asparagine-linked glycosylation overview asparagine-linked glycosylation (alg; also n-linked...

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Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation referenced so far. ALG is the most common type of glycosylation ALG is an essential and highly conserved protein modification pathway in

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Page 1: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Asparagine-linked glycosylation Overview

• Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation referenced so far.

• ALG is the most common type of glycosylation

• ALG is an essential and highly conserved protein modification pathway in eukaryotic cells

Page 2: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

N-linked Glycosylation

pathway

Page 3: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Glycosylation level of Carboxy Peptidase Y (CPY)

The Elo3H2 chimera can partially restore CPY glycosylation

1: the strain has been grown in YPD4% (repressive condition)2: the strain has been grown in YPGal2%glu0,05% (permissive condition) indicates the fully glycosylated form of CPY

W1536 Δrft1::KanMx / PMC195 GAL RFT1*

1 2

W1536 Δrft1::KanMx / pYES Elo3H2

1 2W1536 wild type

1 2

* RFT1 expressed from the GAL promoter (gift from Mingjie Cai, Singapore)

• •

Page 4: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

If the microsomal chain elongation does not seem to be affected by the RFT1 deletion, is glycosylation

affected by VLCFA synthesis?

Page 5: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Effect of inactivation of VLCFA elongation enzyme

Enzymes involved in the fatty acid elongation are unlikely to play a role in CPY glycosylation

1 2 3 4 5 6 7 8

1-2 : W1536 Δelo2::KanMx Δelo3::KanMx / YCp GAL ELO3

3-4 : W1536 Δrft1::KanMx / PMC195 RFT1*

5-6 : W1536 Δtsc13::KanMx / YCp GAL TSC13

7-8 : W1536 Wild type

Odd number: strains grown in YPD4% (repressive condition)Even number: strains grown in YPGal2%glu0,05% (permissive condition)

Page 6: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Test if enzymatic activity of the hydratase 2 domain was required for “complementation” (point mutations in active site)

Result: activity not important!

Page 7: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Controls made to check which part of the construct is responsible for the complementation:

Test: Result:

Over expression of the full length Elo3p (single or multicopy plasmid)

No complementation

Over expression of only ELO3 transmembrane domains used to make the chimera

No complementation

Over expression of the hydratase2 in the cytosol

No complementation

Over expression of a chimera made from the ELO3 part and GST1

Complementation

Over expression of a chimera made from the ELO3 part and hFilaminC

Complementation

Over expression of a chimera made from the ELO3 part and GFP

Complementation

Page 8: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Collaboration with Markus Aebi at the ETH Zurich

Measuring of glycans in rft1 mutants complemented with the Elo3-H2 chimera

Partial restoration!Also: screen for rft1 mutation*

suppressors yielded in MANY suppressing library plasmids (all of which were expressing truncated ER transmembrane proteins

* An rft1Δ strain can grow weakly on 1 M Sorbitol media; suppressors were identified that improved growth of the mutants

Page 9: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Investigation of the glycan flipping specificity

In contrast to Rft1p, the truncated Flc2* protein can flip an immature glycan precursor

* FLC2: truncated form of the Flc2p putative FAD transporter; has similar suppressor activity as the Elo3-H2 chimera

Page 10: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Rft1p and the ERAD

• What is the relation between the ER associated degradation machinery and glycan flipping?

30°C 37°C

wt

Drft1

Dubc7

Dubc7 Drft1

Deleting UBC7 suppresses the Drft1 phenotype

(Ubc7p: Ubiquitin conjugating enzyme, involved in the ER-associated protein degradation pathway)

Page 11: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Drft1 Drft1 Dubc7

Dubc7wt

Cells were grown at 37°C (to stress the ERAD)

Indirect evidence for improved flipping in Drft1 with membrane proteins by blocking the ERAD

24°C 24°C28°C 28°C34°C 34°C37°C 37°C

Drft1 Drft1Dubc7

aCPY western

mCPY-1-2-3-4

Page 12: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Hypothesis

• Whenever a complex is not fully assembled its components are targeted for degradation

• If the degradation process is not working properly, we have accumulation of incomplete protein complex on the membrane

