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2.4.1.143: alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

This is an abbreviated version!
For detailed information about alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, go to the full flat file.

Word Map on EC 2.4.1.143

Reaction

UDP-N-acetyl-alpha-D-glucosamine
+
beta-D-GlcNAc-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-beta-D-GlcNAc-N-Asn-[protein]
=
UDP
 +
beta-D-GlcNAc-(1->2)-alpha-D-Man-(1->3)-[beta-D-GlcNAc-(1->2)-alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-beta-D-GlcNAc-N-Asn-[protein]

Synonyms

2-beta-N-acetylglucosaminyltransferase, acetylglucosaminyltransferase II, alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein beta-1-2-N-acetylglucosaminyltransferase, beta-1,2-N-acetylglucosaminyltransferase II, BmGnTII, EC 2.4.1.51, GlcNAc-T II, GlcNAcT-II, GlcNAcTase-II, GnT II, Gnt II-A, Gnt II-B, GnT-II, GnTII, Mgat2, N-acetylglucosaminyltransferase II, N-acetylglucosaminyltransferase-II, N-acetylglucosaminyltransferases II, N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase II, TbGnTII, TbGT15, TTV/DEXT1, UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II, UDP-GlcNAc:mannoside alpha1-6 acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-alpha-1,6-mannosylglycoprotein beta-1-2-N-acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-alpha-D-mannoside beta1-2-acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mannoside alpha1->6-acetylglucosaminyltransferase

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.1 Hexosyltransferases
                2.4.1.143 alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

Cloned

Cloned on EC 2.4.1.143 - alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
a transformed insect cell line (Tn5beta4GalT) constitutively expresses a mammalian beta-1,4-galactosyltransferase and human beta-1,2-N-acetylglucosaminyltransferase II under the control of an immediate-early promoter. The baculovirus-host system can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans
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construction of a Gnt II-expressing alg3 mutant strain of Pichia pastoris. Engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly
construction of a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system, and functional expression of His-FLAG-tagged human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The enzyme is secreted. The enzyme expressed in silkworm is glycosylated, the structure of N-glycans in the recombinant hGnTII is suggested to be of the complex type, pauci-mannosidic-type glycans, and the removal of the glycans does not affect the enzymatic activity
DNA and amino acid sequence determination and analysis, TbGT15 is present as a single copy per haploid genome, recombinant overexpression of full-length TbGT15 with a C-terminal 3x HA epitope tag, semi-quantitative RT-PCR enzyme expression analysis
expressed in Sf9 insect cells
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expressed in Spodoptera frugiperda and Trichoplusia ni cells by using a baculovirus expression vector
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expression as fusion protein vHis-GlcNAc-TII in Spodoptera frugiperda Sf-9 cells
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expression in Escherichia coli
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expression in Spodoptera frugiperda Sf-9 cells
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gene GnTII, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic tree, functional expression of the enzyme using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system, the enzyme is secreted. The recombinant enzyme exhibits similar pH and temperature dependency and the same substrate specificity as human GnTII expressed by the same system, but deglycosylation with peptide:N-glycanase F does not affect its enzymatic activity. Construction of a vector including the N-terminally FLAG (DYKDDDDK)-tagged lumenal region (Leu27eAla498) including the stem domain and the catalytic domain of BmGnTII together with a signal peptide sequence from bombyxin and transformed into Escherichia coli BmDH10Bac-CP-/Chi- competent cells, which contain the cysteine protease- and chitinase-deficient BmNPV bacmid. RT-PCR expression analysis. The cDNA templates are synthesized from total RNAs extracted from whole bodies of first to fifth-instar larvae and pupa and tissues of fifth-instar larvae. Tissues used are as follows: fat body, midgut, silk gland, epidermis, and Malpighian tubule. To produce the soluble form of BmGnTII n hemolymph, chitosan/BmNPV bacmid nanocomplexes are prepared and then injected into fifth-instar silkworm larvae. The bacmid-injected larvae are reared on an artificial diet
heterologous expression in insect cells
transient expression in COS-7 cells, linked in frame to a cDNA segment encoding the cleavable signal sequence of the human IL-2 receptor
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transient recombinant expression of wild-type and mutant His-tagged MGAT2s' catalytic domain (residues 29-447) in HEK293S (GnTI-) or HEK293F (wild-type) cells. The fusion protein construct encodes an NH2-terminal signal sequence, His8-tag, AviTag, superfolder GFP, the TEV protease recognition site, and the truncated MGAT2 coding region behind a CMV promoter