O-GalNAc Glycans

! Dr. Lianchun Wang

Outline

! O-GalNAc Glycans

! Mucin Glycoproteins

! Function of Mucin Glycoproteins

! O-GalNAc Glycan Structures and Tissue Distribution

! Modification of O-GalNAc Glycans

! O-GalNAc Glycan Analysis

! O-GalNAc Glycan Biosynthesis

! Functions of O-GalNAc Glycans

Common Classes of Animal Glycans

• O-glycosylation is a common covalentmodification of serine and threonine residues ofmammalian glycoproteins.

• O-GalNAc glycans are covalently !-linked viaan N-acetylgalactosamine (GalNAc) moiety tothe -OH of serine or threonine by an O-glycosidicbond

• Core structure: !-linked N-GalNAc residuelinked to serine or threonine. The further additionof Gal, GlcNAc or GalNAc defines Cores 1-8.

• O-GalNAc glycans are antigenic

• Modification of O-GalNAc glycans: O-acetylation of sialic acid and O-sulfation ofgalactose and N-acetylglucosamine, leading tostructural heterogeneicity.

•Other O-glycans: !-linked O-fucose, "-linked O-xylose, !-linked O-mannose, "-linked O-GlcNAc,!- or "-linked O-galactose, and !- or "-linked O-glucose glycans

•Shield the epithelial surfaces against physicaland chemical damage and protect againstinfection by pathogens

O-GalNAc Glycans

• O-glycosylation is a common covalentmodification of serine and threonine residues ofmammalian glycoproteins.

• O-GalNAc glycans are covalently !-linked viaan N-acetylgalactosamine (GalNAc) moiety tothe -OH of serine or threonine by an O-glycosidicbond

• Core structure: !-linked N-GalNAc residuelinked to serine or threonine. The further additionof Gal, GlcNAc or GalNAc defines Cores 1-8.

• O-GalNAc glycans are antigenic

• Modification of O-GalNAc glycans: O-acetylation of sialic acid and O-sulfation ofgalactose and N-acetylglucosamine, leading tostructural heterogeneicity.

•Other O-glycans: !-linked O-fucose, "-linked O-xylose, !-linked O-mannose, "-linked O-GlcNAc,!- or "-linked O-galactose, and !- or "-linked O-glucose glycans

•Shield the epithelial surfaces against physicaland chemical damage and protect againstinfection by pathogens

O-GalNAc Glycans

Mucin Glycoproteins• Mucin: Large glycoprotein with a high content

of serine, threonine, and proline residues andnumerous O-GalNAc-linked saccharides, oftenoccurring in clusters on the polypeptide.

• In mucous secretions and as transmembraneglycoproteins.

• Gel-forming mucins: large, mainly produced in the goblet or mucous cells of thetracheobronchial, gastrointestinal, and genitourinary tracts.• In goblet cells, mucins are stored intracellularly in mucin granules and can be quicklysecreted upon external stimuli.• Hallmark: Repeated peptide stretches [Variable number of tandem repeat (VNTR)]

– Rich in serine and/or threoninn– Abundant O-GalNAc glycans– “bottle brush” conformation– Rich in proline that facilitates O-GalNAc glycosylation

• Secreted mucins have cysteine-rich regions and cystine knots that are responsible for theirpolymerization and the formation of extremely large molecules of several million daltons.• Cell-surface mucins contain an extracellular domain with a central VNTR region that carriesO-GalNAc glycan chains, a single transmembrane domain, and a small cytoplasmic tail at thecarboxyl terminus.• About 20 mucin genes, express in tissue-specific fashion and vary in the number andcomposition of the peptide repeats in their VNTR regions. Within the same mucin, the repeatsusually vary in their amino acid sequences.• The expression of mucin genes is regulated by a large number of cytokines and growthfactors, differentiation factors, and bacterial products.

Function of Mucin Glycoproteins

• Viscoelastic properties that contribute to the high viscosity of

mucous secretions

• Hydrophilic and contain charges that attract water and salts

• Trap Bacteria, viruses, and other microbes.

