A protein, p33/41, expressed in bovine kidney and many other tissues was identified as a lectin which binds to sialoglycoproteins and glycosaminoglycans in a calcium-dependent manner. Partial amino acid sequences of p33/41 are highly homologous to those of a calcium/phospholipid-binding annexin protein, annexin IV (endonexin), p33/41 exhibited similar calcium-dependent phospholipid binding activity (Kojima, K., Ogawa, H., Seno, N., Yamamoto, K., Irimura, T., Osawa, T., and Matsumoto, I. (1992) J. Biol. Chem. 267, 20536-20539). To further characterize p33/41, we cloned the p33/41 cDNA and characterized the recombinant protein encoded by this cDNA. Oligonucleotide probes were synthesized based on partial amino acid sequences of p33/41 and used for screening. A p33/41 cDNA clone was isolated encoding a protein of 319 amino acids with a calculated molecular mass of 35,769 Da, The deduced amino acid sequence was identical to that of bovine annexin IV except for one amino acid substitution. The recombinant protein gave two 33-kDa (p33) and 41-kDa (p41) bands on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and only one 33-kDa band under reducing conditions, as did the native protein, Mass spectrometric analysis combined with site-directed mutagenesis of each of the four cysteine residues of the recombinant protein revealed that p41 is a dimer of p33 cross-linked at Cys-198 via a disulfide bond, The recombinant protein bound to columns of heparin and fetuin glycopeptides in a calcium dependent manner and to phospholipid vesicles composed of phosphatidylserine (PS)/phosphatidylcholine (PC), phosphatidylethanolamine (PE)/PC or phosphatidylinositol (PI)/PC, Furthermore, concurrent binding assays showed that the binding of the recombinant protein to phospholipid vesicles was not affected by heparin, whereas that to heparin was influenced by the phospholipid composition of the vesicles; the highest binding was observed with vesicles composed of PE/PC. These results suggest that p53/41 binds two types of ligands via different sites and that phospholipids modulate the carbohydrate binding activity of p33/41.
