Glycoproteins

1. Glycoproteins

2. Glycoproteins • Mammalian glycoproteins contain three major types of oligosaccharides (glycans): • N-linked, • O-linked, and • glycosylphosphatidyl- inositol (GPI) lipid anchors.

3. Glycoproteins N-Linked glycans are linked to the protein backbone via an amide bond to asparagine residues in an Asn-X-Ser/Thr motif, where X can be any amino acid, except Pro. O-Linked glycans are linked to the hydroxyl group of serine or threonine. GPI-anchored proteins are attached at their carboxy-terminus through a phosphodiester linkage of phosphoethanol- amine to a trimannosyl glucosamine core structure. The reducing end of the latter moiety is bound to the hydrophobic region of the membrane via a phosphatidyl- inositol group.

4. Chemical diversity of glycans

5. Symbolic Representation of Oligosaccharides

6. Symbol and Text Nomenclature for Representation of Glycan Structure Nomenclature Committee Consortium for Functional Glycomics The Nomenclature committee evaluated symbol nomenclatures in wide use, consulted with a variety of interested parties, and selected a version adapted by the Editors of “Essentials of Glycobiology” from that originally put forward by Stuart Kornfeld, and further modified for the upcoming second edition. The selected version fits Consortium needs based on the following criteria:

7. Consortium for Functional Glycomics • The nomenclature must be convenient for the annotation of mass spectra. To this end, it was decided that: • Each sugar type (eg sugars of the same mass: hexose, hexosamine and N-acetylhexosamine), should have the same shape. • Isomers of each sugar type (eg mannose/galactose/glucose) should be differentiated by color or shading. • Where possible, the same color or shading should be used for derivatives of hexose (eg the corresponding N-acetylhexosamine and hexosamine). • Using the same shape but different orientation to represent different sugars should be avoided so structures can be represented either horizontally or vertically.

8. Consortium for Functional Glycomics • The color version of the nomenclature should appear indistinguishable from the black and white version when copied or printed in black and white. • Because 10% of the population is color blind, the use of both red and green for the same shaped symbols should be avoided. • If desired, linkage information can be represented in text next to a line connecting the symbols (e.g. α4, β4).

11. Consortium for Functional Glycomics Text nomenclature • The committee recommends a “modified IUPAC condensed” text nomenclature which includes the anomeric carbon but not the parentheses, and which can be written in either a linear or 2D version. The Committee felt that: • Including the anomeric carbon is important, and is likely to become increasingly more so in the future as more complicated structures are discovered. • The presence of parentheses (which then necessitates the use of brackets to indicate branching structures) is unnecessarily cumbersome, particularly when representing the structure in 2D form.

12. Consortium for Functional Glycomics

13. Consortium for Functional Glycomics Examples of symbol nomenclature used to illustrate N- and O-linked glycans written in the 2D version of the text nomenclature. Note that symbol structures will be used to annotate data where linkages have not been defined (e.g MALDI profiling), and if linkages between monosaccharides are known, they can be added above or to the side of the line connecting the symbols if desired (e.g. α6 or β4).

14. Consortium for Functional Glycomics

15. Objections to USA Scheme The linkage information is only conveyed by the use of numeric notation . This makes the symbols clumsy and when the size is reduced the numeric notation becomes impossible to read. using the angle and dotting of the lines to represent linkage information this can be displayed clearly • The symbols and shadings/colours are arbitrary.   A scheme where derivatives of the basic monosaccharide are filled or shaded is clearer • A scheme where all basic monosaccharides have different shapes is clearer in print and reduced size

16. Comparison of Symbols

17. Simplified Text and Symbolic Representation of Glycosaminoglycans (GAGs) GalNAc4Sb4GlcAb3GalNAc4Sb4IdoAa3GalNAc4Sb4IdoA2Sa3GalNAc4Sb4GlcAb3 Chondroitin/Dermatan Sulfate

18. EuroCarbDB

21. GlycanBuilder • Users can select from five graphical display schemes for glycan structures. As an example structure a complex N-glycan is shown in • The IUPAC Text mode, • the CFG symbolic format with linkage labels, • the CFGL format with linkage positions shown geometrically, • the Oxford black & white (UOXF) • and color (UFOXCOL) schemes, where linkage positions are shown by geometry and anomeric configurations are denoted by dashed (α) or solid (β) lines.

22. GlycanBuilder Note that this structure is indefinite since the linkage positions of the terminal GalNAc residues are not defined.

23. Glucuronic acid Iduronic acid Fucose Glucose Mannose Galactose N-Acetylglucosamine Xylose N-Acetylneuraminic acid (Sialic Acid)

24. N-linked Glycoproteins All eukaryotic cells express N-linked glycoproteins. Protein glycosylation of N-linked glycans is actually a co-translational event, occurring during protein synthesis. N-linked gly- cosylation requires the consensus sequence Asn-X-Ser/Thr. Glycosylation occurs most often when this consensus sequence occurs in a loop in the peptide.

