Expression of carbohydrate-binding protein p33/41 in human tumor cell lines.
We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant protein and raised monoclonal antibodies against the protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).
Satoh
A
A
Department of Chemistry, Ochanomizu University, Tokyo.
Takayama
E
E
Kojima
K
K
Ogawa
H
H
Yamori
T
T
Sato
S
S
Kawaguchi
T
T
Tsuruo
T
T
Katsura
Y
Y
Kina
T
T
Matsumoto
I
I
D000230:Adenocarcinoma / Q000378:metabolism; D017314:Annexin A4; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D037102:Lectins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
D017314:Annexin A4; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D037102:Lectins; C098880:annexin A4, Bos taurus