Synthesis and characterization of a bioactive 82-residue sphingolipid activator protein, saposin C.
The sphingolipid activator protein, saposin C (also termed SAP 2), was chemically synthesized, purified, and characterized. The fully protected 82-residue protein was synthesized by automated solid-phase methods, 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 (HPLC) and its purity determined by capillary electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The composition of the synthetic saposin C was determined by amino acid analysis. Its sequence was verified by Edman sequence analysis of overlapping peptide fragments generated by chymotryptic and Staphylococcus aureus V8 digestions. The sequence at the C-terminus was determined by digestion 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 two 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 glucocerebrosidase 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, synthetic and native saposin C share similar K(act) values (0.5 and 1.5 microM respectively) indicating that they bind to glucocerebrosidase with similar affinities.
Weiler
S
S
Department of Pediatrics, University of Southern California, School of Medicine, Childrens Hospital, Los Angeles.
Carson
W
W
Lee
Y
Y
Teplow
D B
DB
Kishimoto
Y
Y
O'Brien
J S
JS
Barranger
J A
JA
Tomich
J M
JM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004586:Electrophoresis; D005260:Female; D005962:Glucosylceramidase; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011487:Protein Conformation; D049231:Saposins
D006023:Glycoproteins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D049231:Saposins; D005962:Glucosylceramidase