25. Basic N-linked Structure

26. N-linked Glycoproteins In the Golgi, high mannose N-glycans can be converted to a variety of complex and hydrid forms which are unique to vertebrates.

27. N-linked Glycoproteins • The diverse assortment of N-linked glycans are based on the common core pentasaccharide, Man3GlcNAc2. • Further processing in the Golgi results in three main classes of N-linked glycan sub-types; • High-mannose, • Hybrid, • and Complex..

28. High-Mannose Structure

29. Hybrid Structure

30. Complex Structure (tetraantennary)

31. N-linked Glycoproteins The oligosaccharide precursor is transferred en bloc from dolichol to Asn residues in the sequence Asn -X-Ser/Thr by oligosaccharyltransferase.

32. N-linked Glycoproteins

33. N-linked Glycoproteins Complex glycans contain the common triman- nosyl core. Additional monosaccharides may occur in repeating lactosamine units. Additional modifications may include a bisecting GlcNAc at the mannosyl core and/or a fucosyl residue on the innermost GlcNAc. Complex glycans exist in bi-, tri- and tetraantennary forms

34. Different N-linked glycans structures Human proteins α(2,3) β(1,4) β(1,2) α(1,6) α(1,6) β(1,4) β(1,4) α(1,3) β(1,4) β(1,2) α(2,6) Plant proteins α(1,4) β(1,2) α(1,6) β(1,4) β(1,4) β(1,3) α(1,3) α(1,3) β(1,2) α(1,4) β(1,2) β(1,3) ‘Lewis a’ epitope Xyl Gal NeuAC Fuc Man GlcNAc

35. Examples of oligosaccharides found in N-linked glycoproteins

36. Example oligosaccharides found in N-linked glycoproteins

37. : Folded peptide chain : GlcNAc : Mannose : Galactose : Sialic acid : Phosphoric acid : Glucose Different N-glycosylation in Golgi-complexes Ribosome Dolichol mRNA -Glucosidase I -Glucosidase II Oligosaccharyl Transferase Calnexin Folding Endoplasmic Reticulum ER -Mannosidase Glc3Man9GlcNAc2 Man8GlcNAc2 Plant Golgi Yeast Golgi Mammalian Golgi -1,2-Mannosidase I -1,6-Mannosyl Transferase (OCH1) -1,2-Mannosidase I N-acetyl-glucosaminyl-transferase I (GnT-I) UDP-GlcNAc Transporter UDP-GlcNAc Transporter GnT-I -1,3-Mannosyl Transferase (MNN1) -Mannosidase II ⇒Fuc-Transferase -Mannosidase II -1,2-Mannosyl Transferase (MNN2,5?) F Mannose-6- Phosphate Synthesis (MNN4,6 and others) GnT-II GnT-II F UDP-Gal Transporter Xyl-Transferase GalT n X F CMP-NeuAc synthetase N-acetyl-Glucosaminidase SAT X F Mannan Type Complex Type Complex Type

38. Mammalian N-glycans

40. O-linked Glycoproteins Function in many cases is to adopt an extended conformation These extended conformations resemble "bristle brushes" Bristle brush structure extends functional domains up from membrane surface

41. O-linked Glycoproteins O-Linked glycans are usually attached to the peptide chain through serine or threonine residues. O-Linked glycosylation is a true post-translational event and does not require a consensus sequence and no oligosaccharide precursor is required for protein transfer. The most common type of O-linked glycans contain an initial GalNAc residue (or Tn epitope), these are commonly referred to as mucin-type glycans. Other O-linked glycans include glucosamine, xylose, galactose, fucose, or manose as the initial sugar bound to the Ser/Thr residues.

42. O-linked Glycoproteins Glycosylation generally occurs in high-density clusters and may contribute as much as 50-80% to the overall mass. O-Linked glycans tend to be very heterogeneous, hence they are generally classified by their core structure.

43. Di- and Trisialated O-Linked Core

44. O-Linked Core 2 Hexasaccharide

45. Core 2 Core 1 Core 3 Core 4

46. Core 5 Core 6 Core 7 Core 8

47. Proteoglycans Extracellular aggregate of protein and glycosaminoglycans Core protein Oligosaccharide glycosidic bond to O of Ser or Thr

48. Blood Group Substances O-Linked glycans are involved in hematopoiesis, inflammation response mechanisms, and the formation of ABO blood antigens.

49. Blood Group Substances Membranes of animal plasma cells have large numbers of relatively small carbohydrates bound to them • these membrane-bound carbohydrates act as antigenic determinants • among the first antigenic determinants discovered were the blood group substances • in the ABO system, individuals are classified according to four blood types: A, B, AB, and O • at the cellular level, the biochemical basis for this classification is a group of relatively small membrane-bound carbohydrates

50. Type A Blood