Download O-GalNAc Glycans

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-GalNAc Glycans
• O-glycosylation is a common covalent
modification of serine and threonine residues of
mammalian glycoproteins.
• O-GalNAc glycans are covalently !-linked via
an N-acetylgalactosamine (GalNAc) moiety to
the -OH of serine or threonine by an O-glycosidic
bond
• Core structure: !-linked N-GalNAc residue
linked to serine or threonine. The further addition
of Gal, GlcNAc or GalNAc defines Cores 1-8.
• O-GalNAc glycans are antigenic
• Modification of O-GalNAc glycans: Oacetylation of sialic acid and O-sulfation of
galactose and N-acetylglucosamine, leading to
structural heterogeneicity.
•Other O-glycans: !-linked O-fucose, "-linked Oxylose, !-linked O-mannose, "-linked O-GlcNAc,
!- or "-linked O-galactose, and !- or "-linked Oglucose glycans
•Shield the epithelial surfaces against physical
and chemical damage and protect against
infection by pathogens
O-GalNAc Glycans
• O-glycosylation is a common covalent
modification of serine and threonine residues of
mammalian glycoproteins.
• O-GalNAc glycans are covalently !-linked via
an N-acetylgalactosamine (GalNAc) moiety to
the -OH of serine or threonine by an O-glycosidic
bond
• Core structure: !-linked N-GalNAc residue
linked to serine or threonine. The further addition
of Gal, GlcNAc or GalNAc defines Cores 1-8.
• O-GalNAc glycans are antigenic
• Modification of O-GalNAc glycans: Oacetylation of sialic acid and O-sulfation of
galactose and N-acetylglucosamine, leading to
structural heterogeneicity.
•Other O-glycans: !-linked O-fucose, "-linked Oxylose, !-linked O-mannose, "-linked O-GlcNAc,
!- or "-linked O-galactose, and !- or "-linked Oglucose glycans
•Shield the epithelial surfaces against physical
and chemical damage and protect against
infection by pathogens
Mucin Glycoproteins
•
Mucin: Large glycoprotein with a high content
of serine, threonine, and proline residues and
numerous O-GalNAc-linked saccharides, often
occurring in clusters on the polypeptide.
•
In mucous secretions and as transmembrane
glycoproteins.
• Gel-forming mucins: large, mainly produced in the goblet or mucous cells of the
tracheobronchial, gastrointestinal, and genitourinary tracts.
• In goblet cells, mucins are stored intracellularly in mucin granules and can be quickly
secreted 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 their
polymerization and the formation of extremely large molecules of several million daltons.
• Cell-surface mucins contain an extracellular domain with a central VNTR region that carries
O-GalNAc glycan chains, a single transmembrane domain, and a small cytoplasmic tail at the
carboxyl terminus.
• About 20 mucin genes, express in tissue-specific fashion and vary in the number and
composition of the peptide repeats in their VNTR regions. Within the same mucin, the repeats
usually vary in their amino acid sequences.
• The expression of mucin genes is regulated by a large number of cytokines and growth
factors, 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"13GalNAc-Ser/Thr, found in many glycoproteins and
mucins
•
Core 2 O-GalNAc glycans: contains a branching Nacetylglucosamine attached to core 1 & found in both
glycoproteins and mucins from a variety of cells and tissues
•
•
Cores 3 and 4 O-GalNAc glycans: found only in
secreted mucins of certain mucin-secreting tissues,
such as bronchi, colon, and salivary glands
Core 5-8 O-GalNAc glycans: Extremely restricted
occurrence
- Core 5 : human meconium and intestinal
adenocarcinoma tissue
- Core 6 : human intestinal mucin and ovarian cyst
mucin
- 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, and
sialic acid in !-linkages, Nacetylgalactosamine in both !- and "linkages, and sulfate.
Many of these terminal sugar
structures are antigenic or represent
recognition sites for lectins.
- the sialylated and sulfated Lewis
antigens 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 Oglycosylation has a critical role in determining the range of O-glycans synthesized by a
cell
ppGalNAcT expression levels vary considerably between cell types and mammalian
tissues
•
All ppGalNAcTs bind UDP-GalNAc (the donor of N-acetylgalactosamine), but they often
differ in the protein substrates to which they transfer N-acetylgalactosamine. Such
differences allow ppGalNAcTs to be distinguished
•
Many ppGalNAcTs appear to have a hierarchical relationship with one another, such that
one enzyme cannot attach an N-acetylgalactosamine until an adjacent serine or
threonine 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 Nacetylgalactosamine addition are usually favorable to mucin O-glycosylation, whereas
charged amino acids may interfere with ppGalNAcT activity.
O-GalNAc Glycan Biosynthesis: Core 1 and 2
• Initiated by ppGalNAcT transfering the
first sugar from UDP-GalNAc directly to
serine or threonine in a protein, creating
the Tn antigen.
• Tn antigen is uncommon in normal
mucins, but is often found in mucins
derived from tumors.
• Sialyl-Tn antigen: contains a sialic acid
residue linked to C-6 of N-GalNAc of Tn,
commonly associated with cancer.
• The subsequent sugar addition
determines the formation of different Oglycan 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 further
modification
- 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 Nacetylglucosaminyltransferase ( C2GnT)
transfers a GlcNAc from GlcNAc-UDP to
from GlcNAc"1-6(Gal "1-3)GalNAcSer/Thr
• Produced in many tissues
• Synthesis is regulated during
lymphocyte activation, cytokine
stimulation, and embryonic development.
• Altered in leukemia, cancer and other
diseases
• Synthesis is correlated with tumor
progression
• Block the exposure of mucin peptide
epitopes
• C2GnT1-3.
- L type (leukocyte type, C2GnT-1 & -3); synthesize only the core 2 structure, active
in many cell types and tissues
- M type (mucin type, C2GnT-2): Also involved in the synthesis of core 4 and other
GlcNAc "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 certain
tumors
O-GalNAc Glycan Biosynthesis: Core 3 and 4
• Core 3: core 3 "1-3 Nacetylglucosaminyltransferase
(C3GnT) transfers a GlcNAc from
GlcNAc-UDP to from GlcNAc"13GalNAc-Ser/Thr .
• Restricted mostly to mucous
epithelia from gastrointestinal,
respiratory tracts and salivary glands
• The activity of C3GnT is especially
low in colonic tumors and absent in
tumor cells in culture.
• Core 4: synthesis by the M-type
"1-6 Nacetylglucosaminyltransferase
(C2GnT-2) and requires the prior
synthesis of a core 3 O-GalNAc
glycan
Synthesis of Complex O-GalNAc Glycans
• Elongation
–
Elongation of Gal residue of Core 1 and Core 2 by O-GlcNAC specific "1-3 Nacetylglucosaminyltransferase.
–
Extended by N-acetylglucosaminyltransferases and galactosyltransferases to form repeated
GlcNAc"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 Nacetylglucosaminyltransferase family, resulting in the large I antigen
–
Less common elongation reactions are the formation of GalNAc"1-4GlcNAc- (LacdiNAc) and
Gal"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 main
factors determining the structures of O-GalNAc glycans found in mucins, and these
specificities restrict the high number of theoretically possible O-glycans to “only” a few
hundred.
• The specificities also direct the pathways that are feasible
•The relative activities of the glycosyltransferases determine the relative amounts of OGalNAc 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 and
membrane 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 acid
sequence 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 selectins
to mediate leukocyte trafficking.
• Sialyl Lewisx-selectin is involved in tumor metastasis
• Fertilization