BioScience Trends. 2013;7(5):221-229. (DOI: 10.5582/bst.2013.v7.5.221)

Characterization of anti-Tn-antigen MLS128 binding proteins involved in inhibiting the growth of human colorectal cancer cells.

Zamri N, Masuda N, Oura F, Kabayama K, Yajima Y, Nakada H, Yamamoto K, Fujita-Yamaguchi Y


MLS128 monoclonal antibody, which binds an epitope consisting of two or three consecutive Tn-antigens, inhibits colon cancer cell growth by binding to a 110 kDa glycoprotein (GP). Previous studies suggested a possible association of insulin-like growth factor-I receptor (IGF-IR) signaling in the inhibition of colon cancer cell growth by MLS128 (Morita et al. Biosci Trends. 3, 32-37, 2009; Zamri et al. ibid. 6, 303-312, 2012). The current study thus investigated the nature of 110 kDa GP and its possible association with IGF-IR. MLS128 treatment for 3 days caused down-regulation of IGF-IR and disappearance of 110 kDa GP in HT29 colon cancer cells. Immunoprecipitation/immunoblotting experiments did not reveal a direct association between the two molecules in HT29 cells. In LS180 and HT29 cells, however, 110 kDa GP and IGF-IR were found in microdomains. Treatment of these cells with MLS128 for 3 days caused a reduction in the IGF-IR and 110 kDa GP associated with microdomains. Two-dimensional gel electrophoresis/MLS128 immunoblotting of HT29 and LS180 cell lysates and immunoprecipitates revealed three spots, from which tryptic peptides were recovered for protein sequencing. Identification of 110 kDa GP was unsuccessful due to its heterogeneity and resistance to tryptic digestion. During this study, however, limited proteolysis of 110 kDa GP was observed in the microdomain-associated 110 kDa GP from HT29 and LS180 cells, suggesting that protease-susceptible sites or domains exist in the middle of 110 kDa GP. This information on limited proteolysis may provide a clue to identifying 110 kDa GP.

KEYWORDS: Mucin-type O-glycans, microdomains, limited proteolysis, immunoprecipitation, immunoblotting, two-dimensional gel electrophoresis

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