Structural and functional studies of mucin-interacting adhesion domains from Candida glabrata and Helicobacter pylori [Elektronische Ressource] / Manuel Maestre Reyna. Betreuer: Lars-Oliver Essen

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Structural and functional studies ofmucin-interactingadhesion domainsfrom Candida glabrata& Helicobacter pyloriA dissertation submitted in fulfillment of the requirements for the degree of DOCTOR IN NATURAL SCIENCES (Dr. Rer. Nat.)Presented atthe Department of ChemistryPhilipps University MarburgbyManuel Maestre ReynafromValencia, SpainMarburg/Lahn 2011Received on the ________________________________ at the Chemistry Department, Philipps University Marburg.First advisor: Prof. Dr. La‐rOs liver Essen ( Department of Chemistry, Marburg).Second advisor : Prof. Dr. Hans-Ulrich Mösch (Department of Biology , Marburg).Date of Examination : ________________________________I declare that t o the best of my knowledge and belief, this thesis with the title:“Structural and functional studies of mucin-interacting adhesion domains from Candida glabrata & Helicobacter pylori”contains no material previously published or written by another person, except where due reference has been made. It also does not contain any material which has been accepted for the award of any other degree or diploma in any University .thMarburg, March 29 2011,_________________________________________Manuel Maestre ReynaFurther publications:Psakis, G., Nitschkowski, S., Holz, C., Kress, D., Maestre-Reyna, M. , Polaczek, J., Illing, G., Essen, L.-O. (2007). Expression screening of integral membrane proteins from Helicobacter pylori 26695. Protein Sci.

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Structural and functional studies of
mucin-interacting
adhesion domains
from Candida glabrata
& Helicobacter pylori
A dissertation submitted
in fulfillment of the requirements for the degree of
DOCTOR IN NATURAL SCIENCES
(Dr. Rer. Nat.)
Presented at
the Department of Chemistry
Philipps University Marburg
by
Manuel Maestre Reyna
from
Valencia, Spain
Marburg/Lahn 2011Received on the ________________________________ at the Chemistry Department, Philipps
University Marburg.
First advisor: Prof. Dr. La‐rOs liver Essen ( Department of Chemistry, Marburg).
Second advisor : Prof. Dr. Hans-Ulrich Mösch (Department of Biology , Marburg).
Date of Examination : ________________________________I declare that t o the best of my knowledge and belief, this thesis with the title:
“Structural and functional studies of mucin-interacting adhesion domains from Candida
glabrata & Helicobacter pylori”
contains no material previously published or written by another person, except where due reference
has been made. It also does not contain any material which has been accepted for the award of any
other degree or diploma in any University .
thMarburg, March 29 2011,
_________________________________________
Manuel Maestre ReynaFurther publications:
Psakis, G., Nitschkowski, S., Holz, C., Kress, D., Maestre-Reyna, M. , Polaczek, J., Illing, G.,
Essen, L.-O. (2007). Expression screening of integral membrane proteins from Helicobacter pylori
26695. Protein Sci. 16,12,2667-76
Posters:
Psakis, G., Nitschkowski, S., Hölscher, S., Neuhaus, C., Kress, D. , Maestre ‐Reyna, M. , Essen,
L.‐O. (2007). High‐throughput expression and crystallisation of membrane proteins from
Helicobacter pylori and homologues. Structural Proteomics of Membrane Proteins ,
Rauischholzhausen.To my family and
*other animals
* As Gerald Durrell would have put it.Table of Contents
1 Summary/Zusammenfassung ..................................................................................1 .........................
1.1 Epithelial adhesinsCandi fromda gl abrata .................................................. 1 ..........................
1.2 Adhesins of Helicobacter pylor i...................................................................................2 ...........
1.3 Epitheladhäsine von C. glabrata......................................................................2 ........................
1.4 Adhäsine von H. pylori ...............................................................................................3 ..............
2 Introduction................................................................................................................5 ......................
2.1 Mucins are primary targets for pathogenic adhe............................................sins 5 ....................
2.1.1 Mucin structure and functi.............................................................................on 5 ................
2.1.2 Mucin tissue localiza.........................................................................tion 6 ..........................
2.1.3 Mucin glycosylati.........................................................................................................on 9 ..
2.2 Candida glabrat....................................................................................................a 12 .................
2.2.1 The genetic background of C. glabrata......................................................12 .....................
2.2.2 The cell waC. glll of abrata..............................................................................14 ...............
2.2.3 The adhesins of abrata..................................................................................15 ...........
2.2.4 PathogenicityC. gl of abrata...................................................................................17 .........
2.3 Helicobacter pyl .........................................................................................ori 19 ........................
2.3.1 Colonization and pathogenesis of H. pylori ...............................................................19 ......
2.3.2 Adhesion iHn . pylori ...........................................................................................23 ............
2.4 The Structural features of glycan binding proteins from pathoge....................nic origin 25 .......
2.4.1 The PA14 domain........................................................................................................26 .....
2.4.2 Type V protein secretion: autotra............................................................nsport. 30 ..............
3 Aim...............................................................................................................................33 ..................
4 Material.............................................................................................................................s 34 ............
4.1 Chemicals, consumables and equipme..............................................................nt 34 ..................
4.2 Vectors and Microorganisms ......................................................................38 ............................
4.2.1 Vectors.........................................................................................................................38 .....
4.2.2 E. col sitrains...............................................................................................................39 .....
4.3 Prime..........................................................................................................................rs 41 ..........
4.3.1 Primers for de novo cloning:...................................................................................41 .........
4.3.2 Mutagenesis prim.......................................................................................ers: 41 ................
4.4 Media, buffers and Stock solut..........................................................ions 42 ..............................
