Table of Contents Information . . . . . . . . . . . . . . . . . . . . . . . . . 1
Deglycosylation Strategies . . . . . . . . . . . 69
General Information . . . . . . . . . . . . . . . . . . . . 2
Chemical Deglycosylation . . . . . . . . . . . . . . . 69
Basic Monosaccharides. . . . . . . . . . . . . . . . . . . 4
Enzymatic Deglycosylation . . . . . . . . . . . . . . 72
Glycan Component Classes . . . . . . . . . . . . 7
Glycan Metabolism. . . . . . . . . . . . . . . . . . 84
N-Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Carbohydrate-active Enzymes. . . . . . . . . 85
O-Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 GPI Anchored Glycoproteins . . . . . . . . . . . . . 20
Glycan Sequencing Using Exoglycosidases . . . . . . . . . . . . . . . . 85
Glycosphingolipids . . . . . . . . . . . . . . . . . . . . . 21
Endoglycosidases . . . . . . . . . . . . . . . . . . . . . . 87
Glycosaminoglycans and Proteoglycans . . . . 23
Glycosyltransferases . . . . . . . . . . . . . . . . . . . . 87
Bacterial Components . . . . . . . . . . . . . . . 27
Product Directory . . . . . . . . . . . . . . . . . . . 90
Lipopolysaccharides . . . . . . . . . . . . . . . . . . . . 27
Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Peptidoglycans . . . . . . . . . . . . . . . . . . . . . . . . 30
Enzyme Inhibitors. . . . . . . . . . . . . . . . . . . . . 101
Glycan Labeling and Analysis . . . . . . . . . 32 Glycan Labeling . . . . . . . . . . . . . . . . . . . . . . . 32 HPLC Analysis of Glycans . . . . . . . . . . . . . . . . 39 Mass Spectrometry of Glycans. . . . . . . . . . . . 42 NMR Analysis of Glycans . . . . . . . . . . . . . . . . 49 Electrophoresis of Glycans . . . . . . . . . . . . . . . 49
Enzyme Detection Substrates . . . . . . . . . . . 103 Monosaccharides . . . . . . . . . . . . . . . . . . . . . 107 Disaccharides. . . . . . . . . . . . . . . . . . . . . . . . . 108 Carbohydrate Metabolites and Cofactors . . . . . . . . . . . . . . . . . . . . . 109 Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . 110
Enzymatic Glycan Analysis . . . . . . . . . . . . . . . 50
Polyacrylamide (PAA)-based Glycoconjugates . . . . . . . . . . . . . . . . . . . 113
Kits for Carbohydrate Analysis . . . . . . . . . . . 50
Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Glycan Recognizing Proteins . . . . . . . . . . 51
Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . 115
Galectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Neoglycoproteins . . . . . . . . . . . . . . . . . . . . . 117
Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Software and Books . . . . . . . . . . . . . . . . . . . 118
Glycoprotein Purification . . . . . . . . . . . . . 61
Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . 120
m-Aminophenylboronic Acid Matrices . . . . . 61
Indexes . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Lectin Matrices . . . . . . . . . . . . . . . . . . . . . . . . 61
Risk & Safety . . . . . . . . . . . . . . . . . . . . . . . . . 123
Glycoprotein Detection . . . . . . . . . . . . . . 63
Trademarks . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Fluorescent Detection on SDS-PAGE Gels . . . . . . . . . . . . . . . . . . . 63 Detection of Biotin Labeled Glycoproteins on Western Blots . . . . . . . . 65 Colorimetric Detection on PAGE and Western Blots . . . . . . . . . . . . . . . . . . . 67 Glycoprotein Standards . . . . . . . . . . . . . . . . . 68
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General Information
Opening a Sigma-Aldrich Account
Pricing
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General Information
Return Policy Our Customer Service Department is available to assist you should a problem arise with your order. Please inspect your packages immediately upon receipt and notify us promptly of any damage or discrepancies. Should an item be shipped to you incorrectly, as the result of an error on our part, we will take quick and appropriate action to correct the problem. Prior to returning any items, please contact the Customer Service Department to obtain a Return Material Authorization (RMA) and shipping instructions. A return authorization will ensure the safe and proper handling of material and enable us to expedite a resolution. Items returned without prior authorization may not be accepted. Shipment of authorized returns should be made within 30 days of the issuance of the RMA.
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Basic Monosaccharides Monosaccharides, also known as simple sugars, are the core building blocks used to assemble complex glycans. Monosaccharides can be classified according to the number of carbon atoms they contain (trioses, tetroses, pentoses, hexoses, and heptoses). The sugars are further classified by specific functional groups or sugar modifications that they contain, including loss of hydroxyl groups (deoxy, dideoxy) and substitution (amino sugars, sugar alcohols, sugar acids, ketoses, lactones). In addition to functional substitution, the chirality of the monosaccharide is of fundamental importance. With some exceptions, the D-enantiomers primarily occur in nature.
Key to Monosaccharide Symbols, Abbreviations, and Projections The following table shows abbreviations, structure projections using 3-D Chair, Haworth, and Fischer images, and accepted symbols of the monosaccharides and variants most commonly found in glycobiology. These symbols are used for subsequent structure images in this manual.
Symbols, Structure Projections, and Abbreviations for Monosaccharides Monosaccharide
3-D Chair projection
Haworth projection
CH2OH
OH
O
OH
O HO
OH OH
HO
OH
OH
Fischer projection
HO
H
H
OH
HO
H
H
OH
CH2OH
OH
CH2OH HO
O
O
HO OH
HO
OH
OH
OH
OH
HO
H
HO
H
HO
H
H
OH
CH2OH
OH
OH
O
OH
O
OH OH
HO
OH
HO
H
H
OH
HO
H
HO
H
O
Gal
H
OH
CH2OH
β-D-Galactose (Gal)
HO
CH2OH
OH
O
OH
O HO
OH OH
HO
OH
NHAc
H
H
NHAc
HO
H
H
OH
O
GlcNAc
H
NHAc
β-D-N-Acetylglucosamine (GlcNAc)
CH2OH
HO
CH2OH
OH
OH
HO
O
O
OH HO
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Man
CH2OH
HO
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O
H
β-D-Mannose (Man)
4
Glc
H
OH
β-D-Glucose (Glc)
β-D-N-Acetylgalactosamine (GalNAc)
O
Symbol
OH NHAc
NHAc
OH
H
H
NHAc
HO
H
HO
H
O
GalNAc
H CH2OH
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Basic Monosaccharides Symbols, Structure Projections, and Abbreviations for Monosaccharides Monosaccharide
3-D Chair projection
Haworth projection
Fischer projection HO
H
H
OH
HO
H
H
OH
OH
O O HO
OH OH
HO
OH
OH OH
O
Xyl
H
β-D-Xylose (Xyl)
H
HO
HO
CH2OH
OH CO2H
AcHN HO
O
AcHN
OH OH OH
OH
CO2H
H
H
H
OH
O O
CO2H
AcHN
NeuNAc
H H
OH
HO
H
OH
OH
H
α-N-Acetylneuraminic acid Sialic Acid (NeuNAc)
OH CH2OH
CO2H CO2H
O
O
OH
H
H
OH
HO
H
H
OH
O
OH
OH
HO
HO OH
HO
OH
GlcA
H
OH
CO2H
β-D-Glucuronic acid (GlcA) HO O
O
CO2H OH
HO OH
HO HO2C
OH
H
H
OH
HO
H
H
OH
O
OH
OH OH
H
O OH H3C HO HO
CH3
O OH
OH
HO
H
HO
H
H
OH
O
OH OH
H
OH
OH
Fuc
H CH3
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IdoA
CO2H
α-L-Iduronic acid (IdoA)
α-L-Fucose (Fuc)
Symbol
5
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GLY FINAL 1207 PGD cleanxrefs2
N-Glycans
Glycan Component Classes N-linked glycosylation, modification, and degradation are involved in a wide variety of processes in all organisms from archaea to eukaryotes. It is the most common covalent protein modification in eukaryotic cells. No other post-translational protein modification is as chemically complex or serves as many diverse functions.
Structures
α1,6
All N-linked glycans are based on the common core pentasaccharide, Man3GlcNAc2 (see Figure 1). Further processing in the Golgi results in three main classes of N-linked glycan classes:
α1,6
α1,3
• High-mannose • Hybrid • Complex
β1,4
β1,4
β1,4
β1,4
β1,4
Glycan Component Classes
N-Glycans
Hybrid glycans are characterized as containing both unsubstituted terminal mannose residues (as are present in high-mannose glycans) and substituted mannose residues with an N-acetylglucosamine linkage (as are present in complex glycans) (see Figure 3). These GlcNAc sequences added to the N-linked glycan core in hybrid and complex N-glycans are called “antennae”. The uppermost structure shown in Figure 3 is a biantennary glycan with two GlcNAc branches linked to the core. The lower structure is a triantennary glycan with three GlcNAc branches.
Asn
α1,3 β1,2
α1,6
β1,4
Asn
β1,4
α1,6 α1,6
α1,3
α1,3 α2,6
β1,4
β1,4
Figure 1. Core structure for all N-linked glycans.
