A PEPTIDYL-PROLYL CIS TRANS-ISOMERASE (CYCLOPHILIN-G) IN REGULATED SECRETORY GRANULES/

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts wi th antiserum against a beta-glucan-sensitive protease zymogen was puri fied to homogeneity, and its cDNA was cloned, The 1.7-kilobase pair cD NA contains an open reading frame of 660 base pairs, encoding a 23 ami no acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activ ity with a k(cat)/K-m of 0.18 mu m(-1) s(-1) for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK5 06. Although the p27 precursor possesses an amino-terminal secretory h ydrophobic signal sequence, unlike other cyclophilin B molecules, it l acks a conserved carboxyl-terminal endoplasmic reticulum retention sig nal and it contains a central 8-amino acid insertion, Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma brit rather is loc alized to the hemocyte large granules, the regulated secretory granule s that are exocytosed upon stimulation. These results indicate that p2 7 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secret ory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe c rab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granule s.