EVALUATION OF ANOMERICRECOGNITION IN GALACTOSE BINDING LECTINS USING CROSSLINKED HEMICELLULOSE: A COMPARATIVE STUDY THROUGH AFFINITY CHROMATOGRAPHY

The isolation of lectins by affinity chromatography with crosslinked hemicelluloses has been a common practice because of the variety of glycosides that they present, improving the isolation of different kinds of lectins, such as the galactose ligands.Lectins affinity for carbohydrates is so specific that a simple configuration of the chiral carbon can affect affinity, and there are lectins that are more related to alfa-galactosidic than beta-galactosidic residues, setting up that way, an anomeric recognition.The anomeric configuration of galactose residuesseems to have biological importance related to the behavior of some diseases and physiological processes. This work aimed to assess the anomeric recognition of two lectins reported as β-galactose ligands (PNA and ricin) and two lectins reported as α-galactose ligands (frutalin and jacalin) in two types of hemicellulose (xyloglucan of Tamarindus indica and galactomannan of Caesalpinia pulcherrima), subsequently crosslinked and used as chromatographic matrices. As a result,chromatographic profiles and retained fractions suggested preferential anomeric recognition by lectins for the hemicelluloses crosslinked. The galactomannan matrix retained 0,5mg of PNA lectin and 2,3mg of ricin lectin; meanwhile, the xyloglucan matrix retained 3,4mg of PNA and 3,2mg of ricin; results obtained by applying 5 mg of lectin. Ricin expresses a visible flexibility in anomeric recognition, while PNA shows a restricted recognition of β-galactose residues.Frutalin and jacalin did not show recognition of the xyloglucan matrix. This work proposes using hemicellulose reticles with epichlorohydrin as affinity chromatographic matrices for anomeric studies on recognizing galactose binding lectins.

233 STUDY OF CORTICAL GRANULE CONTENT IN IN VITRO-MATURED PORCINE OOCYTES BY MEANS OF PNA LECTIN PRECIPITATION

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida and thus blocking polyspermy. Once released, CG are not renewed. Research on cortical reaction and putative CG enzymes has progressed slowly because mammalian eggs contain only picogram quantities of CG-derived proteins (Moller and Wassarman 1989 Dev. Biol. 132, 103–112; Green 1997 Rev. Reprod. 2, 147–156), so the protein(s) responsible for the physiological changes in ZP after cortical reaction are not well known. The objective of this project was to study porcine CG content in in vitro-matured porcine oocytes by means of lectin precipitation with peanut agglutinin (PNA), since this lectin binds to porcine CG (Yoshida et al. 1993 Mol. Reprod. Dev. 36, 462–468). Immature porcine cumulus–oocytes complexes (COCs) from Landrace � Large White gilts were in vitro-matured for 44 h in NCSU-37 medium. After IVM period, COCs were stripped of cumulus cells, washed in PBS, and quickly washed through purified water. Then oocytes were lysed in a fresh water droplet by gentle pipetting using a narrow-bore glass pipette. Once lysed, zonae pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate from 1000 IVM-oocytes was incubated under continuous shaking (2 h, room temperature) with 100 µL PNA-agarose (Sigma, St. Louis, MO, USA) so that proteins bound to PNA could be precipitated by centrifugation. After lectin precipitation, proteins were detached from PNA-agarose beads by boiling in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5 min, 100�C) (Laemmli 1970 Nature 227, 689). Samples were then centrifuged (5 min, 7000g), the pellet containing PNA-agarose beads was discarded, and the supernatant containing the proteins was collected and further separated by SDS-PAGE. The silver staining of electrophoresis gels revealed eleven bands from 37 to 180 kDa, so a second gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (100V, 1 h) and incubated with PNA-horseradish peroxidase (PNA-HRP, 10 µg mL–1) for 1 h. Visualization was accomplished using the enhanced chemiluminiscence (ECL plus) method and Typhoon 9410 following the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany); only four bands of approximately 57 kDa, 60 kDa, 70 kDa, and 80 kDa were observed. These bands will be cut and processed for proteomic analysis for further studies. Preliminary results show that porcine CG-derived proteins can be studied by PNA lectin precipitation. These results could be employed in the future to develop specific antibodies against porcine CG.

The role of the glycoconjugates in the migration of anuran amphibian germ cells

1. The presence of a large amount of glycoconjugates on the anuran amphibian germ cells was demonstrated using fluorescein isothiocyanate lectins binding specifically to D-galactose and at a lower level, by other lectins binding specifically to N-acetyl-galactosamine. 2. Glycoconjugates including D-galactose were found near the pseudopodial expansions and in the extracellular space, between germ cells and follicular cells. They were also disseminated in the cytoplasm. 3. The injection of PNA lectin (from Arachis hypogea) into the endoderm inhibited the migration of 90 % of the germ cells. This inhibition was lectin-concentration dependent. Ultrastructural study of germ cells, the migration of which was inhibited, showed that they were degenerating. These results suggest that glycoconjugates are related to the migratory activity of germ cells.