-sometimes specific O-GalNAc glycans serve as receptors

• Mucins regulate signal transduction and cell adhesion

– Immune response: GlyCAM-1, CD34, and PSGL-1

– fertilization, blastocyst implantation

– Abnormal structure with human diseases

O-GalNAc Glycan Structures and Tissue Distribution• Tn antigen: GalNAc!-Ser/Thr.• Core 1 O-GalNAc glycan (T antigen): Gal"1-

3GalNAc-Ser/Thr, found in many glycoproteins andmucins

• Core 2 O-GalNAc glycans: contains a branching N-acetylglucosamine attached to core 1 & found in bothglycoproteins and mucins from a variety of cells and tissues

• Cores 3 and 4 O-GalNAc glycans: found only insecreted mucins of certain mucin-secreting tissues,such as bronchi, colon, and salivary glands

• Core 5-8 O-GalNAc glycans: Extremely restrictedoccurrence

- Core 5 : human meconium and intestinaladenocarcinoma tissue- Core 6 : human intestinal mucin and ovarian cystmucin- Core 7: bovine submaxillary mucin- Core 8: human respiratory mucin

Modification of O-GalNAc Glycans

• Sialylation: All cores.• Only Cores 1-4 & 6 occur as

extended complex O-glycans.• The terminal structures of O-GalNAc

glycans may contain fucose,galactose, N-acetylglucosamine, andsialic acid in !-linkages, N-acetylgalactosamine in both !- and "-linkages, and sulfate.

• Many of these terminal sugarstructures are antigenic or representrecognition sites for lectins.

- the sialylated and sulfated Lewisantigens are ligands for selectins

O-GalNAc Glycan Analysis

• Release– "-elimination: N-acetylgalactosamine to N-acetylgalactosaminitol– N-acetylgalactosaminidase:Unsubstituted N-acetylgalactosamine

residues– O-glycanase: unsubstituted Gal"1-3GalNAc (core 1)

• Purification: gel filtration, anion-exchange chromatography,HPLC.

• Analysis: composition, linkages, and structure.- MS, NMR- Exoglycosidase- Antibody: N-acetylgalactosamine (anti-Tn) and core 1 (anti-T)- Lectin- Prediction based on presence of active glycosyltransferase

O-GalNAc Glycan Biosynthesis:Polypeptide-N-Acetylgalactosaminyltransferases (ppGalNAcT)

O-GalNAc Glycan Biosynthesis:Polypeptide-N-Acetylgalactosaminyltransferases (ppGalNAcT)

• ppGalNAcT transfer N-acetylgalactosamine from UDG-GalNAc to Ser/Thr residues.• > 21 members (ppGalNAT-1 to -21).• Localize in Golgi• Type type II membrane protein, may have a distinct lectin-like domain at the carboxyl

terminus• The subcellular localization of ppGalNAcTs and other glycosyltransferases involved in O-

glycosylation has a critical role in determining the range of O-glycans synthesized by acell

• ppGalNAcT expression levels vary considerably between cell types and mammaliantissues

• All ppGalNAcTs bind UDP-GalNAc (the donor of N-acetylgalactosamine), but they oftendiffer in the protein substrates to which they transfer N-acetylgalactosamine. Suchdifferences allow ppGalNAcTs to be distinguished

• Many ppGalNAcTs appear to have a hierarchical relationship with one another, such thatone enzyme cannot attach an N-acetylgalactosamine until an adjacent serine orthreonine is glycosylated by a different ppGalNAcT

• Defined amino acid sequons that accept N-acetylgalactosamine have not been identified,certain amino acids are preferred in the substrate. Proline residues near the site of N-acetylgalactosamine addition are usually favorable to mucin O-glycosylation, whereascharged amino acids may interfere with ppGalNAcT activity.

O-GalNAc Glycan Biosynthesis: Core 1 and 2

• Initiated by ppGalNAcT transfering thefirst sugar from UDP-GalNAc directly toserine or threonine in a protein, creatingthe Tn antigen.

• Tn antigen is uncommon in normalmucins, but is often found in mucinsderived from tumors.

• Sialyl-Tn antigen: contains a sialic acidresidue linked to C-6 of N-GalNAc of Tn,commonly associated with cancer.

• The subsequent sugar additiondetermines the formation of different O-glycan core structures.

• Core 1: core 1 "1-3 galactosyltransferase (T synthase or C1GalT-1)transfers a Gal from Gal-UDP to from Gal"1-3GalNAc-Ser/Thr (T antigen).

