Glycoconjugate Journal

, Volume 18, Issue 11–12, pp 915–923 | Cite as

Novel adapter protein AP162 connects a sialyl-Lex-positive mucin with an apoptotic signal transduction pathway

  • Sabine Hartel-Schenk
  • Alexei Gratchev
  • Marie-Luise Hanski
  • Dieter Ogorek
  • George Trendelenburg
  • Michael Hummel
  • Michael Höpfner
  • Hans Scherübl
  • Martin Zeitz
  • Christoph Hanski


Glycoproteins modified with a sialyl-Lex-moiety are important sensors for extracellular signals regulating cellular recognition, adhesion and migration. The transduction pathways and signals mediated by these glycoproteins within the cell are largely unknown. In search of novel glycoproteins modified with sialyl-Lex-moiety, we screened a human colonic cDNA expression library with a rabbit antiserum produced against sialyl-Lex-positive mucins. The antiserum detected a new protein, named B2, which was cloned and characterised in detail.

The analysis of the B2 gene revealed a 5.7 kb RNA transcript detectable in all investigated tissues and a complete coding sequence of 2778 bp. The B2 protein exhibited two putative PH (pleckstrin homology) domains and a leucine zipper motif but no homology to any known proteins.

Monospecific antibodies against the B2-protein precipitated from the solubilised membrane fraction of the colon carcinoma cell line LS 174T a protein with an apparent Mr = 162 kDa and, additionally, a mucin-like glycoprotein with an apparent Mr = 220 kDa. Protein fractionation on a CsCl gradient, Western blots and sandwich ELISA showed that the 220 kDa mucin carries the sialyl-Lex moiety and is tightly bound to the 162 kDa protein.

The expression of the recombinant B2-protein enhanced staurosporine-induced apoptosis in epithelial cancer cell lines.

These data indicate that B2 is a novel, ubiquitously expressed protein with a putative adapter function. The protein has been named AP162 (adapter protein 162). In colon carcinoma cells B2-protein is tightly associated with a sialyl-Lex-positive mucin and has a potential for involvement in sialyl-Lex-mediated transduction of apoptotic signals.

