Protein Interactions of Epithelial Neutral Amino Acid Transporters

316 Pages·2017·14.74 MB·English

by  Stephen Fairweather

Protein Interactions of Epithelial
Neutral Amino Acid Transporters

Stephen James Fairweather

November, 2017

A thesis submitted for the degree of Doctor of Philosophy in
Biochemistry of The Australian National University
Protein Interactions
of Epithelial Neutral
Amino Acid
Transporters
Stephen Fairweather
A thesis written for candidature to the Doctorate of
Philosophy in Biochemistry at the Australian National
University

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Declaration
The research described in this thesis is my own work except where stated otherwise. The
thesis was undertaken in the Research School of Biology, Division of Biochemistry and
Biomedical Science, at The Australian National University. No portion of the work presented
in this thesis has been submitted for another degree.

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Acknowledgments
Thanks personally, and mostly, to min kaerster Christina, and also to my Dad, Mum, Jan and
Belinda for putting up with me for many years now. Mange tak to Eric and Lise for making
me most welcome in your family.
Thanks scientifically most of all to Stefan my supervisor: if I have acquired any expertise at
all as a biochemist it is mostly due to him through the agency of many directives, pieces of
advice and discussions. In addition, I would like to thank Angelika, our laboratory manager
and technician who showed me most of the basic techniques I now profess to know. Thanks
to Associate Professor Sasha Mikheyev for his advice in designing the phylogenetic analysis
of collectrin.
Thank you to all the many lab members, colleagues, support staff and equipment technicians
who have assisted me in innumerable ways during the years of my doctorate. The number is
too many to list, and most have been acknowledged previously in publications and
presentations, either as co-authors or as assistants. I enjoy doing science because of all of you.
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Abbreviations
AA Amino Acid
AA0 Neutral Amino Acid
AASR Amino Acid Starvation Response
ACE2 Angiotensin Converting Enzyme 2
ADAM-17 A Disintegrin And Metalloproteinase-17
ADHD Attention Deficit Hyperactivity Disorder
APN Aminopeptidase N
ASCT2 Alanine Serine Cysteine Transporter 2
ATF4 Activating Transcription Factor 4
ATP Adenosine Tri-Phosphate
BBMV Brush Border Membrane Vesicles
B0AT1 Broad Neutral Amino acid Transporter 1
B0AT3 Broad Neutral Amino acid Transporter 3
BSA Bovine Serum Albumin
cDNA Complementary DNA
CD147 Classification Determinant
CHO Chinese Hamster Ovary
CKD Chronic Kidney Disease
DCA Direct Coupling Analysis
EBI European Bioinfomatics
EDTA Ethylenediaminetetraacetate
EGTA Ethylene Glycol tetraacetate
ELM Eukaryotic Linear Motif
ER Endoplasmic Reticulum
FBA Feedback Amplifier
FGF21 Fibroblast Growth Factor 21
GABA Gamma Amino Butyric Acid
GCN2 General Control Nonderepressible 2
GFP Green Fluorescent Protein
GFR Glomerular Filtration Rate
GHK Goldman-Hodgkin-Katz (Equation)
GLP-1 Glucagon Like Peptide-1
GTP Guanosine Tri-Phosphate
HBSS Hank’s Buffered Saline Solution
HGNC Human Genome Naming Consortium
HMM Hidden Markov Model
HNF Hepatic Nuclear Factor
IUBMB International Union of Biochemistry and Molecular Biology
JTT Jones-Taylor-Thornton (model)
LAP Leucine Aminopeptidase (E.coli)
LB Lysogeny Broth (media or agar)
LC-MS/MS Liquid Chromatography-Mass Spectrometry/Mass Spectrometry
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MCF Mitochondrial Carrier Fold
MCT Monocarboxylate Transporter
MDMA Methylenedioxyamphetamine
MES 2-(N-morpholino)ethanesulfonic acid
MFS Major Facilitator Superfamily
MOPS 3-(N-morpholino)propanesulfonic acid
MSA Multiple Sequence Alignment
M.W. Molecular Weight
NCBI National Centre for Biotechnology Information
NMDG N-methyl-D-glucamine
NSS Neurotransmitter Sodium Symporters
OCD Obsessive Compulsive Disorder
ORF Open Reading Frame
pCMBS p-chloromercuribenzenesulfonate
PBS Phosphate Buffer Saline
PCSK Proprotein Convertase Subtilisin Kexin
pGHJ pGem-He-Juel
PSSM Position Specific Scoring Matrix
PTM Post-translational modification
RPMI Roswell Park Memorial Institute (media)
RT Room Temperature
SARS Severe Acute Respiratory Syndrome
SDS Sodium Dodecyl Sulfate
SNARE Soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor
SNP Single Nucleotide Polymorphism
SOX9 SRY (Sex Determining Region Y) Box 9
SSRI Serotonin Selective Reuptake Inhibitors
SVM Support Vector Machines
TCA Tricyclic Antidepressants
TCDB Transporter Classification Database
TMPRSS2 Transmembrane protease, serine 2
TM Trans-membrane
WT Wild Type

