A glycosyltransferase-enriched reconstituted membrane system for the synthesis of branched O-linked glycans in vitro

AbstractMimicking the biochemical reactions that take place in cell organelles is becoming one of the most important challenges in biological chemistry. In particular, reproducing the Golgi glycosylation system in vitro would allow the synthesis of bioactive glycan polymers and glycoconjugates for many future applications including treatments of numerous pathologies. In the present study, we reconstituted a membrane system enriched in glycosyltransferases obtained by combining the properties of the wheat germ lectin with the dialysable detergent n-octylglucoside. When applied to cells engineered to express the O-glycan branching enzyme core2 beta (1,6)-N-acetylglucosaminyltransferase (C2GnT-I), this combination led to the reconstitution of lipid vesicles exhibiting an enzyme activity 11 times higher than that found in microsomal membranes. The enzyme also showed a slightly higher affinity than its soluble counterpart toward the acceptor substrate. Moreover, the use of either the detergent re-solubilization, glycoprotein substrates or N-glycanase digestion suggests that most of the reconstituted glycosyltransferases have their catalytic domains in an extravesicular orientation. Using the disaccharide substrate Galβ1-3GalNAc-O-p-nitrophenyl as a primer, we performed sequential glycosylation reactions and compared the recovered oligosaccharides to those synthesized by cultured parental cells. After three successive glycosylation reactions using a single batch of the reconstituted vesicles and without changing the buffer, the acceptor was transformed into an O-glycan with chromatographic properties similar to glycans produced by C2GnT-I-expressing cells. Therefore, this new and efficient approach would greatly improve the synthesis of bioactive carbohydrates and glycoconjugates in vitro and could be easily adapted for the study of other reactions naturally occurring in the Golgi apparatus such as N-glycosylation or sulfation

Development of double porous poly (ε - caprolactone)/chitosan polymer as tissue engineering scaffold

Polymer blend made from poly( - caprolactone)/chitosan (PCL/CHT) offers interesting opportunities for biological applications. The paper presents a new way to fabricate PCL/CHT double-porosity (macrovoids with interconnected microporosity) membrane materials from a chemical optimization of the solvent and non-solvent phases and from a modified phase inversion technique. By varying the PCL/CHT proportion, it is shown that it is possible to improve the chemical and physical properties of the CHT carbohydrate polymer. The PCL/CHT membranes are fully characterized in term of physico-chemical properties (ATR-FTIR, XRD and DSC) to understand the miscibility of the two-polymer blend. Morphological characterization by SEM shows that by increasing CHT wt% in the blend, the size of the macrovoids was increasing. Rapid enzymatic degradation of PCL from all the blend was found by using lipase (from P. cepacia). The mechanisms at the origin of the morphological structuration of the material is also discussed. To test the ability to operate these materials as small diameter vascular scaffolds, cell culture with human umbilical vein endothelial cells (HUVECs) were carried out on the membrane and the results analyzed with laser scanning confocal microscopy (LSCM). Data suggest that the blend membrane with higher concentration of CHT polymer wt% have suitable properties that promote high number of cells on the surface by maintaining cellular cytoskeleton integrity within 3 days. The blend membrane with a double porous morphology could be potentially applicable in future for small diameter vascular graft application. The surface macrovoids (20–90 μm) could be useful for three-dimensional cellular adhesion and proliferation and interconnected microporous spongy network (7–20 μm) is expected to transfer essential nutrients, oxygen, growth factor between the macrovoids and the supernatant

Artificial membranes tuning for lymphatic wall repair

Chylothorax is an uncommon form of pleural effusion, which generally occurs after cardiac surgery and almost any surgical operation in the chest. The aim of this project is to develop a bioresorbable vascular patch for lymphatic wall repair [1]. Here, we project to develop new materials (i.e. membranes) having two different levels of porosity [2]. First short tests made with Polycaprolactone (PCL) membranes and PCL was blended with different biocompatible, bioresorbable membranes. It shows that human dermal lymphatic endothelial (HDLEC) cells can bind and spread on certain membrane and not on others suggesting that the chemical structure and the morphology of the membranes is important

Reconstitution of a glycosylation cellular system : application of glycan synthesis