β1,4
High-mannose glycans contain unsubstituted terminal mannose sugars (see Figure 2). These glycans typically contain between five and nine mannose residues attached to the chitobiose (GlcNAc2) core. The name abbreviations are indicative of the total number of mannose residues in the structure.
β1,4
Asn
α1,3
β1,2
Figure 3. Examples of biantennary (top) and triantennary (bottom) hybrid glycans.
α1,6
α1,3
α1,6
Asn β1,4
Key to Monosaccharide Symbols
α1,3
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
α1,6
α1,2 α1,3
α1,6
α1,2 Asn β1,4
β1,4
α1,3 α1,2
α1,2
Figure 2. Examples of high mannose glycans Man-5 (top) and Man-9 (bottom).
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GLY FINAL 1207 PGD cleanxrefs2
tetraantennary forms and make up the majority of cell surface and secreted N-glycans. Complex glycans commonly terminate with sialic acid residues. Additional modifications such as the addition of a bisecting GlcNAc at the mannosyl core (as indicated in Figure 4) and/or a fucosyl residue on the innermost GlcNAc are also possible.
Complex N-linked glycans differ from the high-mannose and hybrid glycans by having added GlcNAc residues at both the α-3 and α-6 mannose sites (see Figure 4). Unlike the high-mannose glycans, complex glycans do not contain mannose residues apart from the core structure. Additional monosaccharides may occur in repeating lactosamine (GlcNAc-β(1→4)Gal) units. Complex glycans exist in bi-, tri- and Sialic Acid Terminus
Lactosamine
α2,3
β1,4
α2,6
β1,4
β1
,4
,2
β1
α1,6
β1,4
β1,4
Asn
β1,4
α1
,3
β1,4
,6
α2,6
Man3GlcNAc2Core
α1
Glycan Component Classes
N-Glycans
β1
,4
β1,4
α2,3
β1
,2
Figure 4. Example of tetraantennary complex glycan that contains terminal sialic acid residues, a bisecting GlcNAc on the pentasaccharide core, and fucosylation on the core GlcNAc.
Biosynthesis and Degradation Protein glycosylation of N-linked glycans is actually a co-translational event, occurring during protein synthesis. N-linked glycosylation occurs at the consensus sequence Asn-X-Ser/Thr, where the glycan attaches to the amine group of asparagine and X represents any amino acid except proline. Glycosylation occurs most often when this consensus sequence occurs in a loop within the peptide. Oligosaccharide intermediates destined for protein incorporation are synthesized by a series of transferases on the cytoplasmic side of the endoplasmic recticulum (ER) while linked to a dolichol phosphate (P-Dol) base. Following the addition of mannose and N-acetyl-D-glucosamine molecules, typically using GDP-Man and UDP-GlcNAc as glycan donors, the final construct prior to movement from the cytoplasmic side of the ER is Man5GlcNAc2-P-Dol (see Figure 5).
This dolichol precursor with its attached glycan is shifted to the lumen of the ER (“flipped”) for further enzymatic modification. Processing includes trimming of glucose and mannose residues by glycosidases and addition of new residues via glycosyltransferases in the ER and, to a great extent, in the Golgi. In the Golgi, high-mannose N-glycans can be converted to a variety of complex and hybrid forms that are unique to vertebrates. The completed oligosaccharide is then transferred from the dolichol precursor to the Asn of the target protein by oligosaccharyltransferase (OST).
Lumen
Cytosol A
B
C
D
Figure 5. General process of N-linked glycan construction in the ER. The glycan precursor is assembled on a dolichol phosphate base while in the cytoplasm of the ER (A). Once the glycan is switched to the lumen side (B), the glycan has additional sugars added and removed (trimmed) before being attached to the protein (C), where additional processing takes place (D).
Cleaved high-mannose glycans serve as substrates in the Golgi where additional modification and diversification occurs. High-mannose structures are trimmed by the action of mannosidases, removing the mannose extensions and making the glycan available for conversion to hybrid and complex glycans by subsequent addition of GlcNAc sugars (“antennae”) by the actions of N-acetylglucosyl transferases. The mammalian gene Mgat1 codes for the enzyme N-acetylglucosyl transferase-I (GlcNAcT-I), which is responsible for the addition of one GlcNAc in the β(1→2) linkage. The subsequently modified glycan 8
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becomes a substrate for α-mannosidase II which removes the α(1→3) and α(1→6) mannose residues and results in a molecular structure that is subject to glucosyl group donation by GlcNAcT-II (N-acetylglucosyl transferase-II), encoded by the Mgat2 gene. Catalysis by GlcNAcT-II converts the hybrid glycans to the complex forms by attaching additional GlcNAc moieties to the hybrid structure.
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GLY FINAL 1207 PGD cleanxrefs2
N-Glycans
Inhibition or elimination of glycosylation in the study of N-linked glycans can be brought about by a number of compounds. N-glycosylation is strongly inhibited in the presence of compactin, coenzyme Q, or exogenous cholesterol. Treatment with tunicamycin completely blocks glycosylation as tunicamycin inhibits GlcNAc C-1‑phosphotransferase, an enzyme that is critical in the formation of the dolichol precursor.
N-Linked High Mannose Glycans MAN-3 Glycan (Man)3(GlcNAc)2; Mannotriose-di-(N-acetyl-D-glucosamine) C34H58N2O26 FW 910.82
≥80% (MS) store at: 2-8°C
α-Mannosidase, β-mannosidase, sialidase and α-fucosidase are the primary exoglycosidases involved in N-linked glycan trimming and degradation. Insect cells express an N-acetylglucosminidase that cleaves terminal GlcNAc residues from N-linked glycans.
Glycan Component Classes
Fucosylation at the core GlcNAc residue following GlcNAcT-I modification in both hybrid and complex N-linked synthesis is also a common occurrence. In vertebrates, fucose is added in an α(1→6) linkage, while in plants and invertebrates, fucose is added in an α(1→3) linkage.
Functions During development, the intermediate structures of a glycoprotein perform specific functions. In the early phases of glycoprotein evolution, different core oligosaccharide structures are necessary for proper protein folding and functional group orientation. Improperly folded proteins are either reglycosylated and refolded, or deglycosylated and degraded. In later phases, the oligosaccharide moiety is required for intracellular transport and targeting of the glycoprotein in the endoplasmatic reticulum, Golgi complex, and trans-Golgi network. In the final phase, the N-linked glycan undergoes extensive modification in the Golgi complex, resulting in a mature glycoprotein.
M8418-10UG
10 μg
M8418-50UG
50 μg
MAN-9 Glycan (Man)9(GlcNAc)2; Mannonanose-di-(N-acetyl-D-glucosamine) C70H118N2O56 FW 1883.67 [75558‑03‑1]
≥80% store at: 2-8°C
The protein moiety and the attached hydrophilic N-linked glycans are relatively independent, despite being covalently linked, i.e. glycans can be modified without significantly affecting the protein structure and function. Proteins may contain multiple glycosylation sites that are modified with any of the three classes of N-linked glycans. Vertebrates have been found to possess a diverse compliment of complex and hybrid glycoproteins due to the broad variety of glycosidases and glycosyltransferases coded within the genome. While these three classes of Nlinked glycoproteins are also present in lower organisms, there is less diversity of structure than is found in vertebrate glycoproteins. In addition to a specific glycoprotein being able to contain multiple glycan structures, different molecules of the same glycoprotein may have different glycan structures attached to the identical substitution site. Modifications in the glycan structures provide identity characteristics to different cell types and to the same cell type at stages of development, differentiation, transformation, maintenance, and aging. This variation in protein glycosylation is known as microheterogeneity and contributes to the difficulty in identifying and isolating specific glycoproteins. The congenital disorders of glycosylation (CDG) are a series of diseases associated with errors of metabolism due to enzyme deficiencies. The majority of identified CDGs are due to failures in the biosynthesis or degradation of N-glycans because of absence of one of the enzymes involved in N-glycosylation, primarily the exoglycosidic enzymes αmannosidase, β-mannosidase, sialidase, or α-fucosidase.
