Isolation and characterization of plant N-acetyl glucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants

Abstract

We report on the isolation and characterization of full-length cDNA sequences coding for N-acetylglucosaminyltransferase I (GnTI) from potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), and Arabidopsis. The deduced polypeptide sequences show highest homology among the solanaceous species (93% identity between potato and tobacco compared with about 75% with Arabidopsis) but share only weak homology with human GnTI (35% identity). In contrast to the corresponding enzymes from animals, all plant GnTI sequences identified are characterized by a much shorter hydrophobic membrane anchor and contain one putative N-glycosylation site that is conserved in potato and tobacco, but differs in Arabidopsis. Southern-blot analyses revealed that GntI behaves as a single-copy gene. Northern-blot analyses showed that GntI-mRNA expression is largely constitutive. Arabidopsis cgl mutants deficient in GnTI activity also possess GntI mRNA, indicating that they result from point mutations. GntI-expression constructs were tested for the ability to relieve the GnTI block in protoplasts of the Arabidopsis cgl mutant and used to obtain transgenic potato and tobacco plants that display a substantial reduction of complex glycan patterns. The latter observation indicates that production of heterologous glycoproteins with little or no antigenic glycans can be achieved in whole plants, and not in just Arabidopsis, using antisense technology.

Figure 1

Characteristics of the isolated plant GntI-cDNA clones. A, Schematic representation of the deduced polypeptide sequences of the isolated plant GntI-cDNA clones. Potato leaf (A1 St-L) and tuber clones (A6 and A8 St-T), tobacco leaf (A4 and A9 Nt-L), and the assembled cDNA clone from Arabidopsis whole-plant tissue (At-W). Color code: black, cytosolic region; dark gray, membrane anchor; light gray, stalk region; white box, catalytic domain. The N-glycosylation sites are indicated by asterisks. Intron sequences in A6 and A8 are depicted as triangles. B, Homology matrix of the deduced amino acid sequences of the complete plant GntI-cDNA clones. Paired alignments were conducted with the GAP option of the Genetics Computer Group software package (Devereux et al., 1984). Percentage values for identical positions are shown in bold and those for similar ones in brackets. St, S. tuberosum (potato); Nt, N. tabacum (tobacco); At, Arabidopsis.

Figure 2

Alignment of the deduced plant GntI-polypeptide sequences with human GnTI. Amino acid residues identical in all sequences are highlighted by a black background, those conserved in most of the sequences are marked by a gray background. Hydrophobic regions in the membrane-anchor domains are indicated by a bold line above (plant GnTI) and underneath (human GnTI) the NH₂-terminal sequence block. Primer binding sites are shown as arrows above (sense primers) and underneath (antisense primer) the sequence blocks. Sites of introns in tuber clones A6 and A8 are indicated by triangles. The N-glycosylation consensus sites in the plant sequences are marked by asterisks. St, S. tuberosum (potato); Nt, N. tabacum (tobacco); At, Arabidopsis (Arabidopsis); Hs, Homo sapiens (man). Arrows indicate positions of amino acid differences described in the text. [→] A6 and A8 compared with A1; [--▸] A6 compared with A8; [→] A4 compared with A9.

Figure 3

Assessment of GntI-copy number in Arabidopsis. Total DNA was isolated from Arabidopsis leaves and 10-μg aliquots were digested overnight with the restriction enzymes (100 units of each) indicated above the blots. Samples were concentrated by sodium acetate-ethanol precipitation prior to separation in a 0.7% (w/v) agarose gel and subjected to Southern-blot analyses. A fragment comprising the entire Arabidopsis GntI cDNA was radioactively labeled and used as hybridization probe. Sizes of the molecular mass standard (λPstI) are indicated. A, Southern-blot analysis of Arabidopsis wild type; B, comparison of the restriction patterns in Arabidopsis wild type and in cgl mutant lines, C5 and C6. B, BamHI; E, EcoRI; P, PstI.

