The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3,4-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lysoanalogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent.Phospholipases B catalyze the hydrolytic cleavage of both acylester bonds of glycerophospholipids. Products of phospholipase B activity are fatty acids and water-soluble glycerophosphodiesters. In yeast, soluble degradation products are released to some extent into the culture medium, and are thus an indicator of cellular phospholipase B activity. The only acylester-hydrolyzing enzyme from yeast characterized so far at the molecular level is a phospholipase B encoded by the PLB1 gene (1). The highly glycosylated enzyme of about 220 kDa (73 kDa for the protein part, predicted from the sequence) is enriched in the yeast plasma membrane but was also found in the periplasmic space and in the culture supernatant. The lysophospholipase activity of Plb1p greatly exceeds the activity catalyzing the first step of hydrolysis; thus, lyso-phospholipids do not accumulate as intermediate products of Plb1p activity (2-4). In addition, this enzyme has transacylase activity, catalyzing the synthesis of phosphatidylcholine (PtdCho) 1 from two molecules of lyso-phosphatidylcholine. The physiological function of yeast phospholipase B is still unclear; neither disruption of the PLB1 gene nor its overexpression result in detectable growth phenotypes. However, the amount of glycerophosphocholine (GroPCho) and glycerophosphoethanolamine released into the culture su...

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Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Aleuria aurantia lectin (AAL) were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody)—whose affinity is mediated through core fucosylation—and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

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