20 μg
M9037-20UG
Key to Monosaccharide Symbols
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
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9
GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
N-Glycans
N-linked Complex Glycans
A3 Glycan
A1F Glycan ammonium salt
Mannotriose-di-(N-acetyl-D-glucosamine), tris(sialyl-galactosyl-N-acetyl-Dglucosaminyl)- ammonium salt; (NeuNAc-Gal-GlcNAc)3Man3(GlcNAc)2; Trisialylated, galactosylated, triantennary N-glycan C109H178N8O80 FW 2880.59 [145164‑24‑5]
Mannotriose-(fucosyl-di-[N-acetylglucosamine]), mono-sialyl-bis(galactosyl-Nacetylglucosaminyl); NeuNAc(Gal-GlcNAc)2Man3(GlcNAc)2Fuc C79H131N5O58 FW 2078.88 [571188‑30‑2]
from bovine, ≥90% store at: −20°C
from human fibrinogen store at: −20°C
20 μg
M3800-20UG
M2925-10UG
10 μg
A2F Glycan ammonium salt (Gal-GlcNAc)2Man3(GlcNAc)2; Mannotriose-di-(N-acetyl-D-glucosamine), bis (galactosyl-[N-acetyl-D-glucosaminyl]) C62H104N4O46 FW 1641.49 [71496‑53‑2]
from porcine thyroglobulin, ≥90%
from human fibrinogen, ≥90%
store at: −20°C
M2800-10UG
10
NA2 Glycan
Disialylated, galactosylated, fucosylated biantennary N-linked glycan; Mannotriose-(fucosyl-di-[N-acetylglucosamine]), bi(sialyl-galactosyl-N-acetylglucosaminyl); (NeuNAc-Gal-GlcNAc)2Man3(Fuc)(GlcNAc)2 C90H148N6O66 FW 2370.14 [108341‑22‑6]
sigma.com/glycobiology
store at: −20°C
10 μg
M5925-50UG
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50 μg
GLY FINAL 1207 PGD cleanxrefs2
N-Glycans
N-Linked Fragments
Asialo, galactosylated, biantennary N-glycan, bisecting GlcNAc; (GalGlcNAc)2(GlcNAc)Man3(GlcNAc)2; Mannotriose-di-(N-acetylglucosamine), bis (galactosyl-N-acetylglucosaminyl)-bisecting N-acetylglucosaminylC70H117N5O51 FW 1844.68 [84632‑71‑3]
N-Asn
from sheep IgG
2‑Acetamido-1‑β-(L-aspartamido)-1,2‑dideoxy-D-glucose; 2‑Acetamido-1‑N(β-L-aspartyl)-2‑deoxy-β-D-glucopyranosylamine; β-D-GlcNAc-(1→N)-Asn C12H21N3O8 FW 335.31 [2776‑93‑4]
≥98%
store at: 2-8°C
store at: −20°C β
N Asn
A6681-5MG
5 mg
A6681-25MG
25 mg
Glycan Component Classes
NA2B Glycan
3α-Fucosyl-N-acetylglucosamine 2‑Acetamido-2‑deoxy-3‑O-α-L-fucopyranosyl-D-glucopyranose; α-L-Fuc(1→3)-D-GlcNAc C14H25NO10 FW 367.35 [24876‑86‑6]
20 μg
M2300-20UG
NA4 Glycan
≥98%
Asialo, tetraantennary N-linked glycan; (Gal-GlcNAc)4Man3(GlcNAc)2; Mannotriose-di-(N-acetyl-D-glucosamine), tetrakis(galactosyl-N-acetyl-Dglucosaminyl) C90H150N6O66 FW 2372.15 [82867‑74‑1]
store at: 2-8°C
α1 3
from human, ≥80% (MS) store at: 2-8°C β1,4
β1
,4
A5065-1MG ,6
β1
α1
,2
β1,4
β1
β1,4
β1
,4
β1,4
,3
β1,4
α1
β1,4
1 mg
,2
M8918-10UG
10 μg
M8918-50UG
50 μg
NGA2 Glycan
Key to Monosaccharide Symbols
Asialo, agalacto, biantennary N-glycan; (GlcNAc)2Man3(GlcNAc)2; Mannotriose-di-(N-acetylglucosamine), bis(N-acetylglucosaminyl)C50H84N4O36 FW 1317.21 [84808‑02‑6]
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
from human store at: −20°C
M1675-20UG
20 μg
Proteomics, glycosylation and other post-translational modifications, visit sigma.com/proteomics
11
GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
N-Glycans
2α-Mannobiose
3α,6α-Mannopentaose
α-D-Man-[1→2]-D-Man; 2‑O-α-D-Mannopyranosyl-D-mannopyranose C12H22O11 FW 342.30 [15548‑39‑7]
α-Man-(1→3)(α-Man-[1→6])-α-Man-(1→6)(α-Man-[1→3])-Man C30H52O26 FW 828.72 [112828‑69‑0]
~95%
≥85%
store at: −20°C
store at: 2-8°C α
2 α1,6
M1050-10MG
10 mg α1,6
α1,3
6α-Mannobiose α-D-Man-(1→6)-D-Man; 6‑O-α-D-Mannopyranosyl-D-mannopyranose C12H22O11 FW 342.30 [6614‑35‑3]
α1,3
≥85% (HPAE) store at: −20°C
α
M0925-5MG
6
5 mg
3α,6α-Mannotriose M7788-1MG
1 mg
M7788-5MG
5 mg
3,6‑Di-O-(α-D-mannopyranosyl)-D-mannopyranose; α-D-Man-(1→3)-[α-DMan-(1→6)]-D-Man C18H32O16 FW 504.44 [121123‑33‑9]
≥95% (HPLC) store at: −20°C
α1,6
α1,3
D5422-5MG
5 mg
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12
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For Technical Service, call your local office or visit sigma.com/techinfo
GLY FINAL 1207 PGD cleanxrefs2
O-Glycans
O-Glycans O-Linked glycoproteins are usually large proteins with a molecular mass of >200 kDa. Glycosylation generally occurs in high-density clusters and may represent as much as 50‑80% of the overall mass.
O-Linked glycans are most commonly attached to the peptide chain through serine (Ser) or threonine (Thr) residues. While O-linkage does link primarily to peptide residues through a hydroxyl group, there is no consensus sequence required. Tyrosine (Tyr), hydroxylysine (Hydroxy-Lys), or hydroxyproline (Hydroxy-Pro) may also be the peptide site of O-linked glycosylation. The most common O-linked glycans are the mucin-type glycans, which contain an initial GalNAc residue. There are eight mucin-type core structures (see Figure 6). Even with common mucin-type cores, O-linked glycans tend to be very heterogeneous, and there are other structures possible, in addition to several sialylated core structures. However, O-linked glycans are commonly linear or biantennary and have comparatively less branching than N-glycans. β1,3
Ser/ Thr
Core 1
β1,6 β1,3
Ser/ Thr
Core 2 β1,3
Ser/ Thr
Core 3
β1,6 β1,3
Ser/ Thr
Core 4 α1,3
Ser/ Thr
Core 5
Glycan Component Classes
Structures
Mucins are glycoproteins that contain large numbers of high-density clusters of O-linked glycans. The mucin-type glycans of these proteins frequently form cross-linked connections in aqueous solutions, resulting in a high viscosity gel (mucus). Mucins can be secreted, but may also be membrane bound and form glycan-dense areas on the cell surface. In addition to mucin-type glycans, O-linked glycans may incorporate sugars other than GalNAc as the initial sugar bound to the serine/ threonine residues. Examples of alternative O-linked glycans are: • Nuclear and cytoplasmic glycoproteins that contain GlcNAc as the initiating sugar. • Fibrinolytic and coagulation factors that contain fucose as the initiating sugar. • Mannoproteins that are typical to yeasts and that incorporate mannose as the initiating sugar. O-Mannosyl glycans are also found in human α-dystroglycan and other nervous system glycoproteins. • Glycosaminoglycans (GAGs) that are components of proteoglycan structures and contain xylose bound exclusively to serine residues. • Plant cell wall extensins that contain both arabinose attached to hydroxyproline and galactose attached to serine. • Plant arabinogalactans that are attached to the hydroxyproline within the peptide backbone through O-linked galactose or glucose. • Galactose and αGlc(1→2)Gal residues that are bound to hydroxylysine within the triple helix structures of collagen. Complement factor C1q also contains αGlc(1→2)Gal-hydroxyproline sequences. • Glycogenin, a protein precursor required for glycogen synthesis, contains glucose O-linked to tyrosine; the initial glucose is subsequently elongated by glycogen synthase to generate glycogen.
Key to Monosaccharide Symbols
β1,6 Ser/ Thr
Core 6
α1,6
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
Ser/ Thr
Core 7 α1,3
Core 8
Ser/ Thr
Figure 6. Core structures of mucin-type O-glycans.
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13
GLY FINAL 1207 PGD cleanxrefs2
O-Glycans
Glycan Component Classes
Biosynthesis and Degradation O-linked glycosylation is a true post-translational event that occurs in the Golgi, and no oligosaccharide precursor is required for protein transfer. The serine/threonine residues are modified directly by covalent addition of N-acetylgalactosamine residues. Initiation of mucin-type O-glycosylation is dependent upon polypeptide N-acetylgalactosyl transferase (ppGalNAcT); at least twelve mammalian ppGalNAcT isozymes have been identified. The first step in the synthesis of mucin-type glycans requires catalysis by a ppGalNAcT in the presence of UDP-GalNAc as the carbohydrate donor. Subsequent elongation and termination of O-linked glycans is carried out by several glycosyltransferases. The relative expression and subcellular distribution of the various glycosyltransferases determine the outcome of O-glycan biosynthesis. For additional discussion of Glycosyltransferases, see page 87. Termination of O-linked glycans usually includes Gal, GlcNAc, GalNAc, Fuc, or sialic acid. By far the most common modification of the core Gal-β(1→3)GalNAc is mono-, di-, or trisialylation (Core 1 and 2) (see Figure 7). A less common, but widely distributed O-linked hexasaccharide structure contains β(1→4)-linked Gal and β(1→6)-linked GlcNAc, as well as sialic acid. α2,3
More complex O-glycans serve other functions. ZP glycoproteins are O-linked glycans present in high concentrations in the zona pellucida surrounding mammalian eggs. Human ZP matrix contains four ZP glycoproteins, while the mouse ZP matrix has only three.3 The roles of ZP glycoproteins have not been fully determined but are thought to be associated with sperm reception. O-Linked glycans are also involved in hematopoiesis and inflammation response mechanisms. The P-selectin glycoprotein ligand 1 (PSGL-1) contains, among other glycans, a Core 2 O-glycan capped with sialyl Lewis X (C2‑O-sLeX, see Figure 8). PSGL-1 is the primary adhesion target for P-selectin and a target for E-selectin, which are involved in leukocyte rolling and recruitment into sites of inflammation.4 β1,4
α2,3
β1,3
β1,4
β1,3
β1,4
α1,3
β1,6 β1,3
β1,3
Ser/ Thr α2,6
α2,3
Proteins that are O-linked with GlcNAc may alternatively be phosphorylated at the same peptide site, and similarly to phosphorylation, O-GlcNAc processing has been associated with cellular signaling events, including insulin signaling and RNA transcription regulation.1,2
β1,3
Ser/ Thr
Figure 8. Structure of the O-linked Core 2 glycan with attached sialyl Lewis X (sLex) moiety.
