Regulation of Protein Specific Glycosylation

Abstract

Glycosyltransferases sequentially add individual sugars to growing carbohydrate chains on glycoproteins, in some cases producing unique carbohydrate structures on specific glycoproteins that are important for the biological function of the glycoprotein. Understanding the basis for protein-specific glycosylation and its regulation is pivotal to determining the biologic significance of these complex structures. Carbonic anhydratase VI (CA-VI), like the glycoprotein hormones lutropin (LH) and thyrotropin (TSH), bears Asn-linked carbohydrates that contain β1,4-linked N-acetylgalactosamine (GalNAc). We previously identified key basic amino acids in the glycoprotein hormone a subunit that are recognized by a protein-specific GalNAc-transferase(s) (GalNAcT). We hypothesized that CA-VI also contains a peptide sequence that is utilized by a protein-specific GalNAcT to selectively add GalNAc to it’s Asn-linked carbohydrate chains. We have tested this possibility by expressing epitope tagged forms of CA-VI in cells that express one or more protein-specific GalNAcTs and determined that CA-VI is selectively and efficiently modified with β1,4-linked GalNAc. Deletion mutagenesis identified a cluster of amino acids that is essential for recognition by the GalNAcT. Our results show that like the glycoprotein hormones: 1) CA-VI contains a peptide recognition determinant that is utilized by a protein-specific GalNAcT and 2) This determinant consists of basic amino acids. Supported by R01-DK41738 and the BioMedRap Program.