La glycosylation des protéines est une modification co/post-traductionnelle, localiséeprincipalement dans l’appareil de golgi, impliquée dans divers processus physiologiques. Àl’inverse de la synthèse des protéines et des nucléotides, celle des sucres est plus complexe,en partie, à cause des nombreux branchements structuraux et de la diversité stéréochimiquedes glycannes. Les glycosyltransférases sont les enzymes responsables de la biosynthèse desoligo- et polysaccharides. Cependant, il existe différents procédés permettant de réaliser lasynthèse in vitro d’oligosaccharides tels que des procédés chimiques, enzymatiques ouchimio-enzymatiques. La Core 2 β(1,6)-N-acétylglucosaminyltransférase I (C2GnT-I) est uneglycosyltransférase (GT) transmembranaire de type II qui crée une liaison β1,6 entre une Nacétylglucosamine(GlcNAc) et la N-acétylgalactosamine (GalNAc) d’un noyau Core 1,formant ainsi une structure branchée de type Core 2. Une fois le branchement Core 2 initié,au moins trois réactions successives de transfert peuvent avoir lieu, impliquant les β(1,4)-galactosyltransférases, les α(2,3)-sialyltransférases et les α(1,3)-fucosyltransférases. Il estadmis que les GTs sont réparties séquentiellement dans les compartiments cis, médian ettrans de l’appareil de Golgi selon leur ordre d’intervention et le type cellulaire. Nous avonsmis au point un procédé de reconstitution membranaire, comportant des glycoprotéinesgolgiennes, dont les GTs, par l’intermédiaire d’une lectine, la wheat germ agglutinin (WGA).Il est admis que la WGA interagit avec les composants de la membrane plasmique et ceux del’appareil de Golgi. L’étude de notre système membranaire reconstitué a mis en évidence uneactivité élevée de la C2GnT-1 in vitro, et son efficacité à synthétiser des oligosaccharidesbranchés Core 2, par glycosylation séquentielle.Protein glycosylation is a co/posttranslational modification, localized in the Golgi apparatus,involved in various physiological processes. Sugar synthesis is more complex than that ofproteins and nucleic acids, in part because of the glycosidic bond and glycan stereochemistrydiversity. Glycosyltransferases are enzymes responsible of oligo- and polysaccharidesbiosynthesis. Various methods are used for the synthesis of oligosaccharides, in vitro, suchas chemical, enzymatic or chemo-enzymatic. Core 2 β(1,6)-N-acetylglucosaminyltransferase I(C2GnT-I) is a type-II transmembrane glycosyltransferase (GT). This enzyme create a β1,6bond between N-acetylglucosamine (GlcNAc) and Core 1 N-acetylgalactosamine (GalNAc),forming a Core 2 branched structure. Once the Core 2 branch is initiated, at least threesuccessive transfer reactions can take place, involving β(1,4)-galactosyltransferases, α(2,3)-sialyltransferases and α(1,3)-fucosyltransferases. It is known that GT are distributedsequentially in the cis, medial and trans Golgi apparatus in order of intervention andaccording to cell type. We have developed a membrane reconstitution process comprisinggolgi glycoproteins, including GT, by the use of a lectin, the wheat germ agglutinin (WGA). Itis known that WGA interacts with plasma membrane and Golgi apparatus components. Ourreconstituted membrane system showed a high C2GnT-1 activity in vitro and its effectivenessin synthesizing Core2 branched oligosaccharides by sequential glycosylation

