A cluster of basic amino acids within an α-helix is essential for α- 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 α subunit. We previously established that a recognition determinant for the N- acetylgalactosaminyltransferase is contained within a 22-amino acid glycopeptide fragment of the α 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 Pro⁴⁰-Leu⁴¹-Arg⁴²-Ser⁴³-Lys⁴⁴-Lys⁴⁵, Lys⁴⁴, and Lys⁴⁵, as well as Arg⁴² of the tripeptide, are essential for recognition. Substitution of the Leu⁴¹ 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 Leu⁴¹-Arg⁴² and Lys⁴⁴-Lys⁴⁵ residues are present on the same surface of an α-helix, which projects from the surface of the α 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.