The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton.
Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5's ability to distinguish different forms of cell surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding protein, as intracellular factors able to alter TEM8 cell surface structure. Overexpression of either of these proteins in cells converted TEM8 from an SB5-exposed to an SB5-masked form and protected cells from SB5-saporin immunotoxins. Because the predominant form of TEM8 on the cell surface is not recognized by SB5, we also developed a new monoclonal antibody, called AF334, which is able to recognize both the SB5-exposed and the SB5-masked forms of TEM8. AF334-saporin selectively killed TEM8-positive cells independent of TEM8 cell surface structure. These studies reveal that TEM8 exists in different forms at the cell surface, a structure dependent on interactions with components of the actin cytoskeleton, and should aid in the rational design of the most effective diagnostic and therapeutic anti-TEM8 monoclonal antibodies.
National Cancer Institute, Frederick, MD 21702-1201, USA.
Mi Young
MY
Yang
Amit
A
Chaudhary
Steven
S
Seaman
Jill
J
Dunty
Janine
J
Stevens
Mohammed K
MK
Elzarrad
Arthur E
AE
Frankel
Brad
B
St Croix
D000199:Actins / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D018189:Immunomagnetic Separation; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C541116:ACTA2 protein, human; C473287:ANTXR1 protein, human; D000199:Actins; D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C051615:transgelin