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Abstract
Neutral amino acids are essential for protein synthesis, energy homeostasis, and many vital
biochemical pathways. As several neutral amino acids are essential and cannot be synthesised
by humans in vivo, their uptake and reuptake by the body’s absorbing epithelial layers is of
major importance for systemic amino acid homeostasis and human health. Two of the major
pathways for the absorption of neutral amino acids in the small intestine and kidney are the
Broad Neutral Amino acid Transporters 1 and 3 (B0AT1 and B0AT3).
B0AT1 is of medical interest as an indirect regulator of blood glycemia via cellular amino
acid starvation response pathways. Absence of B0AT1 results in improved glycemic control
and other metabolic effects, revealing it as a potential pharmacological target to treat type II
diabetes.
Essential to understanding the role of B0AT1 as a global metabolic regulator is its
requirement for plasma membrane expression with heteromeric protein partners: collectrin in
the kidney and Angiotensin Converting Enzyme 2 (ACE2) in the small intestine. Both
proteins are membrane-anchored by a single trans-membrane domain and are homologous.
B0AT3 also requires collectrin or ACE2 for membrane expression. Beyond this requirement
for plasma membrane expression, however, little is known about the underlying mechanisms
of the interaction.
This thesis demonstrates additional protein partners of B0AT1, namely Aminopeptidase N
(APN), syntaxin 1A, and syntaxin 3. These proteins facilitate changes in the kinetic
parameters of neutral amino acid transport, in addition to regulating membrane expression.
Moreover, I demonstrate that collectrin is required for catalytic activation of both B0AT1 and
B0AT3, while both APN and ACE2 increase B0AT1 and B0AT3 substrate affinity,
respectively. ACE2 and APN also form large complexes with B0AT1 at the small intestine
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brush border membrane. A binding site in both transporters for collectrin is identified as lying
in the hydrophobic pocket between TM 5 and 7 of the transporters. Using a mixture of
experimental and bioinformatics tools, I was able to map the interacting domains of collectrin,
which involve multiple regions of the protein, including the TM domain and large regions of
the N-terminus. The ability of collectrin and B0AT1 orthologs to cross-react with each other
and the homologous region of ACE2, suggests a highly conserved structure-function
relationship between them.
In conclusion, B0AT1 and B0AT3 form large, stable protein complexes with collectrin, ACE2
and other proteins, the binding site and function of which are highly conserved. The necessity
of these interactions mean these complexes should be thought of as heteromultimeric
transporter metabolons – multimeric protein units facilitating efficient neutral amino acid
transport in epithelial cells. There is increasing evidence that the formation of stable
heteromeric membrane transport complexes is a common theme underlying the function of
many transporters initially studied in isolation. Understanding the physiology and structure-
function relationships of transporters in their native multimeric states is fundamental to
understanding relevant biological roles. Especially as membrane transporters are heavily
over-represented as causative agents of human disease and as therapeutic drug targets.
Targeting stable, intrinsic protein-protein interactions presents a still unexplored field of drug
treatment in biomedical science.
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Table of Contents
DECLARATION .................................................................................................................................. II
ACKNOWLEDGMENTS .................................................................................................................. III
ABBREVIATIONS ............................................................................................................................. IV
ABSTRACT ......................................................................................................................................... VI
TABLE OF CONTENTS ................................................................................................................. VIII
CHAPTER 1: INTRODUCTION ...................................................................................................... 1
1.1 Neutral Amino Acid Transport in the Physiological Context ................................................................ 1
1.1.1 The importance of neutral amino acids to metabolism and homeostasis ............................................ 1
