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

The glycoprotein hormone N-acetylgalactosaminyltransferase is responsible for synthesis of Asn-linked oligosaccharides terminating with GalNAc-4-SO4 on lutropin, thyrotropin, and the uncombined glycoprotein hormone alpha subunit, We previously established that a recognition determinant 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 recognition determinant. Using site-directed mutagenesis we have examined the role of individual amino acids in recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase. Within the sequence Pro(40)-Leu(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, Substitution of the Leu(41) with other amino acids can either increase or decrease the rate of GalNAc transfer over an 8-fold range, suggesting that the middle amino acid of the tripeptide plays a modulatory role in recognition, The critical Leu(41)-Arg(42) and Lys(44)-Lys(45) residues are present on the same surface of an alpha-helix, which projects from the surface of the alpha subunit, Our results indicate that an essential element of the recognition determinant consists of a cluster of basic residues and that neutral but not negatively charged residues are tolerated within this cluster.