Synthetic Studies of Glycosylphosphatidylinositol (GPI) Anchors and GPI-Anchored Peptides, Glycopeptides, and Proteins

Abstract

Glycosylphosphatidylinositol (GPI) anchorage of proteins and glycoproteins onto the cell surface is ubiquitous in eukaryotes, and GPI-anchored proteins and glycoproteins play an important role in many biological processes. To study GPI anchorage and explore the functions of GPIs and GPI-anchored proteins and glycoproteins, it is essential to have access to these molecules in homogeneous and structurally defined forms. This review is focused on the progress that our laboratory has made towards the chemical and chemoenzymatic synthesis of structurally defined GPI anchors and GPI-anchored peptides, glycopeptides, and proteins. Briefly, highly convergent strategies were developed for GPI synthesis and were employed to successfully synthesize a number of GPIs, including those carrying unsaturated lipids and other useful functionalities such as the azido and alkynyl groups. The latter enabled further site-specific modification of GPIs by click chemistry. GPI-linked peptides, glycopeptides, and proteins were prepared by regioselective chemical coupling of properly protected GPIs and peptides/glycopeptides or through site-specific ligation of synthetic GPIs and peptides/glycopeptides/proteins under the influence of sortase A. The investigation of interactions between GPI anchors and pore-forming bacterial toxins by means of synthetic GPI anchors and GPI analogs is also discussed.

Keywords: Carbohydrate; GPI; Glycolipid; Glycopeptide; Glycosylphosphatidylinositol; Peptide; Protein; Sortase.

Figure 1

The highly conserved core structure (1) of protein/glycoprotein-anchoring GPIs, as well as GPI anchorage of surface proteins and glycoproteins onto cell membranes through the insertion of GPI lipid chains into the cell membrane bilayer.

Figure 2

Representative structure of the human CD52 antigen

Figure 3

Other GPI anchors and GPI derivatives carrying Pd-catalyzed hydrogenolysis-incompatible functionalities synthesized by the convergent strategy using the PMB group for global and permanent hydroxyl protection

Figure 4

Key intermediates involved in the synthesis of functionalized GPI anchor 74

Figure 5

GPI-anchored glycopeptide 98 prepared by chemical coupling of glycopeptide 99 and GPI

Figure 6

The addition of GPIs to target proteins to form GPI-anchored proteins is achieved in the ER lumen via GPI transamidation catalyzed by GPI-T, with both the GPI anchor and the nascent protein, as well as the enzyme, attached to the ER membrane inner surface. This reaction is regulated by a peptide signal near the nascent protein C-terminus, which is cut off during the transamidation process. GPI-anchored proteins formed can be further processed and finally delivered onto the cell surface through the Golgi network.

Figure 7

Sorting pathway of surface proteins in gram-positive bacteria. After surface proteins containing a sorting signal at the C-terminus are synthesized in the bacterial cytoplasm, they are translocated onto the cell surface. Sortase cleaves a peptide bond of the sorting signal to generate a reactive acyl intermediate and then transfers the acyl group to the N-terminus of the pentaglycine cross bridge of the cell wall peptioglycan. As a result, surface proteins are anchored onto the bacterial cell wall.

Figure 8

Inhibition of CAMP factor-induced sheep erythrocyte lysis by 139. After CAMP factor was first incubated with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) alone or with DPPC and the indicated concentrations of 121 (see labels in the figure), its solution was added to the sphingomyelinase-sensitized erythrocytes. Then, hemolysis was observed by decreasing cell turbidity at OD600.

Scheme 1

A widely adopted strategy for the synthesis of GPI anchors

Scheme 2

Synthesis of the inositol derivative 6 suitable for the synthesis of CD52 GPI anchor

Scheme 3

Alternative synthesis of inositol derivatives having differentiated 1,2,6-O-positions

Scheme 4

Synthesis of the glycosaminyl donor 31

Scheme 5

Synthesis of phospholipidated pseudodisaccharide 32

Scheme 6

Convergent assembly of CD52 GPI anchor 43

Scheme 7

Convergent synthesis of GPIs and GPI analogs with phospholipidated psudodisaccharide 44 as key intermediate

Scheme 8

Synthesis of optically pure inositol derivative 50

Scheme 9

Synthesis of PMB-protected trimannosyl donor 63

Scheme 10

Assembly of GPI 72 carrying unsaturated lipid chains

Scheme 11

Synthesis of the natural GPI anchor 73 of the human lymphocyte CD 52 antigen

Scheme 12

Synthesis of 75 carrying a “clickable” azido group

Scheme 13

synthesis of GPI-anchored peptide 97

Scheme 14

Solid-phase synthesis of glycopeptide 99

Scheme 15

Synthesis of glycan-asparagine conjugate 101

Scheme 16

Final assembly of GPI-anchored glycopeptide 98

Scheme 17

A general strategy for GPI-anchored protein synthesis based on sortase-mediated GPI-protein ligation

Scheme 18

SrtA-mediated coupling reactions of peptides and GPI analogs

Scheme 19

SrtA-mediated chemoenzymatic synthesis of CD52 analogs

Scheme 20

SrtA-mediated chemoenzymatic synthesis of CD52 and CD24 analogs

Scheme 21

SrtA-mediated chemoenzymatic synthesis of GPI-anchored glycopeptides