Ser/ Thr α2,6
The human ABO blood antigens are small O-linked glycans that may be attached to membrane glycoproteins or to cell surface glycolipids. The antigens may also be secreted by tissues as free oligosaccharides or as components of soluble glycoproteins and glycosphingolipids. The blood group O(H) determinant does not generate an immune response, but when modified by addition of either α(1→3)GalNAc (blood group A antigen) or α(1→3)Gal (blood group B antigen), the resultant trisaccharide initiates an immune response (see Figure 9).
α2,8 α1,2
Figure 7. Disialylated (top) and trisialylated (bottom) O-linked Core 1 glycans
O-glycan degradation requires α-N-acetylgalactosaminidase in addition to the same exoglycosidases needed for N-glycan degradation.
β1,0
R
A
Functions Secreted mucins at the apical membrane of epithelial cells can link through disulfide bonds and capture water molecules, forming a mucus membrane. The membrane physically protects the cell from hostile environmental factors such as stomach acids and circulating proteases. Mucin secretion by salivary glands provides lubrication for swallowing. Mucins also block infection by pathogens by presenting a wall of O-linked glycans to attract and bind bacterial carbohydrate binding receptors. Many bacterial pathogens express adhesins, carbohydraterecognition proteins specific for cell surface O-glycan structures that function as receptors for binding and infecting host cells. The adhesins bind to the mucin surface glycans, preventing further progress by the pathogen; the bound pathogen can then be eliminated.
α1,2
α1,3
β1,0
R
B
α1,2
α1,3
β1,0
R
C
Glycosylation by a single GlcNAc moiety is a unique form of O-glycosylation, in that it has been shown to be dynamic, rather than static like other types of O-linked glycosylation. This modification is reversible and catalyzed by the enzymes uridine diphospho-N-acetylglucosmine:polypeptide β-N-acetylglucosaminyltransferase (O-GlcNAc transferase) and neutral β-N-acetylglucosaminidase (O-GlcNAcase).
14
sigma.com/glycobiology
Figure 9. Structures of (A) blood group H(O), (B) blood group A, and (C) blood group B antigens. R represents the hydroxyl-containing amino acid or lipid binding site for the antigen.
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GLY FINAL 1207 PGD cleanxrefs2
O-Glycans
Lacto-N-difucohexaose I α-L-Fuc-(1→2)-β-D-Gal-(1→3)[α-L-Fuc-(1→4)]-β-D-GlcNAc-(1→3)-β-D-Gal(1→4)-D-Glc; Leb-lactose; Lewis-b hexasaccharide; LNDFH I C38H65NO29 FW 999.91 [16789‑38‑1]
from human milk, ≥95% (HPAE/PAD) Blood group Leb active hexasaccharide. store at: 2-8°C
Schindler's Disease has been identified as a congenital disorder of glycosylation that affects O-linked glycans and is due to an α-Nacetylgalactosaminidase deficiency. References: 1. Slawson, C., et al., O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks. J. Cell. Biochem., 97, 71‑83 (2006). 2. Wells, L., and Hart, G.W., O-GlcNAc turns twenty: functional implications for posttranslational modification of nuclear and cytosolic proteins with a sugar. FEBS Lett., 546, 154‑8 (2003). 3. Gupta, S.K., et al., Structural and functional attributes of zona pellucida glycoproteins. Soc. Reprod. Fertil. Suppl., 63, 203‑16 (2007). 4. Sperandio, M., Selectins and glycosyltransferases in leukocyte rolling in vivo. FEBS J., 273, 4377‑4389 (2006).
α1 2
β1 3
α1 4
β1 3
L7033-1MG
β1 4
Glycan Component Classes
T antigen and Tn antigen are O-glycans that have incomplete glycosylation. T antigen (tumor-associated or TF (Thomsen-Fridenreich) antigen) is the Core 1 disaccharide (Gal-β(1→3)GalNAc) that results after desialylation by viral or bacterial neuraminidase. T antigen may also result due to changes in the glycosyltransferases available. Tn antigen is an O-linked GalNAc that is not extended by a glycosyltransferase into a complete core structure. These antigens are not expressed on the surface of normal cells, but are commonly present in cancerous cells and may serve as tumor markers.
1 mg
Lacto-N-difucohexaose II β-D-Gal-(1→3)-[α-L-Fuc-(1→4)]-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-[α-L-Fuc(1→3)]-D-Glc; LNH C38H65NO29 FW 999.91 [62258‑12‑2]
from human milk, ~95% Lewis-a trisaccharide linked to 3‑fucosyllactose
O-Linked Neutral Glycans
store at: 2-8°C
N-Acetyl-D-lactosamine 2‑Acetamido-2‑deoxy-4‑O-β-D-galactopyranosyl-D-glucopyranose; N-Acetyl4‑O-(β-D-galactopyranosyl)-D-glucosamine; β-D-Gal-(1→4)-D-GlcNAc; LN C14H25NO11 FW 383.35 [32181‑59‑2]
≥98% Useful in studies of galactosidase, fucosyltransferase, sialyltransferase, and lectin inhibition. Synthetic
β1 3
α1 4
L6645-.5MG
β1 3
α1 3
β1 4
0.5 mg
store at: −20°C β1 4
A7791-5MG
5 mg
A7791-10MG
10 mg
A7791-25MG
25 mg
A7791-100MG
100 mg
2′-Fucosyl-D-lactose
Key to Monosaccharide Symbols
α-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc C18H32O15 FW 488.44 [41263‑94‑9]
from human milk, ~98% (HPAE/PAD)
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
store at: 2-8°C
2
β
4
F0393-1MG
1 mg
F0393-5MG
5 mg
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15
GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
O-Glycans
Lacto-N-fucopentaose I
O-Linked Sialylated Glycans
α-L-Fuc (1→2)-β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-D-Glc; LNFP I C32H55NO25 FW 853.77 [7578‑25‑8]
3′-N-Acetylneuraminyl-N-acetyllactosamine sodium salt α-NeuNAc-(2→3)-β-D-Gal-(1→4)-D-GlcNAc; 3′-Sialyl-N-acetyllactosamine; 3′SLN C25H42N2O19 FW 674.60 [81693‑22‑3]
from human milk, ≥90% (HPAE/PAD) Blood group H-active pentasaccharide.
store at: −20°C
store at: 2-8°C
α2
α1 2
β1 3
β1 3
β1 4
3
β1 4
A6936-.5MG
0.5 mg
A6936-2MG
2 mg
3'-Sialyllactose L5908-5MG
5 mg
3′-N-Acetylneuraminyl-D-lactose sodium salt; NANA-Lactose; α-NeuNAc(2→3)-β-D-Gal-(1→4)-D-Glc; 3'-Sialyl-D-lactose; 3'-SL C23H39NO19 FW 633.55 [35890‑38‑1]
Lacto-N-fucopentaose III β-D-Gal-(1→4)-[α-L-Fuc-(1→3)]-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-D-Glc; Lexlactose; Lewis-X pentasaccharide; LNFP III C32H55NO25 FW 853.77 [25541‑09‑7]
from human milk, ≥95% (HPAE)
α2,3
β1,4
from human milk, ≥98% (HPAE/PAD)
store at: 2-8°C
store at: −20°C A9079-1MG α1,3 β1 4
1 mg
from bovine colostrum, ~98% (HPAE/PAD) β1 3
store at: −20°C
β1 4
A8681-1MG
L7777-1MG
1 mg
6′-Sialyllactose sodium salt
1 mg
6′-N-Acetylneuraminyl-lactose sodium salt; α-NeuNAc-(2→6)-β-D-Gal-(1→4)D-Glc; 6'-SL C23H38NO19Na FW 655.53 [74609‑39‑5]
Lacto-N-tetraose β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-D-Glc; LNT C26H45NO21 FW 707.63 [14116‑68‑8]
α2,6
β1,4
from human milk, ≥95% (HPAE/PAD) store at: 2-8°C β
3
β
3
β
from human milk, ≥95% (HPAE/PAD)
4
store at: −20°C L6770-1MG
1 mg
L6770-5MG
5 mg
Lacto-N-neo-tetraose
1 mg
A9204-5MG
5 mg
from bovine colostrum, ≥97% (HPAE/PAD) 8
β-D-Gal-(1→4)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-D-Glc; LNnT C26H45NO21 FW 707.63 [13007‑32‑4]
A9204-1MG
store at: −20°C A8556-1MG
1 mg
A8556-5MG
5 mg
from synthetic, ≥85% (HPLC) store at: 2-8°C L6543-2MG
16
sigma.com/glycobiology
2 mg
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GLY FINAL 1207 PGD cleanxrefs2
O-Glycans
Globotriose
N-Acetylneuraminyl-lactose; α-NeuNAc-(2→3)- and -(2→6)-β-D-Gal-(1→4)D-Glc; Neuramin-lactose C23H38NO19Na FW 655.53
4‑O-(4‑O-α-D-Galactopyranosyl-β-D-galactopyranosyl)-D-glucopyranose; α-DGal-(1→4)-β-D-Gal-(1→4)-D-Glc C18H32O16 FW 504.44 [66580‑68‑5]
Mixture of (2→6′) and (2→3′) isomers.
