COMPLEX ASSEMBLY OF CALGRANULIN-A AND CALGRANULIN-B, 2 S100-LIKE CALCIUM-BINDING PROTEINS FROM PIG GRANULOCYTES

Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calciu m-binding proteins that in human, bovine and mouse granulocytes are as sociated into a heterocomplex. We have previously identified in pig gr anulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated w ith CAGC, we herein investigate its possible association with other pr oteins. CAGA was purified from pig granulocytes by gel filtration foll owed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass sp ectrometry, chemical cross-linking and hydrophobic interaction chromat ography. The CAGA-associated protein was further characterized by amin o acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protei n; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by in ternal amino acid sequencing as the porcine homologue of CAGE. The sto ichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. U nlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column i n a calcium-dependent manner. Our results suggest that pig granulocyte s contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heter odimer. It is proposed that CAGA/CAGB and the CAGA homodimer may play different roles in vivo.