Enzymatic glycosylation of contulakin-G, a glycopeptide isolated from Conus venom, with a mammalian ppGalNAc-transferase

We have determined that the mammalian uridine diphospho-N-acetyl-D-galactos amine:polypeptide N-acetylgalactosaminyl-transferase T1 (EC 2.3.1.41) has t he appropriate acceptor substrate specificity to recognize the non-glycosyl ated form of contulakin-G (ZSEEGGSNATKKPYIL-OH where Z = pyroglutamic acid) and to transfer GalNAc to the peptide. Both [Thr(10)] contulakin-G and a p re-contulakin-G(30-66) (RGLVPDDITPQLILGSLISRRQSEEGGSNATKKPYIL-OH) were show n to be accepters for the mammalian enzyme. The site of attachment of the G alNAc residue was determined using chemical and radioactive sequencing tech niques. The mammalian enzyme was highly specific for Thr(10) residue, in wh ich the native peptide was found to be glycosylated, compared with either S er(2) or Ser(7). In the case of pre-contulakin-G, the enzyme was also highl y specific for the equivalent threonine residue. These results suggest that the Cone snail uses an enzyme with similar acceptor specificity to that of the mammalian polypeptide N-acetylgalactosaminyltransferase for glycosylat ing contulakin-G. (C) 2001 Elsevier Science Ltd. All rights reserved.