-Techniques in Glycobiology-NHLBI CardioPEG – Gerald W.Hart, September 17, 2013

Funded by NHLBI P01HL107153

Analysis ofGPI-Anchors

VSG’s Critical Role in History of GPI-Anchor Analysis:

2

3

4

T. Brucei 107 VSG per Cell!

In Blood Stream Trypansomes Defeat the Immune System by Switching Their VSG

Coats!

5

6Ferguson, M..A.J. Journal of Cell Science 112, 2799-2809 (1999)

Tightly Packed VSG Shields the Parasite From the Immune System:

7

Relationship of GPI-Anchors to Phosphatidylinositol Ram VishwakarmaCSIR-Indian Institute of Integrative Medicine, JammuNational Institute of Immunology, New Delhi Piramal Life Sciences Ltd, Mumbai

Amide Linkage toCOOH Terminus ofProtein.

Non-Acetylated GlcN

9

Structure of Triton-X114

10n=7 or 8

Triton-X114 Partitioning is a Simple & Powerful Method for Isolation of GPI-Anchor Proteins.

11

Example of the Use of TX-114 Partitioning for GPI-Anchored Proteins:

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TX-114 is a Powerful Tool:

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Tot P insol. Aq pellet final Det phase

Isolation in SDS conserves Anchor, but short incubation endogenous GPI-PLC Releases sVSG

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Coomassie

SDS Hepes Hepes Hepes +NP40 5mM Zn

3H-myr

T T TS S SP PP

Purification of mfVSG and sVSG:

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Wcells CM Ifacial Aq ph Wcells TCA ppt CM:MetTCA of Supnt of 3

1. TCA PPt Cells

2. Extract pellet with Chloroform:Methanol

3. Add NaCl soln. to form 2 phases

4. mfVSG in Aqueous

1. Incubate tyrps in buffer with organic to lyse cells

MfVSG has ONLY Myristic Acid:

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sVSG

mfVSG

Separation of mfVSG and sVSG on 7.5% SDS-PAGE

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mfVSG Mix sVSG

Studying GPI Biosynthesis in vitro

cell membranessalts,buffersradiolabeled Sugar donor

30 °C

add solventsspin

evaporate

F

O

thin layerchromatography

O F

GPI-Anchor Biosynthetic Pathway Was Mostly Elucidated byThin-Layer Chromatography and Pulse-Chase Labelling:

From Varki

Radiolabeling & TLC Were Used to Elucidate the GPI-Anchor Biosynthetic Pathway:

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Incorporation of 3H-Glucosamine

20

Cell, Vol. 56, 793400, March 10, 1989,

UDP-[3H]GlcNAc UDP-[3H]GlcNAc In Vivo

Pulse-Chase Labeling Reveals Intermediates in Pathway:

21Cell, Vol. 56, 793400, March 10, 1989,

Pulse with UDP-3H-GlcNAcChase UDP-GlcNAc & GDP-Mannose

No

GD

P-M

an

3H -Myrstate

No

UD

P-G

lcN

Ac

MPD Trea

ted

with

GP

I-PLC

In Vivo- +PI-PLC

Early Studies SuggestedLipid Remodeling of GPI-AnchorIn

vitr

o U

DP

-Glc

NA

c +

GD

P-M

an

Lyso-Lipid RemodelingIntermediate

Inositol Acylated

A=dimyrisoyl GPI

A’=other F.A. GPI

22Cell, Vol. 56, 793400, March 10, 1989,

Characterization of GPI-Anchor Intermediates:

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Glycolipids A and A’ Have The Same Glycan Structure:

Cell, Vol. 56, 793400, March 10, 1989,

Gel Filtration – P4

Dionex HIPC-ASG

IntermediatesAfter HONO Treatment

Leaves Glycan with AHMAt Reducing Terminus.

Biosynthetic Pathway of VSG GPI

24FEBS Letters 584 (2010) 1670–1677

Insect Form of Parasite

Blood Stream Form

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Schematic Structure & Cleavage Sites of GPI-Anchors:

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Flow Chart For GPI-Anchor Protein Analysis:

Chemical and enzymatic reactions of GPI anchors

Chapter 11, Figure 3Essentials of Glycobiology

Second Edition

Ferguson et al. Partial Structure Elucidation of GPI-

Anchor:

28

JBC Vol. 260, pp. 14547-14555 1985

1. Treatment with HONO releases intact PI.

2. Treatment of sVSG (released by PI-PLC) with Pronase yields glycopeptide.

3. GP insensitive to alkaline phos. & yields myo-inositol 1-P on HONO treatment.

4. Mild acid renders sensitive to alk phos.

5. Subsequent HONO yields myoinositol

6. HONO yields AHM.

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3,4

5 6

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Linkages Were Determined by Methylation Analysis: Glycan Methylate all Free Hydroxyl Groups (Ether Linkages, Acid Stable)Acid HydrolyzeReduce and Per-Acetylate (Alditol Acetates) GC Analysis

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Size of the Glycan Moiety And Structure Was Estimated by Gel Filtration in Combination With Specific Degradation (Easily Resolve a Hexose Difference):

31

NMR Was Used to Determine the Glycan Structure of the GPI:

Proteomics/Glycomics of GPI-Proteins

32Molecular & Cellular Proteomics 2: 1261–1270, 2003.

Isolation of GPI-anchored Proteins:

33Molecular & Cellular Proteomics 2: 1261–1270, 2003.

>CRD Ab Released to Sol by PLC

Example of MS Data:

34Molecular & Cellular Proteomics 2: 1261–1270, 2003.

Band 1

MS/MS Identification:

35Molecular & Cellular Proteomics 2: 1261–1270, 2003.

Algorithms for Predicting GPI-Anchor Attachment:

36

Conclusions: GPI-Anchor’s Unique Structures Require

Unique Approaches. PI-PLC release from membranes is diagnostic

for GPI-Anchors Triton-X114 Partitioning is a Great Method to

Start Purification of GPI-Anchored Proteins Reductive Labeling of Anhydromannose after

nitrous acid treatment is a powerful tool. Aqueous HF releases GPI from protein. Sizing and sequential use of glycosidases use

same methods as other oligosaccharides. Consensus sequence for predicting GPIs.

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