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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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Related in: MedlinePlus

Western blot analysis of protoplasts from two separate co-transfections. Proteins were extracted from the protoplast (p) and the medium (m) from a co-transfection assay with the plant GnTI-CFP and the YFP-Golgi marker (lane 1 and 2) and a co-transfection with HuGnTI-YFP and HuGnTI-CFP (lane 3 and 4) and used for western blotting with antibodies against GFP. The position of the GnTI fusion proteins (a), the YFP-Golgi marker (b) and the free cleaved YFP/CFP (c) is indicated by the arrows on the left. Molecular weight markers are indicated on the left
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Fig7: Western blot analysis of protoplasts from two separate co-transfections. Proteins were extracted from the protoplast (p) and the medium (m) from a co-transfection assay with the plant GnTI-CFP and the YFP-Golgi marker (lane 1 and 2) and a co-transfection with HuGnTI-YFP and HuGnTI-CFP (lane 3 and 4) and used for western blotting with antibodies against GFP. The position of the GnTI fusion proteins (a), the YFP-Golgi marker (b) and the free cleaved YFP/CFP (c) is indicated by the arrows on the left. Molecular weight markers are indicated on the left

Mentions: The fluorescence signal from HuGnTI in protoplasts indicates that, contrary to stably transformed plants, the conditions of the transient expression assay do allow the detection of this protein in plant cells. Indeed, intact HuGnTI fusion protein in protoplasts could now be detected by western blot analysis (Fig. 7, lane 3). Results also show that the ~27 kDa protein band representing free YFP/CFP, is mostly present in the medium fraction, indicating that cleaved YFP/CFP is mostly secreted (Fig. 7, lane 4). In protoplasts transfected with the AtGnTI-CFP and STtmd-YFP, both full length fusion proteins are detected in the protoplast fraction, while free CFP (and/or YFP) was again detected in the medium fraction (Fig. 7, lane 2).Fig. 7


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)

Western blot analysis of protoplasts from two separate co-transfections. Proteins were extracted from the protoplast (p) and the medium (m) from a co-transfection assay with the plant GnTI-CFP and the YFP-Golgi marker (lane 1 and 2) and a co-transfection with HuGnTI-YFP and HuGnTI-CFP (lane 3 and 4) and used for western blotting with antibodies against GFP. The position of the GnTI fusion proteins (a), the YFP-Golgi marker (b) and the free cleaved YFP/CFP (c) is indicated by the arrows on the left. Molecular weight markers are indicated on the left
© Copyright Policy
Related In: Results  -  Collection

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

Fig7: Western blot analysis of protoplasts from two separate co-transfections. Proteins were extracted from the protoplast (p) and the medium (m) from a co-transfection assay with the plant GnTI-CFP and the YFP-Golgi marker (lane 1 and 2) and a co-transfection with HuGnTI-YFP and HuGnTI-CFP (lane 3 and 4) and used for western blotting with antibodies against GFP. The position of the GnTI fusion proteins (a), the YFP-Golgi marker (b) and the free cleaved YFP/CFP (c) is indicated by the arrows on the left. Molecular weight markers are indicated on the left
Mentions: The fluorescence signal from HuGnTI in protoplasts indicates that, contrary to stably transformed plants, the conditions of the transient expression assay do allow the detection of this protein in plant cells. Indeed, intact HuGnTI fusion protein in protoplasts could now be detected by western blot analysis (Fig. 7, lane 3). Results also show that the ~27 kDa protein band representing free YFP/CFP, is mostly present in the medium fraction, indicating that cleaved YFP/CFP is mostly secreted (Fig. 7, lane 4). In protoplasts transfected with the AtGnTI-CFP and STtmd-YFP, both full length fusion proteins are detected in the protoplast fraction, while free CFP (and/or YFP) was again detected in the medium fraction (Fig. 7, lane 2).Fig. 7

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