Minimal catalytic domain of N-acetylglucosaminyltransferase V

Abstract

UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain.

Tryptophanyl-tRNA Synthetase Urzyme

“…We referred to the 130-residue TrpRS Urzyme as a "minimal catalytic domain" (19). At the other extreme, a 527-residue fragment of acetylglucosaminyltransferase V (ϳ70% of the full-length enzyme) was also referred to as a minimal catalytic domain (31). We have coined the term "Urzyme" to distinguish active constructs that retain only sufficient mass to position the active-site residues for catalysis.…”

Increase in β1‐6 GlcNAc branching caused by N‐acetylglucosaminyltransferase V directs integrin β1 stability in human hepatocellular carcinoma cell line SMMC‐7721

“…Reportedly, the minimal catalytic domain of GnT-V was existed in its carboxylterminus, and deletion of as few as 4-8 amino acids from its carboxyl-terminus destroys its catalytic activity [Korczak et al, 2000]. Based on the above characteristic, we produced DcGnT-V with deletion of six amino acids at its C-terminal extreme end (pcDNA3-DcGnT-V), and its cellular transfectant was designated as Dc-7721.…”

“…However, a detailed explanation of how GnT-V promotes integrin b1 glycosylation with integrin b1 stability is not currently available. Reportedly, the minimal catalytic domain of GnT-V was existed in its carboxyl-terminus, and deletion of as few as 4-8 amino acids from its carboxyl-terminus destroys its catalytic activity [Korczak et al, 2000]. To investigate in detail the relationship between the catalytic activity of GnT-V and integrin b1, we constructed a GnT-V inactive mutant DcGnT-V with deletion of six amino acids at C-terminal extreme end of the original GnT-V (pcDNA3-DcGnT-V).…”

Structural requirements for Arabidopsisβ1,2‐xylosyltransferase activity and targeting to the Golgi

“…Deletion of the next 51 amino acids in the stem (DN82 form) as well as truncations at the C-terminus of 5 amino acids (DC5) produced inactive proteins. It has been shown for many animal glycosyltransferases that the integrity of the C-terminal end is absolutely required for their activity (Xu et al, 1996;Korczak et al, 2000;de Vries et al, 2001). AtXylT which is inactivated as soon as the first five amino acids at the C-terminus are deleted is the first example of the requirement for the C-terminus for glycosyltransferase enzymatic activity in plants.…”

“…Consistent with the lack of enzyme activity of the DN82, DN106 and DN143 forms, this proline-rich region may be required for XylT activity. The presence of proline-rich domain in the stem of AtXylT could explain why, in contrast with many animal glycosyltransferases that can be deleted of their N-terminal region without any effect on their catalytic activity (Korczak et al, 2000;de Vries et al, 2001), AtXylT is completely inactivated after deletion of 82 amino acids residues at its N-terminal end.…”