Characterization of human p33/41 (annexin IV), a Ca2+ dependent carbohydrate-binding protein with monoclonal anti-annexin IV antibodies, AS11 and AS17.
p33/41 (annexin IV) is a member of the family of Ca(2+)-dependent phospholipid binding proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca(2+)-dependent carbohydrate binding activity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcinoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca(2+)-dependent carbohydrate binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney protein. We also performed inhibition assays of carbohydrate binding and of phosphatidylserine/phosphatidylcholine liposome binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a carbohydrate binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.
Satoh
A
A
Department of Chemistry, Faculty of Science, Ochanomizu University, Tokyo, Japan.
Takayama
E
E
Kojima
K
K
Ogawa
H
H
Katsura
Y
Y
Kina
T
T
Matsumoto
I
I
D017314:Annexin A4 / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis*; D002118:Calcium / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D008081:Liposomes / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured
D017314:Annexin A4; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008081:Liposomes; D011994:Recombinant Proteins; D002118:Calcium