The Mycobacterial Cell Wall—Peptidoglycan and Arabinogalactan
Arabinogalactan proteins
description
Transcript of Arabinogalactan proteins
Arabinogalactan proteins
Pbio691 - Plant Cell Wall11/05/2010
Laura Cristea
AGPs
Cellulose
Hemicellulose
Proteins
Plant primary cell wall
AGPsOverview
HRGPs Lowest protein content among HRGPs (1-10%) Highest sugar content among HRGPs (90-99%) Complex glycosylation modules Protein backbone O-glycosylated Ara, Gal, Rha, GlcUA, Fuc Soluble or GPI-anchored Associated with plasma membrane, cell wall Regulation, signaling, growth and development Cell-cell interaction, pathogen defense Substrate for pollen growth, wound-induced (gum arabic)
AGPsClassification based on
structure Classical Hyp-rich AGPs (A)
Classical AGPs with Lys-rich domain (B)
AG peptide (12 aa) (C)
Nonclassical AGPs with Asn-rich domain (D)
Proteins with two AGP and two fasciclin domain (E)
Proteins with two AGP and one fasciclin domain (F)
Proteins with one AGP and one fasciclin domain (G)
Albersheim, P. et al. Plant Cell Walls (2010)
AGPs
Gum arabic
Ala-poor, His-rich
Extensin motif
Intermediate between AGPs and extensins
Repetitive consensus motifSer-Hyp-Hyp-Hyp-Thr-Leu-Ser-Hyp-Ser-Hyp-Thr-Hyp-Thr-Hyp-Hyp-Leu-Gly-Pro-His
Sugar composition resembles the AGPs with arabinose and galactose as major ones
Gum arabic glycoprotein
AGPs
Secretory pathway Hydrophobic C-terminal – GPI Ala, Thr, Ser, Pro, Hyp rich ER – protein backbone Prolyl hydroxylase – not all Pro Golgi – Hyp-O-glycosylation –not all Hyp Glycosyltransferases Hyp glycosylation hypothesis
beta glucosyl Yariv reagent – identification problems
Biosynthesis
Buchanan, Gruissem, Jones Biochemistry & Molecular Biology of Plants
AGPsProlyl hydroxylase
Albersheim, P. et al. Plant Cell Walls (2010)
Post-translational Type II integral membrane protein Affinity – > 4 Pro residues Atmosferic oxygen needed O of 4-OH from Hyp – oxygen
AGPs
Contiguous Hyp residues are arabinosylated (extensins)
Noncontiguous Hyp are not glycosylated or just have one arabinose
Clustered Hyp residues are linked to a galactose backbone with arabinose and galactose as major components in the side chains; other types of sugar might be present
Conclusion: the glycosylation pattern of the AGP protein backbone is determined by the amino acid sequence.
Hyp contiguity hypothesis
AGPsEnzymes for degradation
http://www.molbiol.saitama-u.ac.jp/bussitsu/research.html
AGPsO-glycosylation in general
Wilson, Iain BH (2002) Curr. Opinion in Structural Biology 12, 569-577
Plant O-Hydroxyproline Arabinogalactans Are Composed of
Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains
Li Tan, Peter Varnai, Derek T.A. Lamport, Chunhua Yuan, Jianfeng Xu, Feng Qiu,
Marcia J. KieliszewskiJ. of Biol. Chem. Vol. 285, no. 32, 24575-24583 (2010)
Gene design(AP)51
IFNα2-(SP)20Subcloning for gene expressionTobacco extensin signal sequence
CaMV 35S Plant transformation vector pBI121
Agrobacterium LBA4404 transformation
Tobacco suspension cells
transformation
Protein separation
Tobacco suspension cell
culture
NMR structure determination
Protein biochemicalanalysis
Gene design
IFNα2-(Ser-Hyp)20 Note: IFNα2 sequence not detailed
Tan, L. et al. (2003) Plant Physiology 132, 1362-1369
Xu, J. et al. (2007) Biotechnology & Bioengineering
Gene design(AP)51
IFNα2-(SP)20
Protein separation
Hydrophobic Interaction
Chromatography
(HIC)
Tobacco cells media
Protein biochemicalanalysis
Hyp-arabinogalactan
INFα2-(Ser-Hyp)20
Isolation & purification NaOH hydrolysis (108°C, 18 h) Separation on size-exclusion chromatography (Superdex-Peptide column) Hyp analysis - colorimetric Monosaccharide analysis
Total sugar content – colorimetric (anthrone method) Neutral sugar content – gas chromatography (alditol acetates method)
Nuclear Magnetic Resonance (NMR) INF Hyp-polysaccharide 1 AHP-1
(Ala-Hyp)51-EGFP Cation & Size exclusion
chromatography
AHP-1tube 23
Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165
NMR spectroscopyOne-dimensional 1H
Two-dimensional 1H homonuclear COrrelation SpectroscopY (COSY) TOtal Correlation SpectroscopY (TOCSY) ROtating Frame NOE SpectroscopY (ROESY) Nuclear Overhausser Effect SpectroscopY (NOESY)
Two-dimensional 13C, 1H
Heteronuclear Single Quantum Coherence (HSQC) Heteronuclear Multiple Bond Coherence (HMBC)
Two dimensional 13C, 1H heteronuclear HSQC-TOCSY Two dimensional 13C, 1H HSQC—NOESY
NMRPipe NMRView
Standard: 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS)
Chemical shifts of 1H & 13C
Gane, A.M. et al. (1995) Carbohydrate Research 277, 67-85
1H NMR, HSQC, HSMB from a previous paper
One dimensional 1H – AHP-1
Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165
A:B:C:D:E:F:G = 4:1:1:1:4:1:4
A – Ara D – Hyp H-4
B – Ara E – Gal
C – Rha F – Gal
G – Gal & GlcA
HSQC & HMBC
INF Hyp-polysaccharide 1
Sugar ratio & configuration
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Sugar ratio & configuration
A:B:C:D:E:F:G = 6:2:2:1:5:1:4+2A – Ara D – Hyp H-4B – Ara E – GalC – Rha F – Gal linked to Hyp G – Gal + GlcUA
INF Hyp-polysaccharide 1
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H NMR
Hyp-Gal linkage
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HSQC
TOCSY
Hyp-Gal linkage
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HMBC
Gal configurations
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H NMR
Rha residues
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
TOCSY
HSQC
Rha – GlcUAGlcUA - Gal
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HMBC
Ara linkages
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H NMR
Ara linkages
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HSQC
HMBC
INF Hyp-polysaccharide 2
Sugar composition
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Gal:Ara:GlcUA:Rha – 10:5:4:1
1H NMR
Gal linkage & backbone
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HMBC
Side chains
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HMBC
1H NMR
Primary structures
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
IFN-Hyp polysaccharide 2IFN-Hyp polysaccharide 1
Conclusions
Complete structure elucidation by NMR INF Hyp-polysaccharide 1 has six-residue galactan chain with 2 beta
1,3 linked by a beta 1,6 linkage INF Hyp-polysaccharide has four side chains Repetitive trisaccharide with two six-residue bifurcated side chains Six-residue side chain – identical with gum arabic side chain (no ter 5-
Ara) Glycosylation is not determined by the non-glycosylated sequence or
type of peptide Incomplete glycosylation
Molecular Modeling