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Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants.

Henquet M, Heinhuis B, Borst JW, Eigenhuijsen J, Schreuder M, Bosch D, van der Krol A - Transgenic Res. (2009)

Bottom Line: We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events.In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants.Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

ABSTRACT
In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

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Complementation of cgl1-1 complemented plants. Selected AtGnTI-YFPcgl (lane 1–3), ChGnTI-YFPcgl (lane 4–6) and HuGnTI-YFPcgl (lane 7–9) T2 lines were analyzed by immunoblotting (a) or Elisa (b) using a polyclonal anti-HRP antibody that specifically recognizes complex type N-glycans. Molecular weight markers are indicated on the left. Error bars represent SD
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Fig3: Complementation of cgl1-1 complemented plants. Selected AtGnTI-YFPcgl (lane 1–3), ChGnTI-YFPcgl (lane 4–6) and HuGnTI-YFPcgl (lane 7–9) T2 lines were analyzed by immunoblotting (a) or Elisa (b) using a polyclonal anti-HRP antibody that specifically recognizes complex type N-glycans. Molecular weight markers are indicated on the left. Error bars represent SD

Mentions: From each set of transformants, plants with a single locus insert were identified by segregation analysis of T2 seedlings on medium containing kanamycin. For each of the constructs three independent single-locus-insert homozygous lines were developed, with highest, intermediate and with low level of complementation. Progeny seedlings from these homozygous lines were analyzed for levels of glycoproteins modified with complex glycans, both by western blots analysis and by Elisa (Fig. 3a, b). The results from the western blots show no specific qualitative differences in the patterns of proteins labeled with complex glycans and therefore glycosylation levels only differ quantitatively between the lines (Fig. 3a). In the ChGnTIcgl lines the highest signal was slightly lower than of the highest in the AtGnTIcgl lines, but in all three the HuGnTIcgl plants the level of proteins with complex glycans was very low (Fig. 3b).Fig. 3


Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants.

Henquet M, Heinhuis B, Borst JW, Eigenhuijsen J, Schreuder M, Bosch D, van der Krol A - Transgenic Res. (2009)

Complementation of cgl1-1 complemented plants. Selected AtGnTI-YFPcgl (lane 1–3), ChGnTI-YFPcgl (lane 4–6) and HuGnTI-YFPcgl (lane 7–9) T2 lines were analyzed by immunoblotting (a) or Elisa (b) using a polyclonal anti-HRP antibody that specifically recognizes complex type N-glycans. Molecular weight markers are indicated on the left. Error bars represent SD
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902736&req=5

Fig3: Complementation of cgl1-1 complemented plants. Selected AtGnTI-YFPcgl (lane 1–3), ChGnTI-YFPcgl (lane 4–6) and HuGnTI-YFPcgl (lane 7–9) T2 lines were analyzed by immunoblotting (a) or Elisa (b) using a polyclonal anti-HRP antibody that specifically recognizes complex type N-glycans. Molecular weight markers are indicated on the left. Error bars represent SD
Mentions: From each set of transformants, plants with a single locus insert were identified by segregation analysis of T2 seedlings on medium containing kanamycin. For each of the constructs three independent single-locus-insert homozygous lines were developed, with highest, intermediate and with low level of complementation. Progeny seedlings from these homozygous lines were analyzed for levels of glycoproteins modified with complex glycans, both by western blots analysis and by Elisa (Fig. 3a, b). The results from the western blots show no specific qualitative differences in the patterns of proteins labeled with complex glycans and therefore glycosylation levels only differ quantitatively between the lines (Fig. 3a). In the ChGnTIcgl lines the highest signal was slightly lower than of the highest in the AtGnTIcgl lines, but in all three the HuGnTIcgl plants the level of proteins with complex glycans was very low (Fig. 3b).Fig. 3

Bottom Line: We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events.In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants.Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

ABSTRACT
In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

Show MeSH
Related in: MedlinePlus