Cloning and sequence homology of a rat UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase

Abstract

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) cDNA was amplified from rat sublingual, submandibular and parotid glands, brain, skeletal muscle, and liver, using the polymerase chain reaction (PCR) and sequences derived from bovine polypeptide GalNAc transferase-Type 1 (polypeptide GalNAc transferase-T1). The transcripts encoding the rat sublingual gland and bovine enzymes were 91% identical in nucleotide sequence, except in their 5' and 3' untranslated regions. The enzymes encoded by the rat and bovine cDNAs were 559 amino acids in length and were virtually identical (98% amino acid sequence identity and 99.5% homologous overall). Northern blot analysis indicates that the polypeptide GalNAc transferase-T1 transcripts are expressed in many tissues but at widely differing levels. Although the amino acid sequence of polypeptide GalNAc transferase-T1 is conserved among mammals, the pattern of tissue expression varies between rats and humans. For example, the steady-state level of polypeptide GalNAc transferase-T1 transcript is quite low in lung relative to other rat tissues, whereas high expression of this transcript is detected in human lung. Therefore, we surmise that isoforms of polypeptide GalNAc transferase must exist and that isoforms are expressed in a tissue-dependent fashion. Searches of the GenBank database have revealed homologous sequences for several isoforms derived from several human tissues. In addition, hypothetical proteins from C. elegans also display strong homology; evidence suggests six ancestral isoforms of polypeptide GalNAc transferases may exist in C. elegans.

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

“…GalNAc-T11 is the first isoform exhibiting selectively high levels of expression in another organ, the kidney, and there is no expression in any cell compartment of salivary glands as evaluated by immunohistology. Northern analysis of kidney mRNA indicates that GalNAc-T1, -T2, and -T3 are expressed at low to moderate levels (4,5,44,45) and putative GalNAc-T8 is expressed at high levels (46), whereas GalNAc-T4, -T5, -T6, and -T7 and putative GalNAc-T9 and GalNAc-T10 are not expressed (6 -8, 10 -12, 46, 47). Immunohistological analysis showed weak expression of GalNAc-T1; moderate expression of GalNAc-T2; no expression of GalNAc-T3, -T4, and -T6; and high expression of GalNAc-T11 (Fig.…”

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

“…Recent submissions to the DNA databases include multiple sequences that are highly homologous to bovine polypeptide GalNAc-T among organisms including Caenorhabditis elegans and Homo sapiens. The amino acid sequence identity among multiple human entries is 60% in comparison to the human polypeptide GalNAc-T catalytic region, revealing the presence of at least one highly related gene (27). Moreover, this degree of identity is unusually high among comparisons of glycosyltransferase sequences, implying that such putative proteins will exhibit similar enzymatic activities.…”

Initiation of Protein O Glycosylation by the Polypeptide GalNAcT-1 in Vascular Biology and Humoral Immunity

“…Tissues were extracted and analyzed as described previously (18). Enzyme assays for ppGalNAcT-1 activity were performed at 37°C for 1 to 2 h, using the peptide acceptor PRFQDSSSKAPPPLPSPSRLPG in a final volume of 25 l containing 50 M UDP-GalNAc (77,000 cpm 14 C), 5 mM AMP, 10 mM MnCl 2 , 40 mM cacodylate, pH 6.5, 40 mM ␤-mercaptoethanol, and 0.1% Triton X-100.…”