Molecular Analysis of the Unconventional Export Machinery of Galectin-1, a beta-Galactoside-specific Lectin of the Extracellular Matrix

Galectin-1 ist ein β-Galaktosid-spezifisches Lektin der extrazellulären Matrix, das an der Regulation verschiedener zellulärer Prozesse wie Zellproliferation, Differenzierung und Apoptose beteiligt ist. Das extrazelluläre Auftreten von Galectin-1 war zunächst eine überraschende Entdeckung, da das Protein kein Signalpeptid enthält und somit nicht über ER/Golgi-vermittelten Transport sezerniert werden kann. Darüber hinaus ist die Sekretion von Galectin-1 in Gegenwart von Brefeldin A nicht gehemmt, so dass ein unkonventioneller Sekretionsweg postuliert wurde. In der vorliegenden Dissertation wurde ein robustes in vivo Modell zur funk¬tionellen Rekonstitution der Galectin-1 Sekretion etabliert. Es wurden mittels retroviraler Transduktion stabile Zelllinien generiert, die verschiedene Galectin-1 Reportermoleküle als GFP Fusionsproteine in Abhängigkeit von der exogenen Zugabe von Doxicyclin exprimieren. Da die exportierte Population über β-Galaktosid-haltige Rezeptoren an Zelloberflächen bindet, konnte die Galectin-1 Sekretionsrate über verschiedene Methoden wie Durchflusszytometrie, konfokale Laserscanmikroskopie sowie biochemische Zelloberflächenbiotinylierung unter verschiedenen experimentellen Bedingungen quantifiziert werden. Die etablierten Modellsysteme wurden einerseits zur funktionellen Charakteri¬sierung eines in dieser Arbeit identifizierten Galectin-1 Rezeptors genutzt und andererseits zur Analyse der molekularen Sortierungsdeterminanten verwendet, die Galectin-1 zur entsprechenden Exportmaschinerie dirigieren. Eine systema¬tische Mutagenese des offenen Leserasters von Galectin-1 ergab hierbei, dass Mutationen, die zu einer Bindungsdefizienz führen, letztlich auch einen Exportdefekt verursachen. Komplementär hierzu konnte gezeigt werden, dass Galectin-1 von Zelllinien, die nicht zur Expression von β-Galaktosid-haltigen Rezeptoren befähigt sind, nicht exportiert wird. Hieraus wurde der Schluss gezogen, dass β-Galaktosid-haltige Zelloberflächenrezeptoren für den Gesamtprozess der Galectin-1 Sekretion essentiell sind. Dieser Befund konnte durch die Expression des mit Galectin-1 entfernt verwandten Proteins CGL-2 aus dem multizellulären Pilz Coprinopsis cinerea bestätigt werden. CGL-2 wird von Säugetierzellen unkonventionell exportiert, wobei dieser Prozess von der Bindung an β-Galaktosid-haltige Zelloberflächenrezeptoren abhängt. Die beschriebenen Arbeiten haben somit gezeigt, dass das primäre Sortierungssignal für die unkonventionelle Sekretion von Galectin-1 durch die β-Galaktosid-Bindungsstelle definiert ist und haben weitreichende Implikationen für die weitere Analyse der Galectin-1 Exportmaschinerie

A genome-wide CRISPR screen reconciles the role of N-linked glycosylation in galectin-3 transport to the cell surface.

Galectins are a family of lectin binding proteins expressed both intracellularly and extracellularly. Galectin-3 (Gal-3, also known as LGALS3) is expressed at the cell surface; however, Gal-3 lacks a signal sequence, and the mechanism of Gal-3 transport to the cell surface remains poorly understood. Here, using a genome-wide CRISPR/Cas9 forward genetic screen for regulators of Gal-3 cell surface localization, we identified genes encoding glycoproteins, enzymes involved in N-linked glycosylation, regulators of ER-Golgi trafficking and proteins involved in immunity. The results of this screening approach led us to address the controversial role of N-linked glycosylation in the transport of Gal-3 to the cell surface. We find that N-linked glycoprotein maturation is not required for Gal-3 transport from the cytosol to the extracellular space, but is important for cell surface binding. Additionally, secreted Gal-3 is predominantly free and not packaged into extracellular vesicles. These data support a secretion pathway independent of N-linked glycoproteins and extracellular vesicles

Unconventional secretion of Acb1 is mediated by autophagosomes

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Manjithaya et al. in this issue)

CA125/MUC16 Is Dispensable for Mouse Development and Reproduction

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer

Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion

A novel membrane structure called CUPS is assembled during the secretion of unconventional cargo such as Acb1

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation

Galectins and Gliomas

Malignant gliomas, especially glioblastomas, are associated with a dismal prognosis. Despite advances in diagnosis and treatment, glioblastoma patients still have a median survival expectancy of only 14 months. This poor prognosis can be at least partly explained by the fact that glioma cells diffusely infiltrate the brain parenchyma and exhibit decreased levels of apoptosis, and thus resistance to cytotoxic drugs. Galectins are a family of mammalian beta-galactoside-binding proteins characterized by a shared characteristic amino acid sequence. They are expressed differentially in normal vs. neoplastic tissues and are known to play important roles in several biological processes such as cell proliferation, death and migration. This review focuses on the role played by galectins, especially galectin-1 and galectin-3, in glioma biology. The involvement of these galectins in different steps of glioma malignant progression such as migration, angiogenesis or chemoresistance makes them potentially good targets for the development of new drugs to combat these malignant tumors

Coupling of Rotational Motion with Shape Fluctuations of Core-shell Microgels Having Tunable Softness

The influence of shape fluctuations on deformable thermosensitive microgels in aqueous solution is investigated by dynamic light scattering (DLS) and depolarized dynamic light scattering (DDLS). The systems under study consist of a solid core of polystyrene and a thermosensitive shell of cross-linked poly(N-isopropylacrylamide) (PNIPA) without and with embedded palladium nanoparticles. PNIPA is soluble in water, but has a lower critical solution temperature at 32 C (LCST). Below the LCST the PNIPA shell is swollen. Here we find that besides translational and rotational diffusion, the particles exhibit additional dynamics resulting from shape fluctuations. This leads to a pronounced apparent increase of the rotational diffusion coefficient. Above the transition temperature the shell collapses and provides a rather tight envelope of the core. In this state the dynamics of the shell is frozen and the core-shell particles behave like hard spheres. A simple physical model is presented to capture and explain the essentials of the coupling of rotational motion and shape fluctuations.Comment: 9 pages, 7 figure

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design

Deciphering the molecular nature of ovarian cancer biomarker CA125

The ovarian cancer biomarker CA125 has been extensively investigated over the last 30 years. The knowledge about the exact molecular nature of this protein, however, remains fragmented. This review provides an overview of the structural research regarding CA125, and presents an orthogonal verification method to confirm the identity of this molecule. The need for independent identification of CA125 is exemplified by several reports where mutually exclusive data concerning the existence of isoforms and the glycan moieties is presented. Mass spectrometry can overcome the pitfalls of a single detection/identification method such as antibody probing. Independent verification of CA125 identity in characterization studies will help establish a refined model of its molecular structure that will promote the development of new approaches for diagnosis, prognosis and therapy of ovarian cancer.Florian Weiland, Karina Martin, Martin K. Oehler and Peter Hoffman