A CLUSTER OF BASIC-AMINO-ACIDS WITHIN AN ALPHA-HELIX IS ESSENTIAL FORA-SUBUNIT RECOGNITION BY THE GLYCOPROTEIN HORMONE N-ACETYLGALACTOSAMINYLTRANSFERASE

The glycoprotein hormone N-acetylgalactosaminyltransferase is responsi ble for synthesis of Asn-linked oligosaccharides terminating with GalN Ac-4-SO4 on lutropin, thyrotropin, and the uncombined glycoprotein hor mone alpha subunit, We previously established that a recognition deter minant for the N-acetylgalactosaminyltransferase is contained within a 22-amino acid glycopeptide fragment of the alpha subunit, We proposed that the tripeptide Pro-Leu-Arg is an essential element of the recogn ition determinant. Using site-directed mutagenesis we have examined th e role of individual amino acids in recognition by the glycoprotein ho rmone N-acetylgalactosaminyltransferase. Within the sequence Pro(40)-L eu(41)-Arg(42)-Ser(43)-Lys(44)-Lys(45), Lys(44), and Lys(45), as well as Arg(42) of the tripeptide, are essential for recognition, Substitut ion of the Leu(41) with other amino acids can either increase or decre ase the rate of GalNAc transfer over an 8-fold range, suggesting that the middle amino acid of the tripeptide plays a modulatory role in rec ognition, The critical Leu(41)-Arg(42) and Lys(44)-Lys(45) residues ar e present on the same surface of an alpha-helix, which projects from t he surface of the alpha subunit, Our results indicate that an essentia l element of the recognition determinant consists of a cluster of basi c residues and that neutral but not negatively charged residues are to lerated within this cluster.