Structural and functional studies of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts)

National Institute Of Dental & Craniofacial Research

Investigators

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Abstract

GalNAc-Ts are Golgi-membrane anchored enzymes containing 2 luminal domains that dictate substrate specificity: a catalytic domain and a C-terminal lectin domain consisting of 3 repeats (alpha, beta, gamma) that can potentially recognize and bind extant GalNAcs on glycoprotein or glycopeptide substrates. One of our projects focuses on understanding the role of the luminal domains in substrate binding and recognition. We collaborate with Dr. Kelly Ten Hagens group at NIDCR to study how alternative splicing regulates O-glycosylation. Our recent work shed light on the Drosophila melanogaster GalNAc-T isoenzyme PGANT9, which influences secretory granule morphology in the salivary glands. PGANT9 has 2 splicing isoforms PGANT9A and PGANT9B that differ in the alpha repeat of their lectin domain. PGANT9A-alpha has a net positive charge, while PGANT9B-alpha has a net negative charge. We previously showed that PGANT9B prefers to modify a salivary gland mucin peptide from Sgs3 with a net positive charge, consistent with the overlapping in vivo localization of PGANT9B and Sgs3. In contrast, PGANT9A more efficiently modifies negatively charged peptide substrates. The X-ray crystal structures reveal that the charged repeats of PGANT9A and PGANT9B form a loop that extends towards the active site of each enzyme. We thus proposed that the charged repeats of PGANT9A and PGANT9B dictate substrate specificity by recruiting peptides containing oppositely charged residues or discriminating against peptides with similarly charged residues. We tested our hypothesis and verified that while PGANT9A has high specificity towards peptides with net negative charges, PGANT9B was less specific and glycosylated peptides containing both positive and negative net charges. To understand the differences between the isoforms, we modeled Sgs3 peptides into the PGANT9A/B active sites, which contain a gating loop (catalytic flexible loop) that interacts with and aligns the acceptor peptide in the active site for catalysis. In most isoenzymes, including PGANT9A/B, this loop has a positive net charge. Thus, in PGANT9A, the alpha repeat and the gating loop are both positively charged, explaining its strong preference for negatively charged peptides. In contrast, PGANT9B contains positive charges near the N-terminus of the peptide via the catalytic flexible loop, and negative charges near the C-terminus via the alpha repeat, explaining why it can glycosylate peptides with various charges. Overall, these studies highlight a unique mechanism of GalNAc-T substrate specificity that is regulated by alternative splicing and dictated by charges in both the catalytic and lectin domains. We are currently continuing to collaborate with Dr. Kelly Ten Hagen to understand how alternative splicing influences the function of other isoenyzmes in the family. We study the role of deactivating GALNT12 mutations in patients with colorectal cancer (CRC). The association of GalNAc-T12 with cancer is not clear because its substrates have not been identified. To further our understanding of the downstream effects of aberrant O-glycosylation that arise due to mutations in GALNT12, we collaborate with Dr. Benjamin Schumanns group at Imperial college of London to identify the in vivo substrates of human GalNAc-T12. We are also conducting biochemical studies to follow up on published work and further understand how CRC mutations affect the function of GalNAc-T12. We are also collaborating with Dr. Sergio Hassan (BCBB Structural Biology Group/NIAID) to characterize the function of the lectin domain, and are using structural, biochemical, glycoproteomics, and computational methods to assess the role of the lectin domain repeats in substrate recognition by the isoenzyme GalNAc-T1. Finally, we are studying the role of Mucin-type O-glycosylation in Toxoplasma Gondii pathogenesis, in collaboration with Dr. Louis Weiss, Dr. Stacy Malaker, Dr. Thomas Gerken, and Dr. Yong Sok Lee.