pleckstrin homology (PH) domain PIP-signaling sialyl-Lex colon carcinoma 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Hakomori SI, Aberrant glycosylation in tumors and tumorassociated carbohydrate antigens, Adv Cancer Res 52, 257–31 (1989).Google Scholar
  2. 2.
    M¨uthing J, Spanbroek R, Peter-Katalinic J, Hanisch FG, Hanski C, Hasegawa A, Unland F, Lehmann J, Tschesche H, Egge H, Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes, Glycobiology 6, 147–56 (1996).Google Scholar
  3. 3.
    Springer TA, Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm, Cell 76, 301–14 (1994).Google Scholar
  4. 4.
    Picker LJ, Warnock RA, Burns AR, Doerschuk CM, Berg EL, Butcher EC, The Neutrophil Selectin LECAM-1 presents carbohydrate ligands to the vascular Selectins ELAM-1 and GMP-140, Cell 66, 921–33 (1991).Google Scholar
  5. 5.
    Kuijpers TA, Hoogerwerf M, van der Laan LJW, Nagel G, van der Schoot CE, Grunert F, Roos D, CD 66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells, J Cell Biol 118, 457–66 (1992).Google Scholar
  6. 6.
    Maemura K, Fukuda M, Poly-N-acetyllactosaminyl O-glycans attached to Leukosialin, J Biol Chem 267, 24379–86 (1992).Google Scholar
  7. 7.
    Lasky LA, Selectin-carbohydrate interactions and the initiation of the inflammatory response, Annu Rev Biochem 64, 113–39 (1995).Google Scholar
  8. 8.
    Welply_JK, Abbas SZ, Scudder P, Keene JL, Broschat K, Casnocha S, Gorka C, Steininger C, Howard SC, Schmuke JJ, Graneto M, Rotsaert JM, Manger ID, Jacob GS, Multivalent sialyl-Lex: Potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells, Glycobiology 4, 259–65 (1994).Google Scholar
  9. 9.
    Fukuda M, Bothner B, Ramasamooj P, Dell A, Tiller PR, Varki A, Klock JC, Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells, J Biol Chem 260, 12957–67 (1985).Google Scholar
  10. 10.
    Fukushima K, Hirota M, Terasaki PI, Wakisaka A, Togashi H, Chia D, Suyama N, Fukushi Y, Nudelman E, Hakomori SI, Characterization of Sialosylated Lewis x as a new tumor-associated antigen, Cancer Res 44, 5279–85 (1984).Google Scholar
  11. 11.
    Kim YS, Itzkowitz SH, Yuan M, Chung YS, Satake K, Umeyama K, Hakomori SI, Lex and Ley antigen expression in human pancreatic cancer, Cancer Res 48, 475–82 (1988).Google Scholar
  12. 12.
    Magnani JL, Nilsson B, Brockhaus M, Zopf D, Steplewski Z, Koprowski H, Ginsburg V,Amonoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose II, J Biol Chem 257, 14365–9 (1982).Google Scholar
  13. 13.
    Anostario MJ, Li SH, Huang KS, A ligand binding assay for Eselectin, Anal Biochem 221, 317–22 (1994).Google Scholar
  14. 14.
    Sawada R, Lowe JB, Fukuda M, E-selectin-dependent adhesion ef-ficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels, J Biol Chem 268, 12675–81 (1993).Google Scholar
  15. 15.
    Hanski C, Drechsler K, Hanisch FG, Sheehan J, Manske M, Ogorek D, Klussmann E, Hanski ML, Blank M, Xing PX, McKenzie IFC, Devine PL, Riecken EO, Altered glycosylation of the MUC-1 protein core contributes to the colon carcinomaassociated increase of mucin-bound sialyl-Lewisx expression, Cancer Res 53, 4082–8 (1993).Google Scholar
  16. 16.
    Hanski C, Hanski ML, Zimmer T, Ogorek D, Devine PL, Riecken EO, Characterisation of the major sialyl-Lex-positive mucins present in colon, colon carcinoma and sera of patients with colorectal cancer, Cancer Res 55, 928–33 (1995).Google Scholar
  17. 17.
    Fukuda M, Possible roles of tumor-associated carbohydrate antigens, Cancer Res 56, 2237–44 (1996).Google Scholar
  18. 18.
    Nakamori S, Kameyama M, Imaoka S, Furukawa H, Ishikawa O, Sasaki Y, Kabuto T, Iwanaga T, Matsushita Y, Irimura T, Increased expression of sialyl Lewisx antigen correlates with poor survival in patients with colorectal carcinoma: Clinicopathological and immunohistochemical study, Cancer Res 53, 3632–7 (1993).Google Scholar
  19. 19.
    Hoff SD, Irimura T, Matsushita Y, Ota DM, Cleary KR, Hakomori SI, Metastatic potential of colon carcinoma: Expression of ABO/Lewis-related antigens, Arch Surg 125, 206–9 (1990).Google Scholar
  20. 20.
    Grabowski P, Mann B, Mansmann U, L¨ovin N, Foss HD, Berger G, Scher¨ubl H, Riecken EO, Buhr HJ, Hanski C, Expression of sialyl-Lex antigen defined by Mab AM-3 is an independent prognostic marker in colorectal carcinoma patients, Int J Cancer 88, 281–6 (2000).Google Scholar
  21. 21.
    Israel DI, A PCR-based method for high stringency screening of DNA libraries, Nucleic Acids Res 21, 2627–31 (1993).Google Scholar
  22. 22.
    Sambrook J, Fritsch EF, Maniatis T, Molecular cloning. A laboratory manual. (Cold Spring Harbor Laboratory Press., Cold Spring Harbor, New York, 1989).Google Scholar
