonic antigen (CEA), a widely used tumor marker, is a member of a family of cell surface glycoproteins that are overexpressed in many carcinomas. CEA has been shown to function in vitro as a homotypic intercellular adhesion molecule. This correlation of overproduction of an adhesion molecule with neoplastic transformation provoked a test of the effect of CEA on cell differentiation. Using stable CEA transfectants of the rat L6 myoblast cell line as a model system of differentiation, we show that fusion into myotubes and, in fact, the entire molecular program of differentiation, including creatine phosphokinase upregulation, myogenin upregulation, and beta-actin downregulation are completely abrogated by the ectopic expression of CEA. The blocking of the upregulation of myogenin, a transcriptional regulator responsible for the execution of the entire myogenic differentiation program, indicates that CEA expression intercepts the process at a very early stage. The adhesion function of CEA is essential for this effect since an adhesion-defective N domain deletion mutant of CEA was ineffective in blocking fusion and CEA transfectants treated with adhesion-blocking peptides fused normally. Furthermore, CEA transfectants maintain their high division potential, whereas control transfectants lose division potential with differentiation similarly to the parental cell line. Thus the expression of functional CEA on the surface of cells can block terminal differentiation and maintain proliferative potential.

scholarly journals Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen

2007 ◽  
Vol 177 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Thomas B. Nicholson ◽  
Clifford P. Stanners
Keyword(s):  
Amino Acids ◽  
Carcinoembryonic Antigen ◽  
Cell Adhesion Molecule ◽  
Signal Sequence ◽  
Mature Protein ◽  
Functional Specificity ◽  
Gpi Anchor ◽  
Neural Cell Adhesion ◽  
Tumorigenic Activity ◽  
Necessary And Sufficient

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6–10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.

Abstract 3955: Chemokines Fused to a Fractalkine Backbone and a GPI-Anchor Attract Embryonic Endothelial Progenitor Cells to the Site of Ischemia

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chiraz El-Aouni ◽  
Franziska Globisch ◽  
Achim Pfosser ◽  
Georg Stachel ◽  
Rabea Hinkel ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Adhesion Molecule ◽  
Capillary Density ◽  
Gpi Anchor ◽  
Endothelial Progenitor ◽  
Collateral Growth ◽  
Artificial Adhesion

Recruitment of endothelial progenitor cells to the sites of ischemia is a prerequisite for efficient therapeutic neovascularization via vasculogenesis. Chemokines play a major role in the homing of EPCs at the ischemic vasculature, a mechanism fading in chronic ischemia. To overcome this limitation, we constructed an artificial adhesion molecule consisting of a GPI-anchor, a fractalkine-backbone and an SDF-1 head (SDF-1-fra-GPI), which was applied for enhanced recruitment of embryonic EPCs (eEPCs: CXCR4++, Tie2++, Thrombomodulin++, CD34-, MHCI-, vWF inducible, eNOS inducible) in vitro and in vivo . Methods: In a flow chamber adhesion assay, Control plasmids (pcDNA or GPI-SDF-1 cDNA) were compared to the SDF-1-fra-GPI construct for eEPC recruitment 24h after liposomal transfection of rat endothelial cells. In vivo, in rabbits (n=5 per group) at day 7 (d7) after femoral artery excision, 1 mg of the SDF-1-fra-GPI or eGFP cDNA was transfected into the ischemic limb. At d9, ischemic hindlimbs were retroinfused with 5x10 6 eEPCs. Angiography was performed for collateral quantification and frame count score at d9 and d37 (% of d9), capillary density was assessed via PECAM-1-staining (capillaries/muscle fiber = c/mf). Results: In vitro, eEPC adhesion (16±12 cells/field) was increased to a higher extent by SDF-1-fra-GPI (79±13) than SDF1-GPI (54±8) or control vector (37±8). In vivo , eEPC adhesion in the ischemic hindlimb after SDF-1-fra-GPI transfection compared to mock transfection (30±3 vs. 9±1 cells/field). Whereas capillary density was unaffected (1.66±0.30 SDF-1-Fra-GPI vs. 1.56±0.29 eEPCs), collateral growth (152±10% SDF-1-fra-GPI vs. 124±13%) as well as perfusion score (198±17% SDF-1-fra-GPI vs.160±6% eEPCs) further increased after SDF-1-fra-GPI transfection (controls: 1.24±0.12 c/mf, collaterals 105±8%, perfusion score 112±11%). We conclude that recruitment of EPCs expressing CXCR4 (the SDF-1 receptor) may benefit from pre-treatment of the recipient vasculature with SDF-1-Fra-GPI, an artificial adhesion molecule. This approach might be valuable for enhancing EPC recruitment in the scenario of therapeutic neovascular-ization of chronic ischemic syndromes.

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