Comparative study of the subcellular localisation and the N-glycosylation patterns of a recombinant glycoprotein expressed in Oryza sativa, Nicotiana tabacum and Medicago truncatula [Elektronische Ressource] / vorgelegt von Sylvain Marcel

Siham Belabed

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Comparative study of the subcellular localisation and the N-
glycosylation patterns of a recombinant glycoprotein expressed in
Oryza sativa, Nicotiana tabacum and Medicago truncatula



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation


Vorgelegt von

M. Sc. Sylvain Marcel
aus Le Mans, Frankreich



Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer, RWTH Aachen
Universitätsprofessor Dr. rer. nat. Dirk Prüfer, University of Münster


Tag der mündlichen Prüfung: 1 Juli 2005.

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Abstract

Nowadays, plants have become a promising alternative over the traditional expression
systems for the production of biologically active complex recombinant proteins. So far,
research has been focussed on increasing accumulation levels of recombinant proteins
expressed in many plant models. On the other hand, the cell compartment where recombinant
proteins are deposited often refers to protein quality in term of protein folding and post-
translational modification (e.g. N-glycosylation).
Recombinant proteins directed to the secretory pathway in plants require a leader peptide for
translocation into the endoplasmic reticulum. In the absence of further targeting information
such proteins are generally secreted, via the default pathway, to the apoplast. This has been
well-documented in protoplasts and leaf cells, but the trafficking of recombinant proteins in
storage tissues such as endosperm or cotyledons has only received little attention. In addition,
it has been suggested that the high specificity of seed tissue for starch, minerals and proteins
storage might interfere with protein secretion.
We used Aspergillus niger phytase as a model glycoprotein to compare the intracellular fate
and the N-glycosylation profile of a recombinant protein in different plant tissues (seeds and
leaves) in rice, tobacco and Medicago truncatula. After determination of N-glycosylation
patterns and microscopy analyses, we reported that the recombinant protein was preferably
retained in protein storage organelles within rice and tobacco endosperm cells, while it was
efficiently secreted in leaf and cotyledon tissues as expected for protein following the default
secretory pathway. Interestingly, in rice and M. tuncatula leaves, a significant part of A. niger
phytase was misrouted to the vacuole. Besides, we revealed an important heterogeneity in the
N-glycosylation patterns of A. niger phytase when produced in different plants and tissues. In
aaddition, only A. niger phytase extracted from M. truncatula leaves harboured Lewis -bearing
N-glycans known to be potentially highly immunogenic.
Together, these results showed that localisation and N-glycosylation of a recombinant protein
is tissue dependent, with obvious implications for the production of pharmaceutical proteins
by molecular farming.

Acknowledgements

First of all, I want to express my gratitude to Prof. Rainer Fischer and Dr. Eva Stoger for
giving me the opportunity to develop my PhD in this captivating subject.

I am very grateful to my supervisor, Dr. Stoger, for guiding so well this work with precious
help, advices and support. Eva, thank you very much for your availability and your grand
kindness. I learned so much working with you during the last 4 years.

This work would not have been possible without the brilliant expertise in microscopy of Dr.
Elsa Arcalís to whom I am extremely grateful. Many thanks to Julian Rodriguez for his
precious help in tissue culture, plant maintenance and microscopy. I appreciated very much
sharing lab time with you both. I keep great souvenirs of it.

To Dr. Rita Abranches from the ITQB, Portugal, for her essential collaboration. Thank you,
Rita, for your lab experience, your friendship and very nice discussions.
To Prof. Friedrich Altmann and the team of the University of Natural Resources and Applied
Life Sciences, Austria, for their crucial involvement in this work. I attached much value to
their expertise in glycobiology. Thanks to Michael Küpper for his expertise in mass
spectrometry.
Dr. Günter Hollweg and the team at the Pathology Department of the RWTH Aachen are
acknowledged for allowing us to use their electron microscope.