store at: 2-8°C
α2 3/6
β1
α1
4
β1 4
4
G9287-.5MG
~80% (HPAE/PAD), from bovine colostrum
iso-A-Pentasaccharide
store at: −20°C A3307-5MG
5 mg
A3307-25MG
25 mg
α-GalNAc-(1→3)-(α-Fuc-[1→2])-β-Gal-(1→3)-(α-Fuc-[1→4])-Glc; A-LebPentasaccharide C32H55NO24 FW 837.77 [128464‑25‑5]
from human milk, ≥97% store at: −20°C A0828-5MG
5 mg
A0828-25MG
25 mg
0.5 mg
Glycan Component Classes
Sialyllactose sodium salt
from human urine, ≥90% store at: 2-8°C
α1
Blood Group Antigens
α1 3
4β-Galactobiose 4‑O-β-D-Galactopyranosyl-D-galactopyranose; β-D-Gal-(1→4)-D-Gal C12H22O11 FW 342.30 [2152‑98‑9]
A7560-20UG
≥90%
B-Pentasaccharide
2
α1 4
β1 3
20 μg
store at: 2-8°C
α-Fuc-(1→2)-α-Gal-(1→3)-β-Gal-(1→4)(α-Fuc-[1→3])-Glc C30H52O24 FW 796.72 [72468‑43‑0]
β1 4
G9662-2MG
2 mg
G9662-10MG
10 mg
from human urine, ≥85% store at: 2-8°C
Galacto-N-biose
α1 2
2‑Acetamido-2‑deoxy-3‑O-β-D-galactopyranosyl-D-galactopyranose; T Antigen; β-D-Gal-(1→3)-D-GalNAc C14H25NO11 FW 383.35 [20972‑29‑6]
A substrate for N-acetylglucosaminyltransferase,1 fucosyl- and sialyltransferase,2,3 and galactosidase.4 Also used in lectin inhibition studies.5 Lit. cited: 1. Williams, D., et al., J. Biol. Chem. 255, 11253 (1980) 2. Beyer, T.A. and Hill, R.L., J. Biol. Chem. 255, 5373 (1980) 3. Rearick, J.I., et al., J. Biol. Chem. 254, 4444 (1979) 4. Distler, J.J. and Jourdian, G.W., J. Biol. Chem. 248, 6772 (1973) 5. Tollefsen, S.E. and Kornfeld, R., J. Biol. Chem. 258, 5172 (1983)
α1 3
α1 3
β1 4
50 μg
B3791-50UG
Key to Monosaccharide Symbols
store at: −20°C β1 3
A0167-1MG
1 mg
A0167-5MG
5 mg
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
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17
GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
O-Glycans
iso-B-Pentasaccharide
Lewis and Cell Adhesion Glycans
BLeb-Pentasaccharide; α-Gal-(1→3)-(α-Fuc-[1→2])-β-Gal-(1→3)-(α-Fuc[1→4])-Glc C30H52O24 FW 796.72 [128464‑26‑6]
Lewis-b tetrasaccharide
from human urine, ≥85%
α-Fuc(1→2)-β-Gal-(1→3)-(α-Fuc-[1→4])-GlcNAc; Leb glycan C26H45NO19 FW 675.63
Lewis-b human blood group determinant.
store at: 2-8°C
store at: −20°C
α1 2 α1 3
α1 4 3
β1
20 μg
B0799-20UG
A-Trisaccharide
α1 2
β1 3
α1 4
L7659-1MG
1 mg
Lewis-Y hexasaccharide
Blood group A trisaccharide; α-D-GalNAc-(1→3)-(α-L-Fuc-[1→2])-D-Gal C20H35NO15 FW 529.49 [49777‑13‑1] store at: room temp
α-Fuc-(1→2)-β-Gal-(1→4)(α-Fuc-[1→3])-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc; Lacto-N-neo-difucohexaose I; Ley-lactose C38H65NO29 FW 999.91 [62469‑99‑2]
from human urine store at: 2-8°C
α1 2 α1 3
α1 2
250 μg
A7911-250UG
β1 4
α1 3
β1 3
B-Trisaccharide
20 μg
L7401-20UG
Blood group B trisaccharide; α-D-Gal-(1→3)-(α-L-Fuc-[1→2])-D-Gal C18H32O15 FW 488.44 [49777‑14‑2] store at: 2-8°C
β1 4
Lewis-Y tetrasaccharide α-Fuc-(1→2)-β-Gal-(1→4)-(α-Fuc-[1→3])-GlcNAc C26H45NO19 FW 675.63
Lewis-Y blood group antigenic determinant.
α1 2 α1 3
B1422-1MG
store at: 2-8°C
α1 2
1 mg
β1 4
α1 3
H-Trisaccharide Blood group H trisaccharide; α-Fuc-(1→2)-β-Gal-(1→4)-GlcNAc; 2′-FucosylN-acetyllactosamine C20H35NO15 FW 529.49
L7784-1MG
synthetic Human blood group H type trisaccharide. store at: 2-8°C
α1 2
F7297-1MG
18
sigma.com/glycobiology
β1 4
1 mg
For Technical Service, call your local office or visit sigma.com/techinfo
1 mg
GLY FINAL 1207 PGD cleanxrefs2
O-Glycans α-Gal-(1→3)-Gal Antigens
α-NeuNAc-(2→3)-β-D-Gal-(1→3)-(α-L-Fuc-[1→4])-D-GlcNAc; 3'-SLea C31H52N2O23 FW 820.74 [92448‑22‑1]
3‑α-Galactobiose
store at: −20°C
3‑O-α-D-Galactopyranosyl-D-galactose; α-D-Gal-(1→3)-D-Gal C12H22O11 FW 342.30 [13168‑24‑6]
synthetic, ≥90% α2 3
β1 3
store at: 2-8°C
α1 4
α1
S2279-.2MG
0.2 mg
S2279-1MG
1 mg
G7522-5MG
5 mg
3α,4β,3α-Galactotetraose
3′-Sialyl-Lewis-X tetrasaccharide α-NeuNAc-(2→3)-β-D-Gal-(1→4)(α-L-Fuc-[1→3])-D-GlcNAc; 3'-SLeX C31H52N2O23 FW 820.74 [98603‑84‑0] store at: −20°C
3
Glycan Component Classes
3′-Sialyl-Lewis-a tetrasaccharide
3‑O-(4‑O-[3‑O-α-D-Galactopyranosyl-β-D-galactopyranosyl]-α-D-galactopyranosyl)-D-galactopyranose; α-D-Gal-(1→3)-β-D-Gal-(1→4)-α-D-Gal-(1→3)D-Gal C24H42O21 FW 666.58 [56038‑38‑1]
≥90%
store at: −20°C α2 3
β1 4
α1
α1 3
3
G9912-2MG S1782-.2MG
0.2 mg
S1782-1MG
1 mg
β1 4
α1 3
2 mg
Key to Monosaccharide Symbols
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
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19
GLY FINAL 1207 PGD cleanxrefs2
GPI Anchored Glycoproteins
Glycan Component Classes
GPI Anchored Glycoproteins Glycosylphosphatidylinisotol (GPI) anchored proteins have been identified throughout a broad range of eukaryotic species ranging from humans to insects, yeasts, bacteria, and fungi, suggesting they are a very ancient modification. In general, GPI anchored proteins are not as prevalent as other post-translationally modified proteins, but as a class of glycoproteins, they demonstrate considerable homology.
Structure GPI anchored proteins are linked at their carboxy-terminus through a phosphodiester linkage to phosphoethanolamine attached to a trimannosyl-nonacetylated glucosamine (Man3-GlcN) core. The reducing end of GlcN is linked via another phosphodiester linkage to phosphatidylinositol (PI) (see Figure 10).
