Literature summary for 2.4.1.144 extracted from

Karg, S.R.; Frey, A.D.; Kallio, P.T.
Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression (2010), J. Biotechnol., 146, 54-65.

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Cloned(Commentary)

Cloned (Comment)	Organism
construction of genes for expression of native rat GnTIII, human ManII, Arabidopsis thaliana ManII and for the expression of the catalytic domains of rat GnTIII and human ManII fused to the N-terminal region of Arabidopsis thaliana ManII. Binary plasmids for high-level expression of rat GnTIII (pAX67), chimeric rat GnTIIIA.th. (replaced native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II) (pAX70), co-expression of rat GnTIII together with human ManII (pAX73) or Arabidopsis thaliana ManII (pAX100), co-expression of GnTIIIA.th. together with ManIIA.th. (pAX74), and co-expression of GnTIIIA.th. and Arabidopsis thaliana ManII (pAX101). Expression of the transgenes is driven by a chimeric promoter assembled from regulatory elements from mannopine and octopine synthase genes. Expression cassettes comprising the CaMV 35S promoter, GnTIII or ManII and the nos pA, being pSK103 (ManII), pSK124 (ManII-Arabidopsis thaliana), pAX63 (GnTIII-Arabidopsis thaliana) and pAX68 (GnTIII) serve as starting point for further expression constructs. The cassette comprising the CaMV 35 promoter, plant relocalized GnTIII and polyadenylation signal is excised from pAX68 using SbfI, EcoRI and inserted into pAX36 generating pAX90. The construct comprising the rat GnTIII obtained by replacing the XbaI, AflII fragment from pAX90 with the fragment isolated from pAX63, generating pAX91. Mutated fragment from pCLF40 inserted into pAX69 using AvaI, StuI, generating pAX104. The binary expression plasmids for inactive GnTIII and GnTIIIA.th. under the control of the UAS123mas promoter generated by replacing the StuI, EcoRI fragment from pAX104 with that in pAX67 (generating pAX105) and pAX70 (generating pAX106), respectively. The mutated fragment from pCLF40 inserted into pAX91 using Eam1105I, StuI, generating the expression plasmid for inactive GnTIII under the control of the CaMV 35S promoter (pAX108). The exchange of the fragment containing the Arabidopsis thaliana Golgi localization domain from pAX90 in pAX108 using SbfI, StuI yields an expression plasmid for inactive GnTIIIA.th. under the control of the CaMV 35S promoter. Plasmids transferred into Agrobacterium tumefaciens LBA4404 for transformation into tobacco BY-2 cells	Rattus norvegicus

Cloned (Comment)

Organism

construction of genes for expression of native rat GnTIII, human ManII, Arabidopsis thaliana ManII and for the expression of the catalytic domains of rat GnTIII and human ManII fused to the N-terminal region of Arabidopsis thaliana ManII. Binary plasmids for high-level expression of rat GnTIII (pAX67), chimeric rat GnTIIIA.th. (replaced native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II) (pAX70), co-expression of rat GnTIII together with human ManII (pAX73) or Arabidopsis thaliana ManII (pAX100), co-expression of GnTIIIA.th. together with ManIIA.th. (pAX74), and co-expression of GnTIIIA.th. and Arabidopsis thaliana ManII (pAX101). Expression of the transgenes is driven by a chimeric promoter assembled from regulatory elements from mannopine and octopine synthase genes. Expression cassettes comprising the CaMV 35S promoter, GnTIII or ManII and the nos pA, being pSK103 (ManII), pSK124 (ManII-Arabidopsis thaliana), pAX63 (GnTIII-Arabidopsis thaliana) and pAX68 (GnTIII) serve as starting point for further expression constructs. The cassette comprising the CaMV 35 promoter, plant relocalized GnTIII and polyadenylation signal is excised from pAX68 using SbfI, EcoRI and inserted into pAX36 generating pAX90. The construct comprising the rat GnTIII obtained by replacing the XbaI, AflII fragment from pAX90 with the fragment isolated from pAX63, generating pAX91. Mutated fragment from pCLF40 inserted into pAX69 using AvaI, StuI, generating pAX104. The binary expression plasmids for inactive GnTIII and GnTIIIA.th. under the control of the UAS123mas promoter generated by replacing the StuI, EcoRI fragment from pAX104 with that in pAX67 (generating pAX105) and pAX70 (generating pAX106), respectively. The mutated fragment from pCLF40 inserted into pAX91 using Eam1105I, StuI, generating the expression plasmid for inactive GnTIII under the control of the CaMV 35S promoter (pAX108). The exchange of the fragment containing the Arabidopsis thaliana Golgi localization domain from pAX90 in pAX108 using SbfI, StuI yields an expression plasmid for inactive GnTIIIA.th. under the control of the CaMV 35S promoter. Plasmids transferred into Agrobacterium tumefaciens LBA4404 for transformation into tobacco BY-2 cells

Rattus norvegicus

Organism

Organism	UniProt	Comment	Textmining
Rattus norvegicus	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	expression of GnTIII leads to a high proportion of N-glycans of the complex type, modified with the bisecting GlcNAc (median value 37%, q1, 33%, q3, 39%) and a strong reduction of paucimannosidic type N-glycans (median value 2%, q1, 1%, q3.2%) that are abundant in secreted proteins of tobacco BY-2 cells. Native GnTIII expression, the coexpression of Arabidopsis thaliana ManII leads to an increase in the proportion of complex bisected N-glycan structures, whereas the expression of native human ManII does not significantly increase these structures. In the case of chimeric GnTIIIA.th. and chimeric ManIIA.th. expression, reduction in hybrid bisected structures. No viable cultures expressing GnTIIIA.th. and A. thaliana ManII. Plant-specific core fucosylation and xylosylation can be greatly reduced, with up to 59% of N-glycans on endogenous secreted protein lacking the plant-specific modifications	Rattus norvegicus	?	-	?

Synonyms

Synonyms	Comment	Organism
beta1,4-N-acetylglucosaminyltransferase III	-	Rattus norvegicus
GnT-III	-	Rattus norvegicus

General Information

General Information	Comment	Organism
physiological function	levels of fucosylation (79%, median value 80%, q1, 78%, q3, 82%) and xylosylation (94, median value 94%, q1, 93%, q3, 95%) are not significantly reduced in cells expressing GnTIII under the control of the CaMV 35S promoter compared to the levels observed in wild-type cells. Expression of GnTIII under the control of the UAS123mas promoter reduces fucosylation, but not xylosylation	Rattus norvegicus