Hamma

Key to the species of the genus Hamma 1 Posterior pronotal process without terminal spine............................................................ 2 - Posterior pronotal process with a terminal spine............................................................. 3 2 Pronotum with reddish brown blunt tubercles................................................... franciscae n. sp. - Pronotum with black thorn-like tubercles.......................................................... carlini n. sp. 3 Suprahumeral horns prominent, ending in a thorn............................................................ 4 - Suprahumeral horns absent or not prominent, apex blunt...................................................... 14 4 Posterior pronotal process quite slender in lateral view without well evident nodes.................................. 5 - Posterior pronotal process in lateral view with well evident nodes............................................... 6 5 Suprahumeral horns globose (better visible in frontal view)............................................ cinnameus - Suprahumeral horns conical....................................................................... capeneri 6 Suprahumeral horns very large, posterior process heart-shaped in dorsal view............................... fabulosum - Suprahumeral horns smaller, posterior process not heart-shaped in dorsal view..................................... 7 7 Pronotum with reddish tubercles.................................................................... nodosum - Pronotum with black tubercles........................................................................... 8 8 Suprahumeral horns upturned.................................................................... brevicornis - Suprahumeral horns laterad.............................................................................. 9 9 Posterior process with terminal node large and clearly rounded in dorsal view..................................... 10 - Posterior process with terminal node quite slender, not rounded in dorsal view.............................. robustum 10 Suprahumeral horns about half as long as pronotal width (dorsal view).......................................... 11 - Suprahumeral horns about 1 / 4 to 1 / 3 as long as pronotal width (dorsal view)...................................... 13 11 Posterior process V-shaped in lateral view between second and third node........................................ 12 - Posterior process straight in lateral view between second and third node..................................... rectum 12 Suprahumeral horns straight in dorsal view........................................................... grahami - Suprahumeral horns curved backwards in dorsal view..................................................... heimi 13 Posterior process shorter than tegmina, suprahumeral horns with short, stout terminal spine............... boulardi n. sp. - Posterior process as long as the tegmina, suprahumeral horns with slender terminal spine...................... spinosum 14 Posterior process in dorsal view with large terminal node............................................... ugandensis - Posterior process in dorsal view with very small terminal node................................................. 15 15 Posterior process slender in lateral view................................................................... 16 - Posterior process quite strong in lateral view......................................................... pattersoni 16 Flanks of the pronotal helmet and thoracic pleurae covered by white pilosity; posterior process with lateral carinae... simplex - No presence of white pilosity; posterior process without lateral carinae................................... pygmaeumPublished as part of Loudit, Sandrine Mariela Bayendi, Durante, Antonio & Susini, Antonio, 2014, Membracidae of Gabon: the genus Hamma Buckton, 1905 (Hemiptera: Auchenorrhyncha) with description of three new species, pp. 323-346 in Zootaxa 3838 (3) on page 327, DOI: 10.11646/zootaxa.3838.3.5, http://zenodo.org/record/25207

Hamma pygmaeum Capener 1972

9 Hamma pygmaeum Capener, 1972 Hamma pygmaeum Capener, 1972; Entomology Memoir Department of Agricultural Technical Services Republic of South Africa, 24: 50-51. Remarks Species recorded from EQUATORIAL GUINEA, Nkolentangan (type locality, Capener 1972).Published as part of Loudit, Sandrine Mariela Bayendi, Durante, Antonio & Susini, Antonio, 2014, Membracidae of Gabon: the genus Hamma Buckton, 1905 (Hemiptera: Auchenorrhyncha) with description of three new species, pp. 323-346 in Zootaxa 3838 (3) on page 333, DOI: 10.11646/zootaxa.3838.3.5, http://zenodo.org/record/25207

Hamma ugandensis Capener 1971

14 Hamma ugandensis Capener, 1971 Hamma ugandensis Capener, 1971; Journal of entomological Society of South Africa, XXXIV, 1: 27-28. Remarks Species recorded from UGANDA: Mulange (type locality, Capener 1971).Published as part of Loudit, Sandrine Mariela Bayendi, Durante, Antonio & Susini, Antonio, 2014, Membracidae of Gabon: the genus Hamma Buckton, 1905 (Hemiptera: Auchenorrhyncha) with description of three new species, pp. 323-346 in Zootaxa 3838 (3) on page 339, DOI: 10.11646/zootaxa.3838.3.5, http://zenodo.org/record/25207

Hamma carlini Bayendi Loudit, Durante & Susini, n. sp.

