Polypeptide N-acetylgalactosaminyltransferase activity in tracheal epithelial microsomes

Abstract

Pig tracheal epithelium, a site of extensive mucin biosynthesis, contained polypeptide N-acetylgalactosaminyltransferase activity directed towards L-threonine residues. The enzyme preparation was broadly similar in properties to preparations from other tissues, e.g. pig and bovine submaxillary glands, bovine colostrum, BW5147 mouse lymphoma and baby-hamster kidney cells. Enzyme was membrane-bound and was released from microsomal preparations by extraction with Triton X-100. Extracted enzyme had a pH optimum of 7.5, had a requirement for Mn2+ (10 mM) and was inhibited by Na2EDTA. The Km for UDP-N-acetylgalactosamine was 110 microM and that for an octapeptide acceptor (VTPRTPPP) was 3.0 mM at 37 degrees C. Using a range of synthetic peptides of known structure related to TPPP it was established that L-threonine residues were specifically O-glycosylated probably in the alpha-configuration. Synthetic peptides containing the TPPP sequence required a peptide length of five or more for significant acceptor activity. In VTPRTPPP the two threonine residues were similarly glycosylated, as revealed by tryptic cleavage of the glycosylated product and separation of the 3H-labelled fragments. The enzyme preparation also specifically catalysed the transfer of N-acetylgalactosaminyl residues from UDP-N-acetyl[1-3H]galactosamine to bovine submaxillary mucin core protein and to myelin basic protein.

Control of Mucin‐TypeO‐Glycosylation:O‐Glycan Occupancy is Directed by Substrate Specificities of Polypeptide GalNAc‐Transferases

“…The length of acceptor peptides is clearly important and kinetic properties generally increase with increased flanking sequences of acceptor sites 145-471. Thr residues placed in the N-terminus can be glycosylated, but with poor kinetics [45,48,491. Detailed studies of GalNAc-transferase isoform activities with overlapping peptides derived from the tandem repeat sequences of M UC 1 have clearly demonstrated the significance of the flanking sequences and the peptide design 146, 471.…”

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

“…The in silico prediction of which sites may become O-glycosylated proved difficult and only rather vague ‘consensus’ sequences for this modification were proposed ( Wilson et al, 1991; O'Connell et al, 1992; Hansen et al, 1995 ); many assays with partially-purified enzymes were also performed in order to define these sequences by in vitro means ( Cottrell et al, 1992; O'Connell et al, 1992 ). In retrospect, it is obvious that such attempts at defining consensus sequences would fail, due to the subsequent isolation of a large number of cDNAs encoding N -acetylgalactosaminyltransferase isoforms from mammalian, insect and nematode sources; in particular, many GalNAc-T isoforms have been identified by, especially, the Tabak, Clausen and Narimatsu laboratories ( Bennett et al, 2012 ).…”