Christian Pasquali

Differential sorting and fate of endocytosed GPI-anchored proteins

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI‐ APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI‐APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft‐like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI‐APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI‐APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI‐APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI‐APs in different cell types depend on the residence time of GPI‐APs in lipid rafts, and hence that raft partitioning regulates GPI‐APs sorting in the endocytic pathway.

Large-scale amino-acid analysis for proteome studies

Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.

Leukocyte transmigration is modulated by chemokine-mediated PI3Kγ-dependent phosphorylation of vimentin

Phosphoinositide 3‐kinase γ (PI3Kγ) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine‐stimulated WT or PI3Kγ‐null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kγ signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kγ or the kinase activity of PI3Kγ (PI3KγKD/KD), phosphorylation of vimentin was reduced. PI3Kγ‐null macrophages displayed impaired chemokine‐driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N‐terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kγ‐null macrophages with a vimentin mutant mimicking serine phosphorylation of N‐terminal residues rescued the transendothelial migration defect. These results define vimentin N‐terminal phosphorylation and fiber reorganization as a target of chemokine‐dependent PI3Kγ signaling in leukocytes.

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)‐anchored proteins y two‐dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent‐resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI‐anchored proteins. Although the GPI‐anchored proteins were readily separated by one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), these proteins were undetectable on two‐dimensional (2‐D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent‐resistant membranes such as caveolin‐1 and flotillin‐1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI‐anchored proteins required cleavage of the lipid tail with phosphatidylinositol‐specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI‐anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI‐anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N‐glycosylation. Pretreatment with N‐glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N‐linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray‐tandem mass spectrometry as illustrated for the GPI‐anchored protein, Thy‐1.

The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.

Mapping and identification of protein‐protein interactions by two‐dimensional far‐Western immunoblotting

Studies of protein‐protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so‐called “substrate trapping” mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680—1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome‐wide approach to probe for candidate substrates in cellular extracts that were separated by two‐dimensional (2‐D) gel electrophoresis and blotted onto membranes. Protein‐protein interactions were revealed by far‐Western immunoblotting and positive binding proteins were subsequently identified from silver‐stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radiolabeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein‐protein interactions.

Loss of epithelial polarity is accompanied by differential association of proteins with intracellular membranes

Cellular membranes play an important role in the formation and maintenance of epithelial polarity, which is lost early during carcinogenesis. We set out to identify membrane proteins which are altered during loss of cell polarity in mammary epithelium. As a model system we used murine mammary epithelial cells expressing the conditional oncoprotein c‐JunER, which induces a reversible loss of polarity upon β‐estradiol‐driven activation [1]. When grown either in the absence or presence of hormone, these cells exhibit a polarized or unpolarized phenotype, respectively. Different membrane fractions of polarized or unpolarized cells were analyzed by two‐dimensional electrophoresis (2‐DE) and differentially expressed membrane proteins were identified. To distinguish between transmembrane orientation and peripheral attachment of these proteins, were performed extractions with carbonate at high pH or with Triton X‐114. In addition, cytosolic proteins of both states were analyzed to investigate their differential association with distinct membrane fractions. We found ten protein spots preferentially or exclusively in polarized cells and 17 other proteins as being upregulated during loss of polarity. Some of the peripheral membrane proteins were identified by microsequencing. The resident Golgi protein nucleobindin and fructose‐bisphosphate aldolase were preferentially associated with membranes of polarized cells, whereas αB crystallin was detected exclusively and in high amounts in unpolarized cells.

Extracellular lysosome-associated membrane protein-1 (LAMP-1) mediates autoimmune disease progression in the NOD model of type 1 diabetes

Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I‐10, directed against the plasma membrane (pm) form of LAMP‐1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm‐LAMP‐1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4+CD25+) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic‐infiltrating IFN‐γ‐positive cells was significantly reduced in the mAb I‐10‐treated group. These results demonstrate an unexpected role for pm‐LAMP‐1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN‐γ‐driven responses.