Sabine Wegehingel

The cancer antigen CA125 represents a novel counter receptor for galectin-1

CA125 is an ovarian cancer antigen whose recently elucidated primary structure suggests that CA125 is a giant mucin-like glycoprotein present on the cell surface of tumor cells. Here, we establish a functional link between CA125 and β-galactoside-binding, cell-surface lectins, which are components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression. On the basis of mass spectrometry and immunological analyses, we find that CA125 is a counter receptor for galectin-1, as both soluble and membrane-associated fragments of CA125 derived from HeLa cell lysates are shown to bind specifically to human galectin-1 with high efficiency. This interaction is demonstrated (1) to depend on β-galactose-terminated, O-linked oligosaccharide chains of CA125, (2) to be preferential for galectin-1 versus galectin-3 and (3) to be regulated by the cellular background in which CA125 is expressed. Despite lacking a conventional signal peptide, a CA125 C-terminal fragment of 1148 amino acids, representing less than 10% of the full-length protein, retains the ability to integrate into secretory membranes such as the endoplasmic reticulum (ER) and the Golgi, and is targeted to the plasma membrane by conventional secretory transport. As demonstrated by a novel assay that reconstitutes non-conventional secretion of galectin-1 based on fluorescence-activated cell sorting (FACS), we find that tumor-derived HeLa cells expressing endogenous CA125 present more than ten times as much galectin-1 on their surface compared with non-tumor-derived, CA125-deficient CHO cells. Intriguingly, both the galectin-1 expression level and the cell-surface binding capacity for galectin-1 are shown to be similar in CHO and HeLa cells, suggesting that CA125 might be a factor involved in the regulation of galectin-1 export to the cell surface.

Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2

FGF-2 is an unconventionally secreted lectin that transmits proangiogenic signals through a ternary complex with high-affinity FGF receptors and heparan sulfate proteoglycans (HSPGs). Although FGF-2 signal transduction is understood in great detail, its mechanism of release from cells, which is independent of the classical secretory pathway, remains elusive. To test the hypothesis that FGF-2 secretion is linked to its cell-surface ligands, we studied FGF-2 release using mutants defective for HSPG binding and cells with impaired HSPG biosynthesis. Here, we report that a functional interaction between FGF-2 and HSPGs is required for net export of FGF-2 from mammalian cells. FGF-2 release requires extracellular, membrane-proximal HSPGs. We propose that extracellular HSPGs form a molecular trap that drives FGF-2 translocation across the plasma membrane.

Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells

Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of non-classical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N- and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion. In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is rate-limiting.

Unconventional protein secretion: membrane translocation of FGF-2 does not require protein unfolding.

Endoplasmic reticulum/Golgi-dependent protein secretion depends on signal peptides that mediate membrane translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Classical secretory proteins are transported across the membrane of the endoplasmic reticulum in an unfolded conformation, which is similar to protein import into mitochondria. This process is mediated by Sec61, the protein-conducting channel of the endoplasmic reticulum. Employing both FACS-based in vivo transport assays and confocal microscopy, we now show that fibroblast growth factor 2 (FGF-2), a pro-angiogenic mediator exported from mammalian cells by an unconventional secretory pathway, does not need to be unfolded in order to be released into the extracellular space. These findings suggest that the molecular apparatus mediating export of FGF-2 is not only distinct from classical translocation machineries in terms of molecular identity but also operates in a mechanistically distinct manner that allows membrane translocation of FGF-2 in a folded conformation.

Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant

Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.

Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi‐independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5‐biphosphate (PI(4,5)P2) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post‐translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

Rerouting of fibroblast growth factor 2 to the classical secretory pathway results in post-translational modifications that block binding to heparan sulfate proteoglycans

FGF‐2 is a proangiogenic growth factor secreted by unconventional means. It is unknown why FGF‐2 takes an ER/Golgi‐independent secretory route. We find that secretion of FGF‐2 via the ER/Golgi system causes post‐translational modifications that prevent binding to heparan sulfate proteoglycans (HSPGs), an interaction that is critically important for both FGF‐2 storage and signal transduction. This loss of function is due to artificial O‐glycosylation mainly resulting in the addition of glycosaminoglycan chains of the chrondroitin sulfate type. Our findings suggest that the unconventional mechanism of FGF‐2 export is an ancient pathway of protein secretion that, in the course of evolution, has been kept due to the inability of the classical secretory pathway to export FGF‐2 in a functional form.

A Direct Role for ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

Background: Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes. Results: The cytoplasmic domain of ATP1A1 directly interacts with FGF2 and is required for FGF2 secretion. Conclusion: ATP1A1 supports unconventional secretion by recruiting FGF2 to the inner leaflet of plasma membranes. Significance: A new machinery component required for unconventional secretion of FGF2 was identified and validated. Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the β1- and β3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of β-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.

Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces

Background: FGF2 translocation across plasma membranes depends on phosphoinositide-dependent oligomerization and membrane pore formation. Results: Two unique surface cysteines are critical for efficient FGF2 oligomerization, membrane pore formation, and FGF2 secretion from cells. Conclusion: Formation of intermolecular disulfide bridges drives phosphoinositide-dependent FGF2 oligomerization at plasma membranes. Significance: A new cis element critical for unconventional secretion of FGF2 was identified and validated. Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.