PRIMARY STRUCTURE AND BINDING-PROPERTIES OF CALGRANULIN-C, A NOVEL S100-LIKE CALCIUM-BINDING PROTEIN FROM PIG GRANULOCYTES

In this paper we report the biochemical characterization of calgranuli n C, a new member of the S100 protein family. The protein is highly ab undant in the cytosol of pig granulocytes, with relatively small amoun ts in lymphocytes. A simple protocol for the rapid purification of cal granulin C is described. The purified protein mi grates as a single en tity on SDS-polyacrylamide gel electrophoresis while it has two isofor ms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experi ments indicate that calgranulin C is capable of dimerization. The comp lete amino acid sequence was determined by Edman degradation of peptid es generated by trypsin and V8 protease digestion. Calgranulin C consi sts of 91 residues and has a calculated molecular mass of 10,614 dalto ns. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-bin ding motifs, the first having an extended loop that is distinctive of the S100 protein family. The metal-binding properties were studied by means of a direct Ca-45(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn2+-binding site per monomer was evidenced by f luorimetric titration. Zinc binding to calgranulin C induces a remarka ble increase in the protein affinity for calcium; in the absence of zi nc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M(-1), whereas the Zn2+-loaded form binds 2 Ca2+/monomer wit h K-a values of approximately 3 x 10(7) and 6 x 10(4) M(-1). Circular dichroism analysis showed that the binding of calcium to calgranulin C induces a 15% decrease in the apparent alpha-helix content. This resu lt and the calcium dependent binding of the protein to a phenyl-Supero se column strongly suggest that calgranulin C undergoes a gross confor mational change upon calcium binding, thus supporting the idea that th is protein may be involved in Ca(2+)dependent signal transduction even ts.