The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis

Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton.     

A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization

Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of approximately 20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins.     

Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia

A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.     

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.     

Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles

The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.     

The ganglioside GD1α, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer,is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2

The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7×10¹¹ cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the G_M1a-pathway (G_M2, G_M1a and G_D1a) and G_M1b (IV³Neu5Ac-GgOse₄Cer) and GalNAc-G_M1b of the G_M1b-pathway, the dis8aloganglioside G_D1 (IV³Neu5Ac, III⁶Neu5Ac-GgOse₄Cer) was found in equal amounts compared to G_D1a (IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer). All gangliosides were substituted with C_24:0,24:1 and C_16:0 fatty acids, sphingosine andN-acetylneuraminic acid as the sole sialic acid. Abbreviations: FAB-MS, fast atom bombardment-mass spectrometry; GC-MS, gas chromatography-mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycoloylneuraminic acid [57]. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [58] and the nomenclature of Svennerholm [59]. Gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer gangliopentaosylceramide or GgOse₅Cer, GalNAc1-4Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1a, II³Neu5Ac-GgOse₄Cer; G_M1b, IV³Neu5Ac-GgOse₄Cer; GalNAc-G_M1b, IV³Neu5Ac-GgOse₅Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_D1 or G_D1e, IV³Neu5Ac, III⁶Neu5AcGgOse₄Cer; G_D1e, IV³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer.     

Forssman disaccharide is the specific ligand of a galectin from the sponge Geodia cydonium but does not mediate its binding to nuclear protein np56

The galectin from Geodia cydonium (GCA) had previously beenshown to be involved in regulatory mechanisms of cell sortingand adhesion during reaggregation of allogcneic sponge cells.In this contribution the binding specificity of GCA was establishedto be GaINAc     

Human Trefoil Factor 2 Is a Lectin That Binds α-GlcNAc-capped Mucin Glycans with Antibiotic Activity against Helicobacter pylori

Helicobacter pylori infection is the major cause of gastric cancer and remains an important health care challenge. The trefoil factor peptides are a family of small highly conserved proteins that are claimed to play essential roles in cytoprotection and epithelial repair within the gastrointestinal tract. H. pylori colocalizes with MUC5AC at the gastric surface epithelium, but not with MUC6 secreted in concert with TFF2 by deep gastric glands. Both components of the gastric gland secretome associate non-covalently and show increased expression upon H. pylori infection. Although blood group active O-glycans of the Lewis-type form the basis of H. pylori adhesion to the surface mucin layer and to epithelial cells, α1,4-GlcNAc-capped O-glycans on gastric mucins were proposed to inhibit H. pylori growth as a natural antibiotic. We show here that the gastric glycoform of TFF2 is a calcium-independent lectin, which binds with high specificity to O-linked α1,4-GlcNAc-capped hexasaccharides on human and porcine stomach mucin. The structural assignments of two hexasaccharide isomers and the binding active glycotope were based on mass spectrometry, linkage analysis, ¹H nuclear magnetic resonance spectroscopy, glycan inhibition, and lectin competition of TFF2-mucin binding. Neoglycolipids derived from the C3/C6-linked branches of the two isomers revealed highly specific TFF2 binding to the 6-linked trisaccharide in GlcNAcα1-4Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-3)GalNAc-ol(Structure 1). Supposedly, lectin TFF2 is involved in protection of gastric epithelia via a functional relationship to defense against H. pylori launched by antibiotic α1,4-GlcNAc-capped mucin glycans. Lectin-carbohydrate interaction may have also an impact on more general functional aspects of TFF members by mediating their binding to cell signaling receptors.     

Trefoil Factor Family Domains Represent Highly Efficient Conformational Determinants for N-Linked N,N′-di-N-acetyllactosediamine (LacdiNAc) Synthesis

The disaccharide N,N′-di-N-acetyllactose diamine (LacdiNAc, GalNAcβ1–4GlcNAcβ) is found in a limited number of extracellular matrix glycoproteins and neuropeptide hormones indicating a protein-specific transfer of GalNAc by the glycosyltransferases β4GalNAc-T3/T4. Whereas previous studies have revealed evidence for peptide determinants as controlling elements in LacdiNAc biosynthesis, we report here on an entirely independent conformational control of GalNAc transfer by single TFF (Trefoil factor) domains as high stringency determinants. Human TFF2 was recombinantly expressed in HEK-293 cells as a wild type full-length probe (TFF2-Fl, containing TFF domains P1 and P2), as single P1 or P2 domain probes, as a series of Cys/Gly mutant forms with aberrant domain structures, and as a double point-mutated probe (T68Q/F59Q) lacking aromatic residues within a hydrophobic patch. The N-glycosylation probes were analyzed by mass spectrometry for their glycoprofiles. In agreement with natural gastric TFF2, the recombinant full-length and single domain probes expressed nearly exclusively fucosylated LacdiNAc on bi-antennary complex-type chains indicating that a single TFF domain was sufficient to induce transfer of this modification. Contrasting to this, the Cys/Gly mutants showed strongly reduced LacdiNAc levels and instead preponderant LacNAc expression. The probe with point mutations of two highly conserved aromatic residues in loop 3 (T68Q/F59Q) revealed that these are essential determinant components, as the probe lacked LacdiNAc expression. The structural features of the LacdiNAc-inducing determinant on human TFF2 are discussed on the basis of crystal structures of porcine TFF2, and a series of extracellular matrix-related LacdiNAc-positive glycoproteins detected as novel candidate proteins in the secretome of HEK-293 cells.     

Secretome profiling of primary human skeletal muscle cells

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.