SYNTHESIS AND CHARACTERIZATION OF A BIOACTIVE 82-RESIDUE SPHINGOLIPIDACTIVATOR PROTEIN, SAPOSIN-C

The sphingolipid activator protein, saposin C (also termed SAP 2), was chemically synthesized, purified, and characterized. The fully protec ted 82-residue protein was synthesized by automated solid-phase method s, with multiple recoupling steps resulting in a high average coupling efficiency of 98.8%. The overall yield was estimated to be approx 40% . Deprotection and cleavage of the peptide from the resin was followed by folding in the absence of chaotropic agents at pH 8.5. The protein was purified by reversed-phase high pressure liquid chromatography (H PLC) and its purity determined by capillary electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The co mposition of the synthetic saposin C was determined by amino acid anal ysis. Its sequence was verified by Edman sequence analysis of overlapp ing peptide fragments generated by chymotryptic and Staphylococcus aur eus V8 digestions. The sequence at the C-terminus was determined by di gestion with carboxypeptidase P, followed by phenylthiohydantoin (PTH) derivitization and HPLC analysis of the released amino acid residues. Deglycosylated native saposin C appeared as a lower molecular-weight species than synthetic saposin C on SDS-PAGE. This has been explained by amino acid and C-terminal analysis showing native saposin C to be t wo amino acids shorter at the C terminus than a deduced sequence (from cDNA) previously published Synthetic saposin C displayed 85% of full biological activity as determined by its ability to.stimulate glucocer ebrosidase activity in vitro: Synthetic and native saposin C increased glucocerebrosidase catalyzed hydrolysis of 4-methylumbelliferyl beta- D-glucoside by factors of 6.0 and 7.1, respectively. Furthermore, synt hetic and native saposin C share similar K(act) values (0.5 and 1.5 mu M respectively) indicating that they bind to glucocerebrosidase with s imilar affinities.