Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1

Abstract

UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211-->Ala and His344-->Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125-->Ala and His341-->Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.

Structure-Function Analysis of the UDP-N-acetyl-d-galactosamine:PolypeptideN-acetylgalactosaminyltransferase

“…Previously, we had reported that a H125A mutation inactivates recombinant bovine ppGaNTase-T1 (24). With a larger data set of ppGaNTases, it is now clear that this position, albeit highly conserved (found in 10 out of 14 ppGaNTase isoforms), is not invariant.…”

“…The second conserved residue in this motif (H211 for ppGaNTases-T1 and aspartate for most other GT1 family members) is also critical for function. We have previously mutated H211 in bovine ppGaNTase-T1 and demonstrated that a H211A mutant is inactive (24). A common theme among DXD motif-containing proteins is their ability to bind nucleoside diphosphate sugars and coordinate manganese ions (7).…”

“…4C). With the accumulation of a larger sequence data base of ppGaNTase isoforms (1-5), it is now apparent that histidine 341 is not as conserved as previously thought (24). In contrast, histidine 344, is invariant among all known ppGaNTases and is essential for catalytic activity (24).…”

Cloning and Expression of a Novel, Tissue Specifically Expressed Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family

“…This result points out the limitation of using a restricted set of conserved sequences to identify new isoforms; the clones recovered by such methods may be biased against the population of transferases lacking these blocks. When taken together with the results of a site-directed mutagenesis study demonstrating that the histidine residue in this sequence is non-essential (25), it further suggests that this conserved block per se may not play a significant functional role in the transferase activity of these enzymes.…”

Characterization of a Novel Polypeptide N-Acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila

“…32) In addition, it has several Cys residues conserved in the family, most of which are likely involved in forming disulfide bonds necessary for the proper folding of the enzymes. Two Cys residues among them, which are located at the C-terminus of the DXH motif, are also present in this isozyme.…”