PORCINE CEREBROSIDE SULFATE ACTIVATOR - FURTHER STRUCTURAL CHARACTERIZATION AND DISULFIDE IDENTIFICATION

Cerebroside sulfate activator (CS-Act) is a small compact protein which binds and solubilizes certain glycosphingolipids. Following the recent publication of the purification and preliminary of pig kidney CS-Act [Fluharty, A. L., Katona, Z., Meek, W. E., Frei, K., & Fowler, A. V. (1992) Biochem. Med. Metab. Biol. 47, 66-85], we now report the primary sequence of the C-terminal portion of this protein and the assignment of the three disulfide bonds. Cyanogen bromide (CNBr) treatment of native CS-Act produced three major and several minor peptide fragments. Analysis of one HPLC-purified fragment revealed the C-terminus 14 amino acid sequence. This established the length of the native protein at 79 residues. In conjunction with the sequence data for one other major HPLC-purified CNBr fragment, it could be concluded that the three intrachain disulfide bonds were located at half-cystine residues 4 and 77, 7 and 71, and 36 and 47. Mass spectrometry (fast atom bombardment and electrospray ionization) showed the molecular weight of the major component of the CS-Act preparation to be 9720.5 Da, which was in close agreement with the calculated mass of the 79 amino acid peptide with five covalently attached sugar residues and three internal disulfide bonds. The mass spectrometric molecular weight measurements also showed that the CS-Act preparation possessed microheterogeneity in its carbohydrate moiety, as less intense signals corresponded to species containing (in decreasing order of abundance) two, one, four, and three sugar residues. The same type of measurements on native and chemically reduced CS-Act revealed a mean difference of 5.9 +/- 0.2 Da (mean +/- SEM, n = 3) for the three most abundant species in the preparation, confirming that in the native state the molecules were oxidized, with each containing three disulfide bonds. With account made for the contribution from the carbohydrate components, the molecular weight measurements of the CNBr fragments agreed closely with the predicted values, concordant with the disulfide bridge assignments. The secondary structure prediction analysis shows a high preference for alpha-helical content in this protein. The disulfide placement data, the prediction analysis information, and the property of high stability has led us to revise our previously proposed CS-Act structure to one which contains a bundle of four amphipathic alpha-helices forming an internal hydrophobic core.