EXPRESSION OF THE 3 ALTERNATIVE FORMS OF THE SPHINGOLIPID ACTIVATOR PROTEIN-PRECURSOR IN BABY HAMSTER-KIDNEY CELLS AND FUNCTIONAL ASSAYS INA CELL-CULTURE SYSTEM

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with shor t oligosaccharide chains by their respective exohydrolases. Four of th ese (SAP-A to SAP-D or saposins A to D) are derived from a common prec ursor by proteolytic processing. Alternative splicing of the SAP-precu rsor gene results in insertion of additional 6 or 9 bases of exon 8' o r 8, respectively, into the SAP-B coding region of the transcribed mRN As. To examine the features of the three different SAP-precursor prote ins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells a nd endocytosis studies on human fibroblasts showed that synthesis, tra nsport, and maturation of all SAP-precursor led to formation of the fo ur mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficien t human fibroblasts were loaded with recombinant SAP-precursor protein s with or without 2- and 3-amino acid insertions, respectively, purifi ed from the medium of the baby hamster kidney cells. They were found t o stimulate at nanomolar concentrations the turnover of biosynthetical ly labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of su lfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by b eta-galactosidase (EC 3.2.1.23) loading studies with the respective ex ogenously labeled lipids on SAP precursor-deficient fibroblasts were p erformed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lip id degradation by all three SAP-B isoforms derived from the SAP-precur sors. These findings establish that all three forms of the SAP-B can f unction as sulfatide/ globotriaosylceramide activator.