Page 13: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

• If Rft1p is not present the flipping is unspecific, and depends on membrane homeostasis

ERAD is an essential process to maintain ER homeostasis which is a prerequisite for lipid asymmetry in cellular membranes

Page 14: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Summary

• RFT1 mutants were obtained via both EMS and transposon mutagenesis

• Rft1p can be suppressed by overexpression of truncated transmembrane domain proteins

• Glycan flipping is unspecific in the presence of these suppressors

• Our results provide a new insight to the flipping process on membranes

• One of the ERAD function may be to keep membrane homeostasis

Page 15: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

• In humans patients N-glycosylation deficiency lead to different symptoms correlated to the severity of the disease.

Aebi, M. & Hennet, T. (2001) Trends Cell Biol. 11, 136-141

Range of symptoms found in specific types of CDG

• Human RFT1 deficiency leads to a disorder of N-linked glycosylation.

• Haeuptle MA*, Pujol FM*, Neupert C, Winchester B, Kastaniotis AJ, Aebi M, Hennet T. Am J Hum Genet. 2008;82(3):600-6

Page 16: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Postgenomic approaches

Page 17: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Postgenomics?

• Advances in sequencing technology allows rapid sequencing of whole genomes of different organisms

• Large amounts of sequence data now available

• Development of new methods to take advantage of the availability of genomic data

Page 18: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

DNA microarray principles• http://www.transcriptome.ens.fr/sgdb/presentation/principle.php

http://www.bio.davidson.edu/courses/genomics/chip/chip.html

Page 19: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

DNA microarray formats

Size: 12cm x 8cm 2400 clones by membrane radioactive labeling 1 experimental condition by membrane

Size: 5,4cm x 0,9cm 10000 clones by slide fluorescent labelling 2 experimental conditions by slide

Size: 1,28cm x 1,28cm 300000 oligonucleotides by slide fluorescent labelling 1 experimental condition by slide

High Density Filters (macroarrays)

Glass slides (microarrays)

Oligonucleotides chips

(Affymetrix proprietary technology )

Page 20: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Power and Pitfalls of Microarray Transcriptional Analysis

• One microarray experiment = 6100 northern blots (in yeast)

• Entire transcriptional response of the whole cell can be monitored (transcriptional profiling)

• Entire Groups of genes that follow the same transcription pattern can be identified clue on function?

Power:

• Dynamic range limited• Significance of observed changes not always clear• May require further validation of results• Posttranslational events completely unaccounted for• “Guilt by association” assumption often not true

Pitfalls

Page 21: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Correlation of Expression of Mitochondrial FAS Genes During the

Diauxic Shift

Page 22: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Molecular Barcodes

Steinmetz LM, Davis RW Maximizing the potential of functional genomics.Nat Rev Genet. 2004 Mar;5(3):190-201. Review

Page 23: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Large scale synthetic lethal screen

• ~ 1100 gene deletions in yeast result in inviability of the deletion strain (=essential genes)

• 5100 “non-essential” genes – cells with deletion can survive• With which other gene deletions does any one of these

gene deletions display a synthetically lethal or synthetically sick phenotype?

Development of a large-scale method to tackle this question “SGA (synthetic genetic array) Analysis”

BNI1 and BNR1 both encode members of the highly conserved formin family and control the assembly of actin cables, which guide myosin motors that coordinate polarized cell growth and spindle orientation

Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C.Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 2001

Dec 14;294(5550):2364-8.

Page 24: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

- “Query”- strain (gene of interest – BNI1- deleted and replaced with dominant nourseotricin resistance cassette); carries an integrated fusion of HIS3 to the regulatory region of MFA1 (a-specific gene; only expressed in haploid a cells)

- mated against 4700 individual yeast deletion strains; each deletion was accomplished by gene replacement with a different dominant drug resistance cassette (geneticin resistance)

- sporulation- selection of haploids on media

lacking histidine- selection of double mutants on media

containing both drugs noursetricin and geneticin

- Scoring for double mutants unable to grow

Page 25: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

- bnr1, cla4 and bud6 deletions are synthetically lethal/sick with bni1Δ

- bnr1 Δ has previously been known to be synthetically lethal with bni1Δ (positive control)

