Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase.

Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) alpha-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG alpha-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.

Development of a filter assay for measuring homogalacturonan: alpha-(1,4)-Galacturonosyltransferase activity.

Alpha-(1,4)-galacturonosyltransferases (GalATs) catalyze the addition of (1,4)-linked alpha-D-galacturonosyl residues onto the nonreducing end of homogalacturonan chains. The nucleotide-sugar donor for the enzymatic reaction is uridine diphospho-D-galactopyranosyluronic acid (UDP-D-GalpA). Many GalAT activity assays are based on the incorporation of D-[(14)C]GalpA from UDP-D-[(14)C]GalpA onto exogenously added homogalacturonan acceptors. Reactions based on this method can be time-consuming because multiple labor-intensive centrifugations and washes with organic solvents are required to remove the unincorporated UDP-D-[(14)C]GalpA from the (14)C-labeled products. Here we report the development of an alternative GalAT filter assay based on the ability of homogalacturonan to bind to cetylpyridinium chloride (CPC). GalAT assay reaction products made using radish (Raphanus sativus) microsomal membranes or solubilized proteins from tobacco (Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia) were spotted onto Whatman 3MM paper treated with 2.5% (w/v) CPC. Unincorporated UDP-D-[(14)C]GalpA was selectively removed from the filters by washing with 150-250 mM NaCl. The versatility of this assay is demonstrated by using it to identify GalAT activity in fractions obtained during the partial purification of tobacco GalAT by SP Sepharose cation exchange chromatography and by detecting the GalAT-catalyzed incorporation of D-[(14)C]GalpA onto endogenous acceptors from Arabidopsis membranes.

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry.

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.