α1,2
α1,6
α1,4 α1,6
Figure 10. General structure of GPI anchored proteins. The lipid tails of the phosphatidylinositol moiety are shown inserted through the outer cell membrane and held by the lipid bilayer.
Biosynthesis and Degradation The GPI anchor is assembled on the membrane leaflet of the endoplasmic reticulum. Once completed, it is transferred to the lumen and the carboxy terminus of the protein is attached to the GPI anchor. After translocation across the membrane, GPI anchored proteins are bound to the cell membrane by the insertion of the phosphatidylinositol lipid moieties into the hydrophobic lipid bilayer. Once the GPI anchored protein has been produced, the Man3-GlcN oligosaccharide core may undergo additional glycosylation modifications during secretion from the cell. Release of GPI anchored proteins can be accomplished by treatment with Phospholipase C, Phosphatidylinositol-specific (PLC-PI) (Cat. No. P5542 and P8804). The enzyme specifically hydrolyzes the phosphodiester bond of phosphatidylinositol to form a free 1,2‑diacylglycerol and glycopeptide-bound inositol cyclic-1,2‑phosphate; the cleavage site of PCL-PI is shown in red in Figure 10. Enzymatic cleavage will not occur if the inositiol is acylated; pre-treatment with mild alkali conditions is necessary to remove the fatty acid moiety. Following enzymatic
20
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cleavage, the GPI anchors can be recovered from the insoluble pellet/ fraction after membranes have been treated with Triton® X-114 (Cat. No. X114). Enzymatic release of the GPI anchor from the cell membrane may trigger second messengers for signal transduction.
Functions GPI anchored proteins have been involved in membrane protein transportation, cell adhesion, cell wall synthesis, and cell surface protection. In yeast, GPI anchored proteins are components of the cell wall and are necessary for cellular integrity. Some GPI anchored proteins are antigens, such as human carcinoembryonic antigen (CEA), which is used as a cancer marker. Others such as human reversion-inducing cysteine-rich protein with Kazal motifs (RECK) inhibit tumor invasion and metastasis. In mammalian cells, GPI anchored proteins are concentrated in lipid rafts that are involved in receptor-mediated signal transduction pathways and membrane trafficking. The GPI anchor regulates secretion of cryptococcal phospholipase B and is necessary for prostatin to regulate transepithelial resistance and paracellular permeability.1 Reference: 1. Verghese, G.M., et al., Prostasin regulates epithelial monolayer function: cell-specific Gpld1‑mediated secretion and functional role for GPI anchor. Am. J. Physiol. Cell Physiol., 291, C1258‑70 (2006).
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GLY FINAL 1207 PGD cleanxrefs2
Glycosphingolipids
Glycosphingolipids Functions
Glycosphingolipids (sometimes called glycolipids) have been found in lower and higher eukaryotic sources. They are composed of a glycan structure attached to a lipid tail that contains the sphingolipid ceramide. The basic structure for a glycosphingolipid is a monosaccharide, usually glucose or galactose, attached directly to a ceramide molecule and resulting in, respectively, glucosylceramide (glucocerebroside; GlcCer) or galactosylceramide (galactocerebroside; GalCer). The core glycan structure may be extended by additional monosaccharides. This combination structure results in an amphiphilic molecule with a hydrophilic carbohydrate region and a hydrophobic lipid region. In addition to variations in the structure of the glycan, the ceramide structure may also show variation. The fatty acid attached to the sphingosine may contain carbon chain lengths from C14 to C24 and vary in degree of unsaturation and/or hydroxylation.
Glycosphingolipids are minor components of cell membranes and membrane compartments, and are connected to cell growth, differentiation, and viral and oncogenic transformations. Like GPI anchored proteins, glycosphingolipids aggregate to form membrane rafts on the micromembrane and the outer leaflet of the apical membrane. The hydrophobic lipid tail is embedded in the outer leaflet of cell membranes, positioning the glycan structure into the extracellular matrix. Gangliosides are the major constituents of neuronal cell membranes and the endoplasmic reticulum.
Abbreviations for the oligosaccharide chains of glycosphingolipids have been recommended by IUPAC to make the nomenclature less complex.1 The recommended root names are shown in Table 1. Root Structure Root Name
Symbol
Ganglio-
Gg
IV
Galb3GalNAcb4Galb4Glc-
III
II
I
Lacto-
Lc
Galb3GlcNAcb3Galb4Glc-
Neolacto (Lactoneo)
nLc (Lcn)
Galb4GlcNAcb3Galb4Glc-
Globo-
Gb
GalNAcb3Gala4Galb4Glc-
Isoglobo-
iGb
GalNAcb3Gala3Galb4Glc-
Mollu-
Mu
GlcNAcb2Mana3Manb4Glc-
Arthro-
At
GalNAcb4GlcNAcb3Manb4Glc-
Glycan Component Classes
Structure
Glycosphingolipids are thought to provide protection from harsh conditions in the extracellular environment, such as low pH and degrading enzymes. They help cells to interact with extracellular matrices and other cells, and serve as surface receptors for bacterial toxins and possibly endogenous extracellular molecules. In addition to their function as membrane components, glycosphingolipids are precursors for lipids involved in signal transduction and contribute to the water permeability barrier of the skin. The skin and the brain are the key organs in which functional aspects of mammalian glycosphingolipids are important. Galactosylceramide derivatives are necessary key components of myelin and have demonstrated growth factor-like properties. Acidic gangliosides influence the electrical field across the cellular membrane, as well as the concentration of ions on the external surface of the cells. In addition, gangliosides may have a role in electrical insulation in myelin cells in the nervous system.
Table 1. Root Names, Symbols, and Root Structures for Glycosphinoglipid Chains.
The outer extensions of glycosphingolipids contain sugars including fucose, glucuronic acid, and sialic acid, and blood group structures that are similar to those of O- and N-glycans. Like O- and N-glycans, modifications such as 9‑O-acetylation, N-deacetylation of sialic acids, or O-sulfation and O-acylation of galactose residues may also be present. Glucosyl ceramide and galactosyl ceramide are neutral, while glycosphingolipids containing sulfate, phosphate, or sialic acid residues are acidic and have a negative net charge. Gangliosides are glycosphingolipids that specifically contain one or more sialic acid (N-acetylneuraminic acid; NANA) residues.
Biosynthesis and Degradation Most animal glycosphingolipid families are derived from lactosylceramide (LacCer; β-D-galactosyl(1→4)-β-D-glucosyl-ceramide). The first step in lactosylceramide synthesis is the acylation and desaturation of Derythro-sphinganine. The ceramide is then glucosylated on the cytosolic face of the endoplasmatic reticulum (ER) and Golgi membranes followed by β-galactosylation of glucosylceramide on the opposite face of the ER and Golgi membranes to form lactosylceramide. The glycan chain can be elongated by specific glycosyltransferases and sulfotransferases. Glycosphingolipid biosynthesis, degradation, and intracellular transport are highly regulated, and cells demonstrate type-specific expression and stable glycosphingolipid patterns.
Key to Monosaccharide Symbols
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
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21
GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
Glycosphingolipids Some gangliosides, such as those that contain the sialyl Lewis X (sLeX) structure (see Figure 11), are useful as cancer cell markers, since they are elevated in the membranes of certain types of tumor cells such as melanomas and metastatic brain tumors. Purified gangliosides are frequently used as markers for characterization of various cell types. They may also serve as substrates and inhibitors of glycosidases and glycosyltransferases. α1,3
α2,3
β1,4
β1,4
Gangliosides are named in accordance with the IUPAC Guidelines “Nomenclature of Glycolipids, IUPAC Recommendations 1997)”, Pure Appl. Chem., 69, 2475‑2487 (1997). The letter “G” is used to indicate ganglioside and the number of sialic acid residues are abbreviated as M (mono), D (di) or T (tri). Reference: 1. International Union of Pure and Applied Chemistry (IUPAC) and International Union of Biochemistry and Molecular Biology (IUBMB) Joint Commission on Biochemical Nomenclature (JCBN) Nomenclature of Glycolipids, Recommendations (1997). http:// www.chem.qmul.ac.uk/iupac/misc/glylp.html
β1,1 Cer
Figure 11. Structure of the tetrasaccharide sialyl Lewis X (sLex) attached to galactosylceramide (GalCer). Cer represents the ceramide moiety.
Neutral Glycosphingolipids Name
Assay
Cat. No.
Galactocerebrosides from bovine brain
~99%, TLC
C4905-10MG C4905-25MG
Globotriaosylsphingosine from porcine blood
-
G9534-1MG
Glucocerebrosides from human (Gaucher's) spleen
≥98.0%, TLC
49108-1MG
Lactocerebrosides from bovine brain
~95%
C3166-1MG C3166-5MG
Psychosine from bovine brain
≥98%, TLC
P9256-1MG P9256-10MG
Gangliosides Gangliosides are major constituents of neuronal cell membranes and endoplasmic reticulum. They contain a sialylated polysaccharide chain linked to ceramide through a β-glycosidic linkage. For classification of gangliosides see Svennerholm, L., et al. (eds.), Structure and Function of Gangliosides, New York, Plenum, 1980. Name
Assay
Sterilization
Cat. No.