Result of initial “small scale” experiment

Page 26: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Results of large scale approach• 67 potential synthetically lethal/sick interactions

identified• 51 (76%) SL interactions confirmed by tetrad

analysis• Substantial amount of false positives, which can

be reduced by repeated screening or tetrad analysis

• BNI1 interactions were highly enriched for genes with roles in cell polarity (20%), cell wall maintenance (18%), and mitosis (16%)

• In total 43 previously unknown synthetic genetic interactions for bni1 were discovered , including 9 genes of unclassified function

Page 27: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Results displayed as “Interaction Network”

Page 28: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Caveats

• Essential genes not included in analysis• Interactions can be missed:

Goehring AS, Mitchell DA, Tong AH, Keniry ME, Boone C, Sprague GF Jr. Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome

activation. Mol Biol Cell. 2003 Apr;14(4):1501-16.

Page 29: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Systems biologyDefinition of characteristics:• Multidisciplinary research with a close interaction

between experimental research and mathematical modeling and analysis

• Aspires to generation and iterative improvement of mathematical models which describe realistically cellular networks or dynamic biological processes, and which have predictive power

• Requires use of existing and generation of novel data, in particular quantitative data, time courses and spatial information of high definition

http://www.gmm.gu.se/globalysbn/WhitePaperYSB_may2004.pdf

Page 30: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Schematic description

Page 31: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Yeast Systems Biology

• S. cerevisiae as a model to advance understanding of the rules and principles of the dynamic operation of cells

• Recreation of a yeast cell in silico, where a computer model of a yeast cell will predict the reaction of a yeast cell to a certain stimulus

• Currently the ideal eukaryotic organism, because best characterized

Page 32: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Yeast Systems Biology Network

• http://www.gmm.gu.se/YSBN/• Umbrella organization• Research groups from all over the world

contribute• Established to coordinate efforts, make

technologies and data available• Offers training• Public relations tool

Page 33: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Modeling the Budding Yeast Cell Cycle

• Well studied pathway• Conserved• Regulatory network established• Computer modeling will help to query the

existing model of the pathway• Comparison of computational results with

results of experiments will help in the generation of new experiments ( iterative improvements..)

Page 34: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

A “wiring diagram” for yeast cell cycle controlhttp://mpf.biol.vt.edu/research/budding_yeast_model/pp/index.php#

Page 35: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

The rate of Cln2 synthesis, k's,n2 + k''s,n2 · [SBF], includes a constitutive rate, k's,n2 (for simulation of GAL-CLN2 mutants), and a contribution dependent on the relative activity of the transcription factor SBF, controlling expression of the CLN2 gene

Cln2 degradation is assumed to be a simple first-order process, kd,n2 · [Cln2] with half-life = 0.693/kd,n2 = 6 min, in reasonable accord with experiment

Chen, K.C., Calzone, L., Csikasz-Nagy, A., Cross, F.R., Novak, B. and Tyson, J.J. (2004). Integrative analysis of cell cycle control in budding yeast. Mol. Biol. Cell 15:3841-3862.

http://www.molbiolcell.org/cgi/content/full/15/8/3841

Page 36: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Model Versus Reality

• http://mpf.biol.vt.edu/research/budding_yeast_model/pp/problems.php

Page 37: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

New Challenges in Systems Biology

• Genetic approaches have often been of qualitative nature (Yes/No)

• Molecular Biology has to become more quantitative

• Measurement or prediction of Amount, Interactions, Modifications and Spatial movements at each XYZ+T (=AIMS-4D) necessary for a good basis for accurate modeling

Page 38: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

• Many values provided in the literature not accurate (enough?)

• Subcellular localization and concentrations of proteins and substrates in the single cell have to be known

• Need for establishment of accurate kD, kM, kcat, …

Page 39: Asparagine-linked glycosylation Overview Asparagine-linked glycosylation (ALG; also N-linked glycosylation) is one of the 41 different types of glycosylation

Cellular biochemistry will remain to be important!!!