Figure 4

Analysis of GntI-mRNA expression in Arabidopsis wild type and the cgl mutant. Poly(A⁺) mRNA was isolated from 250 μg of total RNA and subjected to northern-blot analysis. The radioactively labeled hybridization probe was the same as described for Southern-blot analyses (Fig. 3). A, Different tissues of Arabidopsis wild-type plants; B, leaf samples of Arabidopsis wild type (WT) and Arabidopsis cgl mutants C5 and C6. The approximate size of the Arabidopsis GntI mRNA is indicated.

Figure 5

Cloning scheme of potato and tobacco GntI-sense and -antisense constructs. The complete potato and tobacco NotI-cDNA fragments (A1, 1,660 bp; A9, 1,720 bp) were inserted between CaMV-35S promoter (CaMV p35S, approximately 550 bp) and octopine synthase termination sequences (OCSpA, approximately 200 bp) in plant expression vector pA35N (see “Materials and Methods”). Clones with GntI-cDNA inserts in sense and antisense orientation, respectively (indicated by arrows), were used for complementation tests (Fig. 6). The EcoRI-HindIII fragments comprising the entire plant expression cassettes of constructs pA35N-A1-las and pA35N-A9as, respectively, were inserted into binary vector pBin19 (Bevan, 1984) and used for the generation of transgenic potato and tobacco plants. LB/RB, Left and right T-DNA borders, respectively; pNOS, nopaline synthase promoter driving the neomycin phosphotransferase gene (NEO); NOSpA, nopaline synthase termination sequences.

Figure 6

Complementation test of potato and tobacco GntI-expression constructs in the Arabidopsis cgl mutant. Protoplasts were isolated from 3- to 4-week-old C5 plants and transformed with plasmid DNA using the polyethylene glycol method. Subsequent cultivation was for 115 h at 25°C in the dark. Afterward, cells were chilled on ice, pelleted, and prepared for SDS-PAGE along with leaf samples harvested from Arabidopsis wild-type and cgl mutant plants. The western blot was stained with Ponceau S to check for comparable transfer and protein amounts (not shown) and developed with a complex glycan antiserum (Laurière et al., 1989). Lanes 1 through 5, Protoplast samples transformed with plasmid DNA of the following plant expression constructs: 1, potato GntI (A1) sense; 2, potato GntI (A1) antisense; 3, tobacco GntI (A9) sense; 4, tobacco GntI (A9) antisense; and 5, mock-transformed control (modified PHA-L; von Schaewen et al., 1993). Note that only one-half of the amount used in lanes 1 through 4 was loaded in lane 5 (PHA ½). Lane 6, Molecular-mass standard (Sigma SDS VII-L). Lanes 7 and 8, Whole-leaf control samples (50 μg of total protein each): 7, Arabidopsis wild type (WT); and 8, Arabidopsis mutant C5 (cgl). Molecular masses are given in kD. The position of Rubisco (large subunit) is indicated by an arrow. High molecular mass signals (≥45 kD bands) resulting from relief of the GnTI block in cgl protoplasts are marked by a dotted bracket on the left side of the panel. For origin of low molecular mass signals (36–45 kD bands) marked by a solid bracket, see explanation in the text.

Figure 7

Analysis of complex glycan patterns in leaf protein extracts of potato wild-type and selected antisense plants growing in tissue culture. A, Coomassie-stained SDS-gel reference (12% [w/v] polyacrylamide, 75 μg of total protein per lane); B, corresponding western blot developed with a complex glycan antiserum. WT (Pot), Untransformed potato wild type; cgl (Ara), Arabidopsis cgl mutant C5; numbers 79 through 512 (antisense), selected potato transformants. The positions of the molecular mass standard (Sigma SDS VII-L) and of Rubisco (large subunit) are indicated.

Figure 8

Progression of complex glycan reduction in terminal leaves of potato GntI-antisense plants after growing for 4 weeks in soil. Western-blot analysis was analogous to Figure 7 (only that a 10% [w/v] polyacrylamide gel was used). Numbers 1, 3, and 6 refer to leaves counted from the top of the respective potato plants. Number 439, strongest; number 512, weakest potato GntI-antisense plant (compare Fig. 7). The position of Rubisco (large subunit) is indicated by an arrow.