Galactose-extended glycans of antibodies produced by transgenic plants

Abstract

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that beta1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human beta1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal beta1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal beta1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human beta1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Figure 1

Correlation of mRNA expression of GalT and RCA binding. (A) Northern blot showing the mRNA expression of GalT in 25 GalT plants and an untransformed control plant (lane 0). (B) Western blot of the same plants showing binding of the Gal-specific lectin RCA to isolated proteins.

Figure 2

MALDI-TOF mass spectra of (A) N-linked glycans isolated from control tobacco leaves, (B) N-linked glycans isolated from leaves of tobacco plants transformed with human GalT, and (C) N-linked glycans affinity purified on RCA. Asterisk, (M + K)⁺ adducts. See Tables 1 and 2 for structures.

Figure 3

Western blot showing sheep-anti-mouse-IgG [polyclonal, directed against both heavy and light chains] (A) and RCA binding (B) to purified antibody produced in hybridoma culture (lane 1), tobacco plants (lane 2), and two tobacco plants (GalT-8 × Mgr-48–11 and -12) coexpressing GalT (lanes 3 and 4). An amount of 0.17 μg hybridoma antibody was used per lane, and equal amounts of the other antibodies as judged from the intensity of the heavy chain bands on Coomassie-stained gels (not shown). H.C., heavy chain; L.C., light chain.

Figure 4

MALDI-TOF mass spectra of N-linked glycans isolated from (A) hybridoma Mgr-48 antibody, (B) tobacco Mgr-48 antibody, and (C) Mgr-48 antibody expressed in tobacco plants transformed with human GalT. Asterisk, (M + K)⁺ adducts. See Tables 1 and 2 for structures.