Asialoganglioside GM1 from bovine brain
~98%
-
G3018-.5MG G3018-1MG
Asialoganglioside-GM2 from bovine brain
~95%
-
G9398-50UG G9398-.1MG
Monosialoganglioside GM1 from bovine brain
≥95%
-
G7641-1MG G7641-5MG G7641-10MG
Monosialoganglioside GM1 from bovine brain
≥95%, TLC
γ-irradiated
G9652-1MG
Monosialoganglioside GM2 from bovine brain
≥95%, TLC
-
G8397-1MG
Monosialoganglioside GM3 from canine blood
≥98%
-
G5642-.5MG G5642-1MG
Monosialoganglioside GM3 from canine blood
~95%
γ-irradiated
G0153-1MG
Disialoganglioside GD1a from bovine brain
≥95%, TLC
-
G2392-1MG G2392-5MG
Disialoganglioside GD1b from bovine brain
~95%
-
G8146-.5MG G8146-1MG
Disialoganglioside-GD2
~95%
-
G0776-.1MG
Trisialoganglioside-GT1b from bovine brain
≥96%, TLC
-
G3767-1MG G3767-5MG
Lysoganglioside-GM1 from bovine brain
≥95%
-
G5660-1MG
semisynthetic
22
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GLY FINAL 1207 PGD cleanxrefs2
Glycosaminoglycans and Proteoglycans
Glycosaminoglycans and Proteoglycans Structures Glycosaminoglycans (GAGs) are large linear polysaccharides constructed of repeating disaccharide units with the primary configurations containing an amino sugar (either GlcNAc or GalNAc) and an uronic acid (either glucuronic acid and/or iduronic acid). There are five identified glycosaminoglycan chains (see Figure 12):
Hyaluronidase
D-Glucuronic acid
C OOH
• Hyaluronan • Chondroitin • Dermatan • Heparin/heparan • Keratan
O OH C OOH O OH
Hyaluronan is not sulfated, but the other glycosaminoglycan chains contain sulfate substituents at various positions of the chain. The sulfate groups as well as the uronic acids result in the glycosaminoglycan chains having a negative charge. GAG polymers are significantly larger than Nglycans or O-glycans and the chains are linear rather than branched like N-glycans.
A
B
4S
4S
4S
4S
6S
6S
OH
O
OH
C H2OH O
C H2OH
O
C H3
HN
O OH
O
O
Glycan Component Classes
Hyaluronan (HA; hyaluronic acid) is composed of alternating residues of β-D-(1→3) glucuronic acid (GlcA) and β-D-(1→4)-N-acetylglucosamine (GlcNAc) (see Figure 13). Unlike the other glycosaminoglycans, hyaluronan does not attach to proteins to form proteoglycans.
N-Acetyl-D-Glucosamine
OH C H3
HN
OH OH
O D-Glucuronic acid
N-Acetyl-D-Glucosamine
Hyaluronic Acid Figure 13. Hyaluronic acid is composed of alternating residues of β-D-(1‑3) glucuronic acid and β-D-(1‑4)-N-acetylglucosamine.
Chondroitin sulfate and dermatan sulfate (chondroitin sulfate B) are composed of disaccharide units containing N-acetylgalactosamine (GalNAc) and an uronic acid joined by β(1→4) or β(1→3) linkages, respectively (see Figure 14, 15, 16). Chondroitins contain glucuronic acid (GlcA) and are 4‑O-sulfated (chondroitin sulfate A) or 6‑O-sulfated (chondroitin sulfate C). Dermatan sulfate also contains N-acetylgalactosamine (GalNAc), but the uronic acid present in dermatan is 1 L-iduronic acid (IdoA). Chondrotinases ABC, AC & C
C
2S
COOH N-acetyl-β-galactosamine-4-sulfate
O
D
2S
6S
2S
O
O
OH
CH2OH
HO3S
O O
OH β-glucuronic acid
E
6S
6S
6S
Figure 12. Carbohydrate sequences of the five types of glycosaminoglycan chains using monosaccharide symbols: (A) Hyaluronan, (B) Chondroitin, (C) Dermatan, (D) Heparin and (E) Keratan. Possible sulfation presence and location (2S, 4S or 6S) is indicated.
CH3
HN
6S
O
Chondroitin Sulfate A
Figure 14. Chondroitin sulfate A consists of an alternating copolymer β-glucuronic acid-(1‑3)-N-acetyl-β-galactosamine-4‑sulfate.
Key to Monosaccharide Symbols
β-D-Glucose (Glc)
β-D-Xylose (Xyl)
β-D-Mannose (Man)
α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)
β-D-Galactose (Gal)
β-D-Glucuronic acid (GlcA)
β-D-N-Acetylglucosamine (GlcNAc)
α-L-Iduronic acid (IdoA)
β-D-N-Acetylgalactosamine (GalNAc)
α-L-Fucose (Fuc)
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23
GLY FINAL 1207 PGD cleanxrefs2
Glycosaminoglycans and Proteoglycans Chondrotinase ABC
Glycan Component Classes
The biosynthesis of hyaluronic acid (hyaluronan) is a stereospecific enzymatic copolymerization of N-acetylglucosamine and glucuronic acid. The hyaluronan synthetases involved in this process require UDP-Nacetylglucosamine and UDP-glucuronic acid as sugar donors, and can generate a 1,000,000 Dalton polymer in less than a minute. Hyaluronan is cleaved by hyaluronidases; testicular hyaluronidases degrade hyaluronan to tetrasaccharides, while bacterial hyaluronidases degrade hyaluronan to disaccharides. In lysosomes, hyaluronan is degraded to the monosaccharides N-acetylglucosamine, which is recycled, and glucuronic acid, which is further metabolized via the pentose phosphate pathway.
N-acetyl-β-galactosamine-4-sulfate
O
CH2OH
O
COOH OH
O
Biosynthesis and Degradation
O
HO3S
O OH β-Iduronic acid
CH3
HN O
Chondroitin Sulfate B
Figure 15. Chondroitin sulfate B (dermatan sulfate) consists of an alternating copolymer β-iduronic acid-(1‑3)-N-acetyl-β-galactosamine-4‑sulfate.
The biosynthesis of the sulfated glycosaminoglycans involves both polymerization and sulfation steps. Sulfation is performed by sulfotransferases utilizing a 3′-phosphoadenyl-5′-phosphate (PAPS) activated sulfate donor. Keratan chains can be over 20,000 Dalton and contain a series of lactosamine (βGal-β(1→4)-GlcNAc) subunits that are nonsulfated, monosulfated (6‑position of GlcNAc), or disulfated (6‑position of both Gal and GlcNAc). In animals, degradation occurs via the sulfatase-catalyzed removal of the terminal sulfate and the sequential action of exoglycosidases. Bacterial keratanases can degrade keratan sulfate at specific positions.
Chondrotinases ABC, AC & C COOH N-acetyl-β-galactosamine-6-sulfate
O
CH2OSO3H
O
O
OH
O
OH
O OH β-glucuronic acid
CH3
HN Chondroitin Sulfate C
O
Figure 16. Chondroitin sulfate C consists of an alternating copolymer β-glucuronic acid-(1‑3)-N-acetyl-β-galactosamine-6‑sulfate.
Heparan and heparin glycosaminoglycans are complex heterogeneous mixtures of repeating disaccharide units consisting of an uronic acid (D-glucuronic or L-iduronic acid) and D-glucosamine or N-acetyl-Dglucosamine (see Figure 17). Various degrees of sulfation occur (at the oxygen and/or nitrogen containing groups) on each monosaccharide unit, ranging from zero to tri-sulfation. Heparan is less sulfated than heparin.2 Heparinase Specificities
Heparinase I & II
Functions
D-glucosamine CH2OSO3 O
D-glucosamine
Heparinase II & III
OH L-iduronic acid O O COOH OH
CH2OH O
N-Acetyl-D-glucosamine
OSO3
D-glucuronic acid COOH
CH2OSO3
O
O OH O
CH3
HN
O HN SO3
O HN
OSO3 SO3
OH O
O
Both chondroitin sulfate and heparan sulfate classes of sulfated glycosaminoglycans link to serine residues in core proteins through a common tetrasaccharide construct (GlcA-Gal-Gal-Xyl-Ser). This process is initiated by the xylosyltransferase-catalyzed attachment of xylose using UDP-xylose as donor. After the attachment of the tetrasaccharide, the proteoglycan structure diverges, since the next carbohydrate linkage determines the glycosaminoglycan class attached. A β-glycosidic bond to N-acetylgalactosamine results in the attachment of chondroitin sulfate to the peptide, while a α-glycosidic bond to N-acetylglucosamine results in the attachment of heparan sulfate.
OH
O
Figure 17. Heparan and heparin glycosaminoglycan consist of heterogeneous mixtures of repeating units of D-glucosamine and L-iduronic acids or D-glucuronic acids, sulfation at each residue may vary.