- Present in most cell type- Requires ER protein Cosmc for full activity in Golgi- Sialic acid modification at C-3 of Gal and C6 of N-GalNAc, prohibiting furthermodification- Leukemia and tumor cells contain large number of sialylated core 1 glycan.- Unsubstituted core 1 T antigen seen in cancer and inflammatory bowel disease

O-GalNAc Glycan Biosynthesis: Core 1 and 2

• Core 2: core 2 "1-6 N-acetylglucosaminyltransferase ( C2GnT)transfers a GlcNAc from GlcNAc-UDP tofrom GlcNAc"1-6(Gal "1-3)GalNAc-Ser/Thr

• Produced in many tissues

• Synthesis is regulated duringlymphocyte activation, cytokinestimulation, and embryonic development.

• Altered in leukemia, cancer and otherdiseases

• Synthesis is correlated with tumorprogression

• Block the exposure of mucin peptideepitopes

• C2GnT1-3.- L type (leukocyte type, C2GnT-1 & -3); synthesize only the core 2 structure, activein many cell types and tissues- M type (mucin type, C2GnT-2): Also involved in the synthesis of core 4 and otherGlcNAc "1-6-linked branches, active only in mucin-secreting cell type- The expression and activity of both the L-a nd M enzymes are altered in certaintumors

O-GalNAc Glycan Biosynthesis: Core 3 and 4

• Core 3: core 3 "1-3 N-acetylglucosaminyltransferase(C3GnT) transfers a GlcNAc fromGlcNAc-UDP to from GlcNAc"1-3GalNAc-Ser/Thr .

• Restricted mostly to mucousepithelia from gastrointestinal,respiratory tracts and salivary glands

• The activity of C3GnT is especiallylow in colonic tumors and absent intumor cells in culture.

• Core 4: synthesis by the M-type"1-6 N-acetylglucosaminyltransferase(C2GnT-2) and requires the priorsynthesis of a core 3 O-GalNAcglycan

Synthesis of Complex O-GalNAc Glycans• Elongation

– Elongation of Gal residue of Core 1 and Core 2 by O-GlcNAC specific "1-3 N-acetylglucosaminyltransferase.

– Extended by N-acetylglucosaminyltransferases and galactosyltransferases to form repeatedGlcNAc"1-3Gal"1-4 (poly-N-acetyllactosamine) sequences that represent the little i antigen.

– Linear poly-N-acetyllactosamine units can be branched by members of the "1-6 N-acetylglucosaminyltransferase family, resulting in the large I antigen

– Less common elongation reactions are the formation of GalNAc"1-4GlcNAc- (LacdiNAc) andGal"1-3GlcNAc- sequences

• Terminal structures in mucin– ABO and other glycan-based blood groups– Sialic acid– Fucose– Sulfate

Control of O-GalNAc Glycan Synthesis• The acceptor specificities of glycosyltransferases and sulfotransferases are the mainfactors determining the structures of O-GalNAc glycans found in mucins, and thesespecificities restrict the high number of theoretically possible O-glycans to “only” a fewhundred.

• The specificities also direct the pathways that are feasible

•The relative activities of the glycosyltransferases determine the relative amounts of O-GalNAc glycans in mucin

– C2GnT-1 and !2-3 sialyltransferase both located in cis- and medial-Golgi compartments,and competes for the common core 1 substrate

•The activities of transferases are controlled by many factors such as metal ions andmembrane components

– "1-4 galactosyltransferase 1: !-lactalbumin– "1-3 galactosyltransferase: coexpression of chaperone Cosmc.

• The first step of O-GalNAc glycosylation is clearly regulated by the amino acidsequence of the acceptor substrate.

• Intracellular localization

• Donor substrate

Functions of O-GalNAc Glycans

• Essential to hydrate and protect the underlying epithelium.

•Physical barrier between lumen and epithelium, protection from protease degradation

• Trap and remove bacteria, virus and other pathogens

• Serve as “decoys,” masking underlying antigens or receptors

• Affect the conformation of the attached protein

• O-glycans of cell-surface receptors regulate receptor stability and expression levels

• O-glycans altered during during lymphocyte activation and in leukemia

• Sialyl Lewisx epitopes attached to core 2 O-GalNAc glycans interacts with selectinsto mediate leukocyte trafficking.

• Sialyl Lewisx-selectin is involved in tumor metastasis

• Fertilization