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Importance of splicing for prosaposin sorting.

ABSTRACT: 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.

SUBMITTER: Madar-Shapiro L

PROVIDER: S-EPMC1219994 | biostudies-other | 1999 Feb

REPOSITORIES: biostudies-other

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Project description:The relative importance of environment and dispersal related processes for community assembly has attracted great interest over recent decades, but few empirical studies from the marine/estuarine realm have examined the possible effects of these two types of factors in the same system. Importance of these processes was investigated in a hypothetical metacommunity of benthic invertebrates in 16 micro-tidal estuaries connected to the same open sea area. The estuaries differed in size and connectivity to the open sea and represented a salinity gradient across the estuaries. The Elements of Metacommunity Structure (EMS) approach on estuary scale was complemented with a mechanistic variance partitioning approach on sample scale to disentangle effects of factors affecting assembly of three trait groups of species with different dispersivity. A quasi-Clementsian pattern was observed for all three traits, a likely response to some latent gradient. The primary axis in the pattern was most strongly related to gradients in estuary salinity and estuary entrance width and correlation with richness indicated nestedness only in the matrix of the most dispersive trait group. In the variance partitioning approach measures of turnover and nestedness between paired samples each from different estuaries were related to environmental distance in different gradients. Distance between estuaries was unimportant suggesting importance of factors characterizing the estuaries. While the high dispersive species mainly were sorted in the salinity gradient, apparently according to their tolerance ranges towards salinity, the two less dispersive traits were additionally affected by estuary entrance width and possibly also area. The results exemplify a mechanism of community assembly in the marine realm where the niche factor salinity in conjunction with differential dispersal structure invertebrates in a metacommunity of connected estuaries, and support the idea that dispersive species are more controlled by the environment than less dispersive species.

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Importance of splicing for prosaposin sorting.

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