Keratan sulfate differs from the other glycosaminoglycan chains in that it does not contain uronic acid residues. Keratan is made up of Nacetyllactosamine (βGal-β(1→4)-GlcNAc) subunits. It may be attached to the protein backbone through either N-linkage or O-linkage.3
Hyaluronan is a major component of the extracellular matrix; it binds and retains water molecules and fills the gaps between collagen fibrils. CD44, a human cell surface glycoprotein that participates in multiple cell functions binds to hyaluronan, and hyaluronan-CD44 interactions have been associated with malignant tumor invasion. The interactions of glycosaminoglycans with a variety of ligands are associated with inflammation, growth, coagulation, fibrinolysis, lipolysis and cell-matrix biology. Proteoglycans are also components of the extracellular matrix, and they interact with a variety of molecules, including cell adhesion molecules and growth factors such as transforming growth factor-β (TGF-β) and basic fibroblast growth factor (bFGF). References: 1. Trowbridge, J.M., and Gallo, R.L., Dermatan sulfate: new functions from an old glycosaminoglycan (review). Glycobiology, 12, 117R-125R (2002). 2. Salmivirta, M., et al., Heparan sulfate: a piece of information (review). FASEB J., 10, 1270‑79 (1996). 3. Funderburgh, J.L., Keratan sulfate: structure, biosynthesis, and function. Glycobiology,10, 951‑8 (2000).
Proteoglycans are the specific group of glycoproteins that have at least one glycosaminoglycan chain attached to the protein; categorization is typically by the GAG chain(s) present. Heparan/heparin sulfate and chondroitin sulfate are the most common GAGs contained by proteoglycans. In addition, most proteoglycans also contain N-linked and O-linked glycans.
24
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GLY FINAL 1207 PGD cleanxrefs2
Glycosaminoglycans and Proteoglycans
Glycosaminoglycans
Heparin sodium salt from porcine intestinal mucosa
Chondroitin 6‑sulfate sodium salt from shark cartilage
[9041‑08‑1]
~90%, balance is chondroitin sulfate A
average mol wt ~3,000 Depolymerized by peroxidolysis (free-radical induced cleavage). activity: <60 units/mg store at: room temp
store at: 2-8°C C4384-250MG
Low molecular weight
250 mg
C4384-1G
1g
C4384-5G
5g
C4384-25G
25 g
H3400-50MG
50 mg
H3400-100MG
100 mg
H3400-250MG
250 mg
H3400-1G
1g
mol wt 4,000‑6,000 Da
Glycan Component Classes
Chondroitin sulfate C sodium salt; Poly[β-glucuronic acid-(1→3)-N-acetyl-βgalactosamine-6‑sulfate-(1→4)] alternating [12678‑07‑8]
store at: room temp
Chondroitin sulfate B sodium salt
H8537-50MG
50 mg
Dermatan sulfate sodium salt; β-Heparin [54328‑33‑5]
H8537-100MG
100 mg
H8537-250MG
250 mg
from porcine intestinal mucosa, ≥90%
H8537-1G
1g
store at: 2-8°C C3788-25MG
25 mg
C3788-100MG
100 mg
Hyaluronic acid sodium salt from bovine vitreous humor
Chondroitin sulfate A sodium salt from bovine trachea Alternating Copoly β-glucuronic acid-(1→3)-N-acetyl-β-galactosamine4‑sulfate-(1→4) [39455‑18‑0]
cell culture tested Approx. 70%; balance is chondroitin sulfate C 5g
C9819-25G
25 g
store at: −20°C H7630-10MG
10 mg
H7630-50MG
50 mg
Poly(β-glucuronic acid-[1→3]-β-N-acetylglucosamine-[1→4]), alternating [9067‑32‑7] store at: −20°C
Heparan sulfate sodium salt from bovine kidney Heparitin sulfate sodium salt [57459‑72‑0]
H5388-100MG
100 mg
H5388-250MG
250 mg
H5388-1G
store at: 2-8°C H7640-1MG
High molecular weight polymer; forms the core of complex proteoglycan aggregates found in extracellular matrix.
Hyaluronic acid sodium salt from rooster comb
store at: 2-8°C C9819-5G
Poly(β-glucuronic acid-[1→3]-β-N-acetylglucosamine-[1→4]), alternating (C14H21KNO11)n [9067‑32‑7]
Hyaluronic acid sodium salt from Streptococcus equi
1 mg
H7640-5MG
5 mg
H7640-10MG
10 mg
Poly(β-glucuronic acid-[1→3]-β-N-acetylglucosamine-[1→4]), alternating [9067‑32‑7] protein ......................................................................................................... ≤1%
Heparin sodium salt from porcine intestinal mucosa [9041‑08‑1]
1g
store at: −20°C 53747-1G
1g
53747-10G
10 g
Grade I-A, activity: ~170 USP units/mg store at: room temp H3393-10KU
10,000 units
H3393-25KU
25,000 units
H3393-50KU
50,000 units
H3393-100KU
100,000 units
H3393-250KU
250,000 units
H3393-500KU
500,000 units
H3393-1MU
1,000,000 units
Your Favorite Gene™, Your Comprehensive Gene Search Tool, visit sigma.com/yfg
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GLY FINAL 1207 PGD cleanxrefs2
Glycan Component Classes
Glycosaminoglycans and Proteoglycans
Proteoglycans and Associated Proteins
Heparan sulfate proteoglycan
Aggrecan from bovine articular cartilage
HSPG
lyophilized powder Major structural proteoglycan of cartilage extracellular matrix. Large proteoglycan with a molecular weight greater than 2,500 kDa. Approximately 100‑150 glycosaminoglycan (GAG) chains are attached to the core protein (210‑250 kDa). The majority of the GAG chains are chondroitin/dermatan sulfate with the remainder being keratan sulfate. This structural molecule produces a rigid, reversibly deformable gel that resists compression. It combines with hyaluronic acid to form very large macromolecular complexes. Addition of small amounts (0.1‑2% w/w) of hyaluronic acid to an aggrecan solution (2mg/ml) results in the formation of a complex with an increased hydrodynamic volume and in a significant increase (30‑40%) in the relative viscosity of the solution. Aggrecan is a critical component for cartilage structure and the function of joints. The synthesis and degradation of aggrecan are being investigated for their roles in cartilage deterioration during joint injury, disease, and aging. Contains three globular domains, G1, G2, and G3, that are involved in aggregation and hyaluronan binding, cell adhesion, and chondrocyte apoptosis. salt ............................................................................................... essentially free store at: −20°C A1960-1MG
1 mg
Biglycan from bovine articular cartilage essentially salt-free, lyophilized powder (from a sterile-filtered solution) Interacts with collagen type I, as well as with fibronectin and TGF-β. mol wt 200‑350 kDa (proteoglycan consisting of a 45 kDa core protein and two chrondroitin/dermatan sulfate glycosaminoglycan chains)
≥400 μg/mL glycosaminoglycan, sterile-filtered Extracellular matrix component that binds to fibroblast growth factors, vascular endothelial growth factor (VEGF) and VEGF receptors through its sugar moiety. Acts as a docking molecule for matrilysin (MMP-7) and other matrix metalloproteinases. For cell culture use. Isolated from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma. Solution in 50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, pH 7.4, containing ≥400 μg protein per ml. Uronic acid ........................................................................................ ≥100 μg/mL ship: dry ice store at: −20°C H4777-.1MG
0.1 mg
Proteoglycan from bovine nasal septum Chromatographically purified using 7M urea on DEAE-cellulose by modified procedure by Antonopoulos.1 Lit. cited: 1. Antonopoulos, C.A., et al., Biochim. Biophys. Acta 338, 108‑119 (1974) store at: −20°C P5864-10MG
10 mg
Proteoglycan from chicken sternal cartilage Chromatographically purified using 7M urea on DEAE-cellulose by modified procedure by Antonopoulos.1 Lit. cited: 1. Antonopoulos, C.A., et al., Biochim. Biophys. Acta 338, 108‑119 (1974) store at: −20°C P5989-2MG
2 mg
store at: −20°C B8041-.5MG
0.5 mg
Decorin from bovine articular cartilage salt-free, lyophilized powder, sterile-filtered Decorin interacts with collagen type I and II, fibronectin, thrombospondin and TGF-β. Decorin is an approx. 100 kDa proteoglycan consisting of a 40 kDa core protein and one chondroitin or dermatan sulfate glycosaminoglycan chain. store at: −20°C D8428-.5MG
Tissue Inhibitor of Metalloproteinase-1 human TIMP-1
recombinant, expressed in CHO cells, ~500 μg/mL protein, buffered aqueous solution TIMP-1 has greater binding efficiency to MMP-9, MMP-1, and MMP-3 than the other MMPs. Supplied in 0.01M sodium phosphate buffer pH 7.3, 0.15M NaCl. mol wt ~29 kDa ship: wet ice store at: −20°C T8947-5UG
5 μg
0.5 mg
Transforming Growth Factor-β Soluble Receptor III human TGF-β sRIII
>97% (SDS-PAGE), recombinant, expressed in mouse NSO cells Lyophilized from a 0.2 μm filtered solution in phosphate buffered saline containing 5 mg bovine serum albumin The receptor-mediated activity is measured by its ability to inhibit the TGF-β2 bioactivity in HT-2 cells. Endotoxin .................................................................................................. tested store at: −20°C T4567-.1MG
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0.1 mg