Importance of splicing for prosaposin sorting.
The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.
Madar-Shapiro
L
L
Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv 69978, Israel.
Pasmanik-Chor
M
M
Vaccaro
A M
AM
Dinur
T
T
Dagan
A
A
Gatt
S
S
Horowitz
M
M
D017398:Alternative Splicing; D015152:Blotting, Northern; D002460:Cell Line; D002553:Cerebroside-Sulfatase / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005822:Genetic Vectors; D005962:Glucosylceramidase / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017354:Point Mutation; D011233:Precipitin Tests; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D014615:Vaccinia / Q000235:genetics
D018076:DNA, Complementary; D006023:Glycoproteins; C483345:PSAP protein, human; D011498:Protein Precursors; D011994:Recombinant Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D012313:RNA; D002553:Cerebroside-Sulfatase; D005962:Glucosylceramidase