I would like to give a special acknowledgment to Duncan keen for his valuable work and
patience regarding rice transformation and tissue culture.
To Prof. Paul Christou, Dr. Georgia Drakakaki and Dr. Liz Nicholson for being so helpful and
supportive at the very beginning and all along of this adventure.

To Prof. Dirk Prüfer and Prof. Fritz Kreuzaler for agreeing to act as co-examiners.

And last but not least, I want to give a huge thank you to Angela, Arianna, Berta, Elisa, Eva,
Florence, Greta, Mariu, Pamela, Siham, Tanya, Jörg, Markus, Martín, Ruwin and all the
people from the Institute of Biologie VII for their helpful discussions and making life easier
and funnier.

Table of contents

I. INTRODUCTION_______________________________________1
I.1. Molecular farming: addressing constraints caused by protein deposition and N-
glycosylation __________________________________________________________________ 1
I.2. Deposition of recombinant proteins in the plant cell ___________________________ 2
I.2.1. The secretory pathway _________________________________________________________ 2
I.2.2. Protein biosynthesis and maturation through the secretory pathway ______________________ 4
I.3. N-glycosylation in plants __________________________________________________ 9
I.3.1. N-glycan maturation within the secretory pathway____________________________________ 9
I.3.2. The impact of N-glycosylation on protein folding and activity _________________________ 10
I.3.3. Allergenicity of plant glycans ___________________________________________________ 12
I.4. Protein storage in seeds 13
I.4.1. General seed structure _________________________________________________________ 13
I.4.2. Seed storage proteins__________________________________________________________ 15
I.5. Objective and strategy of the study ________________________________________ 17
I.5.1. Objective of the study 17
I.5.2. Experimental design 18
II. MATERIALS AND METHODS____________________________21
II.1. Materials ______________________________________________________________ 21
II.1.1. Chemicals and consumables__________________________________________________ 21
II.1.2. Buffers, media and solutions _________________________________________________ 21
II.1.3. Enzymes and reaction kits ___________________________________________________ 21
II.1.4. Primary, secondary antibodies and substrates ____________________________________ 22
II.1.5. Bacterial strains ___________________________________________________________ 22
II.1.6. Plants ___________________________________________________________________ 22
II.1.7. Vectors __________________________________________________________________ 23
II.1.8. Oligonucleotides 23
II.1.9. Resin and membranes_______________________________________________________ 23
II.1.10. Equipment _______________________________________________________________ 24
II.2. Methods 25
II.2.1. Transformation vectors________________________________________________ 25
II.2.1.1. Endosperm-specific rice expression vector ______________________________________ 25
II.2.1.2. Constitutive expression vectors for rice, tobacco and M. truncatula ___________________ 25
i
II.2.2. Basic recombinant DNA techniques______________________________________ 26
II.2.2.1. Isolation of plasmid DNA from Escherichia coli__________________________________ 26
II.2.2.2. Quantification of DNA______________________________________________________ 26
II.2.2.3. Agarose gel electrophoresis __________________________________________________ 26
II.2.3. Preparation and transformation of E. coli ________________________________ 26
II.2.3.1. Preparation of heat-shock competent E. coli DH5 α cells ____________________________ 26
II.2.3.2. Transformation of E. coli DH5 α by heat-shock treatment ___________________________ 27
II.2.3.3. Preparation of electrocompetent E. coli DH5 α cells 27
II.2.3.4. Electroporation of E. coli DH5 α_______________________________________________ 28
II.2.3.5. Quick colony lysis for selection of recombinant colonies 28
II.2.3.6. DNA sequencing __________________________________________________________ 28
II.2.4. Preparation and transformation of Agrobacterium tumefaciens _______________ 28
II.2.4.1. Preparation of heat-shock competent A. tumefaciens cells___________________________ 28
II.2.4.2. Transformation of A. tumefaciens _____________________________________________ 29
II.2.4.3. Preparation of A. tumefaciens glycerol stock _____________________________________ 29
II.2.5. Plant transformation __________________________________________________ 30
II.2.5.1. Rice