Detection of the gangliosideN-glycolyl-neuraminyl-lactosyl-ceramide by biotinylatedEscherichia coli K99 lectin

K99 lectin fromEscherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay. Abbreviations: NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid; PBS, phosphate buffered saline (0.9% NaCl containing 150mm sodium phosphate, pH 7.2); LPS, lipopolysaccharide; BCHZ, biocytin hydrazide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle medium. For the gangliosides, trivial names and structures are given according to the recommendations in [43]. NeuAc2-3Gal1-4Glc1-1Cer (NeuAc-GM₃); NeuGc2-3Gal1-4Glc1-1Cer (NeuGc-GM₃); GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₂); NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer (GD3); Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₁); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GD_1a); Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Gle1-1Cer (GD_1b); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3) Gal1.-4Glc1-1Cer (GT_1b). NeuGc2-3Gal1-4GleNAc1-4Gal1-4Glc1-1Cer (NeuGc-SPG).     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.     

Chasing long-range evolutionary couplings in the AlphaFold era

Coevolution between protein residues is normally interpreted as direct contact. However, the evolutionary record of a protein sequence contains rich information that may include long-range functional couplings, couplings that report on homo-oligomeric states or even conformational changes. Due to the complexity of the sequence space and the lack of structural information on various members of a protein family, it has been difficult to effectively mine the additional information encoded in a multiple sequence alignment (MSA). Here, taking advantage of the recent release of the AlphaFold (AF) database we attempt to identify coevolutionary couplings that cannot be explained simply by spatial proximity. We propose a simple computational method that performs direct coupling analysis on a MSA and searches for couplings that are not satisfied in any of the AF models of members of the identified protein family. Application of this method on 2012 protein families suggests that ~12% of the total identified coevolving residue pairs are spatially distant and more likely to be disordered than their contacting counterparts. We expect that this analysis will help improve the quality of coevolutionary distance restraints used for structure determination and will be useful in identifying potentially functional/allosteric cross-talk between distant residues.     

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) fromSilene alba cells

The peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J 9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc N-acetylglucosamine - PNGase peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin layer chromatography - HPAEC-PAD High pH anion exchange chromatography-pulsed amperometric detection     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Lumican regulates osteosarcoma cell adhesion by modulating TGFβ2 activity

Human osteosarcoma cell lines were recently shown to express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability to regulate the growth and motility of these cells. In this study, lumican-deficient Saos 2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN) (p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2) activity, no difference in the ability of lumican siRNA-transfected and scramble siRNA-transfected Saos 2 cells to adhere onto FN was detected (p=NS). Exogenous TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results therefore, suggest that the inverse correlation existing between lumican expression and Saos 2 cell adhesion is dependent on active TGF-β2 signaling. Furthermore, the significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01) was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FN-dependent adhesion, β1 integrin expression and pFAK activation were likewise identified as downstream TGF-β2 effectors regulated by lumican expression. In conclusion, this study demonstrates a novel out-in signaling circuit in human osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2, integrin β1 and pFAK to regulate osteosarcoma adhesion.     

Molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycan-like glyconectins

Glyconectins (GNs) represent a new class of proteoglycan-like cell adhesion and recognition molecules found in several Porifera species. Physico-chemical properties of GN carbohydrate moieties, such as size, composition, and resistance to most glycosaminoglycan-degrading enzymes, distinguish them from any other type of known glycoproteins. The molecular mechanism of GN-mediated self/non-self discrimination function is based on highly species-specific and Ca(2+)-dependent GN to GN associations that approach the selectivity of the evolutionarily advanced immunoglobulin superfamily. Carbohydrates of glyconectins 1, 2, and 3 are essential for species-specific auto-aggregation properties in three respective Porifera species. To obtain a structural insight into the molecular mechanisms, we performed carbohydrate structural analyses of glyconectins isolated from the three sponge model systems, Microciona prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). The glycan content of all three GNs ranged between 40 and 60% of their total mass. Our approach using sequential and selective chemical degradation of GN glycans and subsequent mass spectrometric and NMR analyses revealed that each glyconectin presents novel and highly species-specific carbohydrate sequences. All three GNs include distinct acid-resistant and acid-labile carbohydrate domains, the latter composed of novel repetitive units. We have sequenced four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2, which represent a heterogeneous but related family of structures, and four sulfated units in GN3.     

Activities of de-N-glycosylation are ubiquitously found in tomato plant

Activities of two de-N-glycosylation enzymes, PNGase (peptide N ⁴(N-acetyl-glucosaminyl)asparagine amidase) and ENGase (endo N-acetyl-β-D-glucosaminidase), involved in the release of N-glycans from N-glycoproteins, were monitored in several organs of tomato plants (Lycopersicon esculentum, Mill., cv. Dombito) with a fluorescence-HPLC procedure using a resofurin-labelled N-glycopeptide substrate. PNGase and ENGase activities were detected in every organ assayed but with quantitative differences. The highest activities were found in the youngest parts of the plant, i.e. apical buds, flowers and leaf blades. PNGase activities were consistently higher than ENGase activities (three-fold in average). Both de-N-glycosylation activities were associated with high levels of proteins and protease activities. During fruit growth and ripening, these three parameters decreased notably. The ubiquitous detection of these enzyme activities in the different organs is probably associated with the previously characterized unconjugated N-glycans in tomato. The possible role of PNGase and ENGase degradation products (i.e. unconjugated N-glycans) are discussed in relation with their biological functions in plant development.