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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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Localization of the GnTI-YFP fusion proteins in leaves of 10 day old transgenic T2 seedlings by whole mount microscopy. Left: light images leaf surface. Middle: fluorescent images taken under equal filter and exposure settings. Right: overlay light and fluorescent images. Seedlings expressing AtGnTI-YFP or ChGnTI-YFP, displayed a punctate fluorescence pattern consistent with a Golgi-localization. In seedlings expressing HuGnTI-YFP either no or only very weak punctuated fluorescence was detected. Similar results were obtained for at least three independent transformed lines expressing the different constructs
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Fig5: Localization of the GnTI-YFP fusion proteins in leaves of 10 day old transgenic T2 seedlings by whole mount microscopy. Left: light images leaf surface. Middle: fluorescent images taken under equal filter and exposure settings. Right: overlay light and fluorescent images. Seedlings expressing AtGnTI-YFP or ChGnTI-YFP, displayed a punctate fluorescence pattern consistent with a Golgi-localization. In seedlings expressing HuGnTI-YFP either no or only very weak punctuated fluorescence was detected. Similar results were obtained for at least three independent transformed lines expressing the different constructs

Mentions: We analyzed the sub-cellular localization of the different GnTI-YFP fusion proteins in cells of leaves from 10-day old transgenic seedlings by whole mount confocal microscopy. As could be expected from the expression levels in leaves, the YFP fluorescence signal was most clearly detected in leaves of cgl1-1 seedlings transformed with AtGnTI-YFP or ChGnTI-YFP. The YFP fluorescence in these plants displayed a punctuated pattern, consistent with a Golgi-localization of the fusion protein (Fig. 5). No fluorescence signal above background levels were detected in leaves of cgl1-1 plants complemented with HuGnTI (Fig. 5), consistent with the lack of detection on western blot of intact HuGnTI-YFP fusion protein in these plants (Fig. 4). The low fluorescence signal in the stably transformed plants with HuGnTI-YFP prevented accurate sub-cellular localization of the gene product(s). Therefore, to confirm that the HuGnTI-YFP expression construct can produce intact fusion protein in plants and to obtain a better signal for sub-cellular localization of this fusion protein in plant cells, a transient protoplast expression assay was used.Fig. 5


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)

Localization of the GnTI-YFP fusion proteins in leaves of 10 day old transgenic T2 seedlings by whole mount microscopy. Left: light images leaf surface. Middle: fluorescent images taken under equal filter and exposure settings. Right: overlay light and fluorescent images. Seedlings expressing AtGnTI-YFP or ChGnTI-YFP, displayed a punctate fluorescence pattern consistent with a Golgi-localization. In seedlings expressing HuGnTI-YFP either no or only very weak punctuated fluorescence was detected. Similar results were obtained for at least three independent transformed lines expressing the different constructs
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902736&req=5

Fig5: Localization of the GnTI-YFP fusion proteins in leaves of 10 day old transgenic T2 seedlings by whole mount microscopy. Left: light images leaf surface. Middle: fluorescent images taken under equal filter and exposure settings. Right: overlay light and fluorescent images. Seedlings expressing AtGnTI-YFP or ChGnTI-YFP, displayed a punctate fluorescence pattern consistent with a Golgi-localization. In seedlings expressing HuGnTI-YFP either no or only very weak punctuated fluorescence was detected. Similar results were obtained for at least three independent transformed lines expressing the different constructs
Mentions: We analyzed the sub-cellular localization of the different GnTI-YFP fusion proteins in cells of leaves from 10-day old transgenic seedlings by whole mount confocal microscopy. As could be expected from the expression levels in leaves, the YFP fluorescence signal was most clearly detected in leaves of cgl1-1 seedlings transformed with AtGnTI-YFP or ChGnTI-YFP. The YFP fluorescence in these plants displayed a punctuated pattern, consistent with a Golgi-localization of the fusion protein (Fig. 5). No fluorescence signal above background levels were detected in leaves of cgl1-1 plants complemented with HuGnTI (Fig. 5), consistent with the lack of detection on western blot of intact HuGnTI-YFP fusion protein in these plants (Fig. 4). The low fluorescence signal in the stably transformed plants with HuGnTI-YFP prevented accurate sub-cellular localization of the gene product(s). Therefore, to confirm that the HuGnTI-YFP expression construct can produce intact fusion protein in plants and to obtain a better signal for sub-cellular localization of this fusion protein in plant cells, a transient protoplast expression assay was used.Fig. 5

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