Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

PEtab—Interoperable specification of parameter estimation problems in systems biology

Reproducibility and reusability of the results of data-based modeling studies are essential. Yet, there has been—so far—no broadly supported format for the specification of parameter estimation problems in systems biology. Here, we introduce PEtab, a format which facilitates the specification of parameter estimation problems using Systems Biology Markup Language (SBML) models and a set of tab-separated value files describing the observation model and experimental data as well as parameters to be estimated. We already implemented PEtab support into eight well-established model simulation and parameter estimation toolboxes with hundreds of users in total. We provide a Python library for validation and modification of a PEtab problem and currently 20 example parameter estimation problems based on recent studies.     

A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.     

Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.     

Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses

The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.     

O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine

The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1-4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS(2) analysis of tetralysine complexes and by MS(n) measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.     

Initiation of mammalian O-mannosylation in vivo is independent of a consensus sequence and controlled by peptide regions within and upstream of the alpha-dystroglycan mucin domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.     

The Geodia cydonium galectin exhibits prototype and chimera-type characteristics and a unique sequence polymorphism within its carbohydrate recognition domain

The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[1-¹⁴C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between G_M1 and G_D1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse₅Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse₅Cer backbone. No cross-reaction with G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).