A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues.

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection.

Polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) isozyme surface charge governs charge substrate preferences to modulate mucin type O-glycosylation.

A large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) initiate mucin type O-glycosylation transferring α-GalNAc from a UDP-GalNAc donor to the hydroxyl groups of Ser and Thr residues of peptides and proteins, thereby defining sites of O-glycosylation. Mutations and differential expression of several GalNAc-Ts are associated with many disease states including cancers. The mechanisms by which these isozymes choose their targets and their roles in disease are not fully understood.

ISOGlyP: de novo prediction of isoform-specific mucin-type O-glycosylation.

Mucin-type O-glycosylation is one of the most common posttranslational modifications of proteins. The abnormal expression of various polypeptide GalNAc-transferases (GalNAc-Ts) which initiate and define sites of O-glycosylation are linked to many cancers and other diseases. Current O-glycosyation prediction programs utilize O-glycoproteomics data obtained without regard to the transferase isoform (s) responsible for the glycosylation.

Differential splicing of the lectin domain of an -glycosyltransferase modulates both peptide and glycopeptide preferences.

Mucin-type -glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. -Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in ) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, β, and γ.

Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3.

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHTR↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates.

The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function.

Polypeptide acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type -glycosylation by catalyzing the transfer of -acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive.

Human red and green cone opsins are -glycosylated at an N-terminal Ser/Thr-rich domain conserved in vertebrates.

There are fundamental differences in the structures of outer segments between rod and cone photoreceptor cells in the vertebrate retina. Visual pigments are the only essential membrane proteins that differ between rod and cone outer segments, making it likely that they contribute to these structural differences. Human rhodopsin is glycosylated on Asn and Asn, whereas human (h) red and green cone opsins (hOPSR and hOPSG, respectively) are -glycosylated at Asn Here, utilizing a monoclonal antibody (7G8 mAB), we demonstrate that hOPSR and hOPSG from human retina also are -glycosylated with full occupancy.

Polypeptide GalNAc-Ts: from redundancy to specificity.

Mucin-type O-glycosylation is a post-translational modification (PTM) that is predicted to occur in more than the 80% of the proteins that pass through the Golgi apparatus. This PTM is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) that modify Ser and Thr residues of proteins through the addition of a GalNAc moiety. These enzymes are type II membrane proteins that consist of a Golgi luminal catalytic domain connected by a flexible linker to a ricin type lectin domain.

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

Mucin-type -glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear.

Evidence for GALNT12 as a moderate penetrance gene for colorectal cancer.

Characterizing moderate penetrance susceptibility genes is an emerging frontier in colorectal cancer (CRC) research. GALNT12 is a strong candidate CRC-susceptibility gene given previous linkage and association studies, and inactivating somatic and germline alleles in CRC patients. Previously, we found rare segregating germline GALNT12 variants in a clinic-based cohort (N = 118) with predisposition for CRC.

Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions.

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate-carbohydrate interactions, CCIs) of Tn (Tn-Tn) and STn (STn-STn) provides a model for their possible roles in cellular transformation.

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences.

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs.

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13.

Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins.

Biochemical and functional characterization of glycosylation-associated mutational landscapes in colon cancer.

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC), and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes (B3GNT2, B4GALT2, ST6GALNAC2) involved in the biosynthesis of N- and Cores 1-3 O-linked glycans in the colon, accounting for ~16% of the CRCs tested.

Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.

A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood.

Single molecule study of heterotypic interactions between mucins possessing the Tn cancer antigen.

Mucins are linear, heavily O-glycosylated proteins with physiological roles that include cell signaling, cell adhesion, inflammation, immune response and tumorgenesis. Cancer-associated mucins often differ from normal mucins by presenting truncated carbohydrate chains. Characterization of the binding properties of mucins with truncated carbohydrate side chains could thus prove relevant for understanding their role in cancer mechanisms such as metastasis and recognition by the immune system.

Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.

N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures.

The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation.

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear.

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level.

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen.

O-glycoprotein biosynthesis: site localization by Edman degradation and site prediction based on random peptide substrates.

The characterization of mucin-type O-glycosylation is fraught with extreme difficulty at almost every level of analysis: from difficulties in obtaining glycopeptides suitable for study, their structural heterogeneity, lack of broad acting glycosidase tools capable of simplifying the glycans, and finally the vast complexity of performing analysis on multiply glycosylated glycopeptides. This, along with a lack of known peptide sequence motif(s) for the transferases that initiate mucin-type O-glycosylation, significantly hinders our understanding of mucin-type O-glycosylation at almost every level from their biosynthesis to their biological and biophysical properties. In this chapter, the use of partial chemical deglycosylation coupled with Edman amino acid sequencing is described to quantify sites of O-glycosylation.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree.

The formation of mucin-type O-glycans is initiated by an evolutionarily conserved family of enzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). The human genome encodes 20 transferases; 17 of which have been characterized functionally. The complexity of the GalNAc-T family reflects the differential patterns of expression among the individual enzyme isoforms and the unique substrate specificities which are required to form the dense arrays of glycans that are essential for mucin function.