  23. 23.
    Nagase T, Ishikawa K, Nakajima D, Ohira M, Seki N, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O, Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro, DNA Res 4, 141–50 (1997).Google Scholar
  24. 24.
    Testa JR, Bellacosa A, AKT plays a central role in tumorigenesis, Proc Nat Acad Sci USA 98(20), 10983–5 (2001).Google Scholar
  25. 25.
    Berrie CP, Falasca M, Patterns within protein/phosphoinositide interactions provide specific targets for therapeutic intervention, FASEB J 14, 2618–22 (2000).Google Scholar
  26. 26.
    Baeckstr¨om D, Zhang K, Asker N, R¨uetschi U, Ek M, Hansson GC, Expression of the leukocyte-associated Sialoglycoprotein CD 43 by a colon carcinoma cell line, J Biol Chem 270, 13688–92 (1995).Google Scholar
  27. 27.
    Schleiffenbaum B, Spertini O, Tedder TF, Soluble L-selectin is present in human plasma at high levels and retains functional activity, J Cell Biol 119, 229–38 (1992).Google Scholar
  28. 28.
    Ingley E, Hemmings B, Pleckstrin homology (PH) domains in signal transduction, J Cell Biochem 56, 436–43 (1994).Google Scholar
  29. 29.
    Hemmings BA, PtdIns (3,4,5) P3 gets its message across, Science 277, 34 (1997).Google Scholar
  30. 30.
    Stokoe D, Stephens LR, Copeland T, Gaffney PRJ, Reese CB, Painter GF, Holmes AB, McCormick F, Hawkins PT, Dual role of Phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B, Science 277, 567–70 (1997).Google Scholar
  31. 31.
    Frech M, Andjelkovic M, Ingley E, Reddy KK, Falck JR, Hemmings BA, High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity,J Biol Chem 272, 8474–81 (1997).Google Scholar
  32. 32.
    Nagel W, Zeitlmann L, Schilcher P, Geiger C, Kolanus J, Kolanus W, PI 3-kinase regulates membrane recruitment of cytohesin-1 via its PH-domain and thereby activates the aLß2 integrin adhesion pathway, Immunobiology 197, 2–4 (1997).Google Scholar
  33. 33.
    Klarlund JK, Guilherme A, Holik JJ, Virbasius JV, Chawla A, Czech MP, Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains, Science 275, 1927–30 (1997).Google Scholar
  34. 34.
    Crockett-Torabi E, Selectins and mechanisms of signal transduction, J Leukocyte Biol 63, 1–14 (1998).Google Scholar
  35. 35.
    Simon SI, Hu Y, Vestweber D, Smith CW, Neutrophil tethering on E-selectin activates beta integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway, J Immunol 164, 4348–58 (2000).Google Scholar
  36. 36.
    Axelsson B, Perlmann P, Persistent superphosphorylation of leukosialin (CD43) in activated T cells and in tumour cell lines, Scand J Immunol 30, 539–47 (1989).Google Scholar
  37. 37.
    Silverman LB, Wong RCK, Remold-O'Donnell E, Vercelli D, Sancho J, Terhorst C, Rosen F, Geha R, Chatila T, Mechanism of mononuclear cell activation by an anti-CD43 (sialophorin) agonistic antibody, J Immunol 142, 4194–200 (1989).Google Scholar
  38. 38.
    Wong RCK, Remold-O'Donnell E, Vercelli D, Sancho J, Terhorst C, Rosen F, Geha R, Chatila T, Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C, J Immunol 144, 1455–60 (1990).Google Scholar
  39. 39.
    Zrihan-Licht S, Baruch O, Elroy-Stein O, Keydar I, Wreschner DH, Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins Cytokine receptor-like molecules, FEBS Letters 356, 130–6 (1994).Google Scholar
  40. 40.
    Pandey P, Kharbanda S, Kufe D, Association of the DF3/MUC1 breast cancer antigen with Grb2 and the Sos/Ras exchange protein, Cancer Res 55, 4000–3 (1995).Google Scholar
  41. 41.
    Kolanus W, Nagel W, Schiller B, Zeitlmann L, Godar S, Stockinger H, Seed B, aLß2 Integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule, Cell 86, 233–42 (1996).Google Scholar
  42. 42.
    Tang D, Okada H, Rulan J, Liu L, Stambolic V, Mak TW, Ingram AJ, Akt is activated in response to an apoptotic signal, J Biol Chem 276, 30461–6 (2001). Received 14 May 2002; revised 29 July 2002; accepted 5 August 2002Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Sabine Hartel-Schenk
    • 1
  • Alexei Gratchev
    • 1
  • Marie-Luise Hanski
    • 1
  • Dieter Ogorek
    • 1
  • George Trendelenburg
    • 2
  • Michael Hummel
    • 1
  • Michael Höpfner
    • 1
  • Hans Scherübl
    • 1
  • Martin Zeitz
    • 1
  • Christoph Hanski
    • 3
  1. 1.Department of GastroenterologyUniversity Clinic Benjamin Franklin Free University BerlinBerlinGermany
  2. 2.Department of NeurologyHumboldt University Clinic CharitéBerlinGermany
  3. 3.Department of GastroenterologyUniversity Clinic Benjamin Franklin Free University BerlinBerlinGermany

Personalised recommendations