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 calgranulin C, a new member of the S100 protein family. The protein is highly abundant in the cytosol of pig granulocytes, with relatively small amounts in lymphocytes. A simple protocol for the rapid purification of calgranulin C is described. The purified protein migrates as a single entity on SDS-polyacrylamide gel electrophoresis while it has two isoforms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experiments indicate that calgranulin C is capable of dimerization. The complete amino acid sequence was determined by Edman degradation of peptides generated by trypsin and V8 protease digestion. Calgranulin C consists of 91 residues and has a calculated molecular mass of 10,614 daltons. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-binding 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 45Ca(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn(2+)-binding site per monomer was evidenced by fluorimetric titration. Zinc binding to calgranulin C induces a remarkable increase in the protein affinity for calcium; in the absence of zinc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M-1, whereas the Zn(2+)-loaded form binds 2 Ca2+/monomer with Ka 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 result and the calcium-dependent binding of the protein to a phenyl-Superose column strongly suggest that calgranulin C undergoes a gross conformational change upon calcium binding, thus supporting the idea that this protein may be involved in Ca(2+)-dependent signal transduction events.