Hamma carlini Bayendi Loudit, Durante & Susini n. sp. (Figs. 12, 13) Holotype ♂ Gabon, Makokou, Ipassa research station (Ivindo National Park), 0° 30 ’ 43 ”N 12 ° 48 ’ 13 ”E, Feb.-Mar. 2011, A. Susini leg. In MSNS. Diagnosis Species similar to H. capeneri and H. nodosum in lateral view, easily distinguishable thanks to the absence of the caudal spine of the posterior process. In addition, H. capeneri has a shorter and thinner posterior process, and H. nodosum presents a clearly larger terminal node of the posterior process in lateral view. Size Total length: 3.4 mm Pronotal length: 2.8 mm Tegminal length: 3.5 mm Description HEAD: black, slightly convex, punctate, with quite dense golden pubescence; vertex 1.63 wider than high; a shallow concavity between the ocelli; upper margin arcuate, slightly sinuate; ventral margin W-shaped with the lower parts non very pronounced; ocelli clearly above the centro-ocular line. Frontoclypeus oval, lateral lobes completely fused to frontoclypeus with margins hardly distinguishable; rostrum brown with black base; antennae brown. PRONOTUM: black punctate, with many small yellow bristles and several thorn-like tubercles, each associated with a single apical seta; metopidium two times wider than high, median carina percurrent; supraocular callosities large, vaguely pentagonal, partially bare, unpunctated; humeral angles prominent and blunt; posterior angle rounded; suprahumeral horns reducerd, tuberculate, projected outwards, with a brown thorn at the top. Posterior process black, strongly produced emerging slightly dorsal to posterior margin and with apex extending little beyond the anal angle of forewing; sinuate in lateral view; with two nodes well observable in dorsal view at the anterior and posterior extremities; no spine at the caudal end; dorsal and ventral carinae percurrent; lateral carinae present only on the area between the nodes. The whole posterior process with few thorn-like tubercles gathered mainly along the carinae and on the nodes. All the tubercles with a small yellow bristle at the apex. SCUTELLUM: entirely black, punctate, with the base longer than the height, emarginated with scutellar apices acute; base swollen except for the corners, with one ogival tubercle on each side of the swelling. The said tubercles with a tuft of small whitish backwards setae. FOREWING: approximately two and half times longer than wide, hyaline, sclerotized basally, punctate, black in colour. Pterostigma sub-triangular, amber in colour, finely edged in black; venation lighter than pterostigma, but end of veins A 1 and A 2 and anal margin deep brown; a brown rectangularish dot at the anal angle. LEGS: black with distal half tibiae and tarsi yellow; praetarsi black. ABDOMEN: gray-brown with punctuation, dorsally covered by sparse whitish setae. Etimology The species is dedicated to Piero Carlino, MSNS, Lecce, Italy, for his valuable help in collecting Membracidae specimens. The surname is latinized into Carlinus, of which the genitive is Carlini (ICZN art. 31.1.1).Published as part of Loudit, Sandrine Mariela Bayendi, Durante, Antonio & Susini, Antonio, 2014, Membracidae of Gabon: the genus Hamma Buckton, 1905 (Hemiptera: Auchenorrhyncha) with description of three new species, pp. 323-346 in Zootaxa 3838 (3) on page 342, DOI: 10.11646/zootaxa.3838.3.5, http://zenodo.org/record/25207

Hamma nigrum Durante & Loudit & Susini 2021, sp. nov.