4.4.1 Medi........................................................................................................a: 42 ......................
4.4.2 Buffe............................................................................................................................rs: 43 .
4.4.3 Stock solut....................................................................................................ions: 45 ............
5 Methods...........................................................................................................................48 ...............
5.1 Molecular biology...............................................................................................................48 ....
5.1.1 DNA synthesis methods .......................................................................................48 ............
5.1.2 Cloni.......................................................................................................................ng 50 ......
5.1.3 DNA Isolation methods..................................................................................................51 ..
5.1.4 Agarose gel electrophore...................................................................sis 52 ..........................
5.1.5 Cell preparation m..............................................................................ethods 52 ...................
5.2 Biochemical methods.............................................................................................................54 .
5.2.1 Recombinant gene expression 54 ...
5.2.2 Cell......................................................................................................................... lysis 55 ..
5.2.3 Protein purific................................................................................ation 57 ..........................
5.2.4 Protein concentration 59 .......................
5.2.5 Protein refo....................................................................................lding 59 ..........................
5.2.6 Estimation of protein concentra........................................................................tion 62 .........
I5.2.7 Protein precipi ................................................................................................tation 63 .........
5.3 Analytical Methods......................................................................................................64 ...........
5.3.1 Western blot..........................................................................................ting 64 .....................
5.3.2 CD spectrosc...........................................................................................opy 65 ...................
5.3.3 Analytical gel fil.............................................................................tration 66 .......................
5.3.4 High throughput glycan binding studies at the Consortium for Functional Gl.yc67omics.
5.3.5 Fluorescence spectroscopy 69 ...
5.4 Crystallographic Me...................................................................................................thods 73 ....
5.4.1 Crystallization screening. 73 ......
5.4.2 Optimizing and reproducing crystalli..................................................................zation 74 ...
5.4.3 Diffractometric measurements and sample prepa................................................ration. 75 ..
5.5 Computer assisted met............................................................................hods 77 ........................
5.5.1 Homology model building methods...............................................................................77 ..
5.5.2 Structure elucidation m.......................................................................................ethods 88 ...
5.5.3 Protein properties calculation m...............................................................ethods 92 .............
6 Result......................................................................................................................s 96 ......................
6.1 Epithelial Adhesins fromCandi da glabrata (Epa)......................................................96 ...........
6.1.1 Purification of the Epa1A domain..................................................................................97 ..
6.1.2 Structure determination of the Epa1A dom............................................................ain. 98 ...
6.1.3 Generation of Epa1A variant..........................................................................s. 102 .............
6.1.4 Expression of Epa varia..........................................................................................nts 104 ...
6.1.5 Purification of Epa varian.............................................................ts. 104 .............................
6.1.6 Structural analyses of subclass-converted Epa1A va...............................riants 105 .............
6.1.7 Carbohydrate binding of Epa A domains...................................................110 ....................
6.1.8 Quantitative carbohydrate binding assays via fluorescence t..................itration 119 ...........
6.2 Lewis antigen binding adhesins from H. py lori ...................................................................122 .
6.2.1 Bioinformatic studi.....................................................................................es 123 ................
6.2.2 Refolding of BabA fragme.....................................................................nts 124 ...................
6.2.3 CD spectroscopy of BabA 235.................................................................................125 ......
6.2.4 pH dependent analytical gel filtration of BabA 235 fra..........................gment 130 .............
6.2.5 High-throughput semi-quantitative binding assays of Ba ..................bA 235 131 ................
6.2.6 Other Strategies for Ba ..................................................................................bA235 135 ......
6.2.7 Recombinant expression and purification of SabA extracellular fra..........gments. 137 .......
7 Discussion .......................................................................................................139 .............................
7.1 H. pylori adhesin BabA................................................................................140 ........................
7.1.1 The BabA extracellular domain can suffer profound pH-dependent conformati onal
changes..............................................................................................................................140 ......
7.1.2 H. pylori interacts with the host in a pH dependent m....................................anner 144 ......
7.1.3 BabA specificity shift towards LewisX and LewisY blood group antige.ns.......145..........
7.1.4 Outl.............................................................................................ook 147 .............................
7.2 Epithelial adhesins fromC. gl abrata.............................................................................148 .......
7.2.1 Comparison of the A domain from Epa adhesins with S. cerevis Fiaelo5 flocculi.n..149...
7.2.2 The binding pocket of Epa......................................................................1A 151 ..................
7.2.3 A structural model for carbohydrate binding in E.................................pa1A 153 ................
7.2.4 The specificity of Epa1A and its vari............................................ants. 154 .........................
7.2.5 Epa A domains do not bind lactose................................................................159 ................
7.2.6 Outlook 160 .............................
8 Acknowledgements.........................................................................................162 .............................
Bibliography .......................................................................................................................................I ..
9 Appendice..............................................................................................................s 1 .........................
II9.1 Appendix I: Glycan A......................................................................................rrays 1 .................
9.1.1 Array V3.1......................................................................................................................1 ....
9.1.2 Glycan array V4.1.............................................................................................13 ...............
9.2 Appendix II: Protein se.........................................................................quences 29 .....................
9.2.1 Sequences of Epa1A and vari....................................................................ants 29 ................
9.2.2 Sequences of BabA extracellular fragmHe. pyntls fromori 26695 ................. 31 ................
9.2.3 SequencesbA extracellular fragmHe. pyntls fromori J 99..............32.......................
9.2.4 Sequences of BabA extracellular fragmHe. pyntlsor fromi P 12 ............................33 .........
9.2.5 SequencesbA extracellular fragmHe. py ntlsor fromi G 27................ 35 ....................
9.3 Appendix III: fluorescence spect.............................................................roscopy data 38 ...........
III