Hamma nigrum sp. nov. urn:lsid:zoobank.org:act: 6A5C5E1E-9D51-41C8-8D1E-75B5C1C458D4 Figs 1, 4B, 5B Diagnosis Species belonging to the genus Hamma with the thorax entirely black and pronotum smooth; suprahumeral horns with a laterally pointing thorn; a posterior process with four nodes, V-shaped in lateral view between the second and fourth nodes; last node roundish in dorsal view, ending in a well-developed terminal spine. Pterostigma about three times as long as broad. Differential diagnosis The general black colour clearly distinguishes this species from the following two (i.e., spinellii sp. nov. and caneparii sp. nov.), although not from Hamma heimi. The roughly triangular shape of the supraocular callosity (Fig. 4B) is also diagnostic with respect to all other species in the group. The anchor-like third node (Fig. 5B) of the posterior process clearly distinguishes it from all other species in the group, being rhomboidal in dorsal view, whereas it is roughly circular in heimi, and truly anchor-like in spinellii sp. nov. and in caneparii sp. nov. Other diagnostic characters with respect to spinellii sp. nov. and caneparii sp. nov. include: – upper margin of the head (Fig. 4B) roughly circular (roughly square in spinellii sp. nov. and caneparii sp. nov.); – metopidium (Fig. 4B) smooth (tuberculate in spinellii sp. nov. and caneparii sp. nov.); – suprahumeral horns (Fig. 4B) with a robust thorn slightly curving dorsally in frontal view (straight in spinellii sp. nov. and caneparii sp. nov.); – pterostigma (Fig. 5B) diagnostic only with respect to caneparii sp. nov.: l/w ratio 2.95 in nigrum sp. nov. (mean value); 2.4 in caneparii sp. nov. Diagnostic characters with respect to heimi include: – upper margin of the head more arcuate than in heimi; – punctation on the pronotum finer than in heimi; – first (proximal) node of the posterior process roughly circular in lateral view, dome-shaped in heimi. Etymology The species takes its name from the Latin adjective ' nigrus ʼ, meaning 'blackʼ, in the neuter form in accordance with the grammatical gender. Material examined Holotype GABON • ♀; Ogooué Ivindo, Mont Sassamongo, Batouala; 1 Dec. 2012; A. Susini leg.; MSNS. Paratypes EQUATORIAL GUINEA • 1 ♀; Nyefang, Mossumu; 5 Oct. 2015; A. Susini leg.; MSNS • 1 ♀; same collection data as for preceding; coll. Susini. Description MEASUREMENTS. Holotype total length: 5.2 mm, average total length: 4.5 mm; holotype pronotal length: 5 mm; average pronotal length: 4.7 mm; holotype tegminal length: 4.2 mm, average tegminal length: 4.2 mm. HEAD. Brilliant black, convex, punctate, with very small and sparse setae; vertex almost twice as wide as high; shallow concavity between the ocelli; upper margin arcuate, rounded, slightly sinuate; ventral margin W-shaped with the lower parts not very pronounced; ocelli clearly above the centro-ocular line. Frontoclypeus pear-shaped, lateral lobes completely fused to frontoclypeus with margins barely distinguishable; rostrum dark brown; antennae light brown. PRONOTUM. Brilliant black, punctate, almost completely naked; metopidium smooth, almost twice as wide as high, median carina percurrent, unpunctate, straight until the base of the posterior process and subsequently sinuate, supraocular callosities barely noticeable, small and unpunctate, roughly triangular; humeral angles prominent and blunt; suprahumeral horns well developed, with a tower-shaped base (width/height ratio: 1.48), slightly tuberculate, sometimes with dorsal tip reddish-brown, with a robust brown thorn projecting outwards and curving upwards slightly in frontal view. Posterior process brilliant black, punctate, emerging posteriorly from the pronotum and continuously from the posterior margin; sinuate in lateral view, with four nodes, the first of which (proximal) almost spherical; second node small, dome-shaped in lateral view (flattened in dorsal view) with four to eight small spines dorsally; third node anchor-like in dorsal view with one to three spines at the end of the lateral arms; fourth node subspherical, almost twice as big as the first, with many spines and a robust terminal spine at the caudal end; dorsal and ventral carinae discontinuous, black, in some parts tending to brown. A few spines dorsally and ventrally along the trunk of the posterior process between the third and fourth nodes. Each spine bears a very thin light apical seta. SCUTELLUM (Fig. 5). Entirely black, punctate, with the base longer than the height, emarginate, with scutellar apices acute with a few translucent setae; base swollen except for the corners, with one ogival tubercle on each side of the swelling, each tubercle with a tuft of small whitish setae. FOREWING. Almost three times as long as wide (l/w ratio 2.94, mean value), hyaline; basally sclerotized, punctate, dark brown-black in colour. Pterostigma sub-triangular with very rounded corners, blackish in colour; venation black to brown; a large sub-pentagonal brown patch extending from costa to inner margin in the median area; a Y-shaped brown patch extending from costa to anal angle, connected with the previous patch at the level of the second discoidal cell. LEGS. Ochreous yellow, praetarsi brown. ABDOMEN. Urites black with punctation (in one specimen with white suffusion), and thin brown caudal border.Published as part of Durante, Antonio, Loudit, Sandrine Mariella Bayendi & Susini, Antonio, 2021, On the genus Hamma Buckton, 1905 (Hemiptera: Auchenorrhyncha) in Equatorial Africa, with descriptions of three new species, pp. 89-107 in European Journal of Taxonomy 748 (1) on pages 94-97, DOI: 10.5852/ejt.2021.748.1345, http://zenodo.org/record/474515