1.1.2 Physiological adaptions of the intestinal and renal epithelium ............................................................ 1
1.1.3 Brush border membrane conditions are optimised for active transport .............................................. 5
1.1.4 Protein digestion and absorption mechanisms at the brush border membrane ................................ 10
1.2 Secondary Active Membrane Transporters ....................................................................................... 16
1.2.1 Mechanisms, diversity, classification .................................................................................................. 16
1.2.2 The Solute Carrier 6 family of secondary active transporters ............................................................. 17
1.3 Broad Neutral Amino Acid Transporters B0AT1 and B0AT3 ................................................................ 21
1.3.1 Overview ............................................................................................................................................. 21
1.3.2 B0AT1 and B0AT3 thermodynamics at the brush border membrane .................................................. 23
1.3.3 Michaelis-Menten kinetics and it assumptions for B0AT1 and B0AT1 ................................................. 24
1.3.4 B0AT1 and B0AT3 structure and substrate binding sites ..................................................................... 28
1.3.5 Clinical relevance of Broad Neutral Amino Acid Transporters ............................................................ 37
1.4 Heteromeric Partners of Secondary Active Transporters ................................................................... 42
1.4.1 What do heteromeric partners of the secondary active transporters do? ......................................... 42
1.4.2 Heteromeric amino acid transporters of the SLC3 and SLC7 families ................................................. 46
1.4.3 Embigin and basigin: monocarboxylate (SLC16) transporter ancillaries ............................................. 48
1.4.4 The sterol transporter family SLC51 heteromer Ost α-Ost β .............................................................. 51
1.5 Heteromeric Partners of Broad Neutral Amino Acid Transporters ..................................................... 52
1.5.1 Collectrin and Angiotensin Converting Enzyme 2 (ACE2) ................................................................... 52
1.5.2 The interactions of B0AT1 and B0AT3 with collectrin and ACE2 .......................................................... 57
1.5.3 Syntaxins ............................................................................................................................................. 59
1.5.4 Other potential heteromeric partners ................................................................................................ 61
1.6 Project Aims ...................................................................................................................................... 62
CHAPTER 2: METHODS ................................................................................................................ 63
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2.1 Xenopus laevis Oocytes ..................................................................................................................... 63
2.1.1 Xenopus laevis oocytes – overview ..................................................................................................... 63
2.1.2 Basic electrical and biochemical parameters of Xenopus laevis membranes ..................................... 63
2.1.3 Limitations of Xenopus laevis oocytes as an experimental system ..................................................... 65
2.1.4 Surgery and preparation of oocytes.................................................................................................... 69
2.1.5 In vitro transcription, microinjection and expression of proteins in Xenopus laevis oocytes ............. 71
2.1.6 Surface biotinylation of proteins expressed in Xenopus laevis oocytes.............................................. 71
2.1.7 Whole membrane isolation of Xenopus laevis oocytes ...................................................................... 72
2.2 Murine Brush-Border Membrane Vesicles ......................................................................................... 73
2.2.1 Brush-Border membrane vesicle isolation principles and properties ................................................. 73
2.2.2 Preparation and characterisation of mouse intestinal BBMVs ........................................................... 74
2.3 Cell Cultures ...................................................................................................................................... 76
2.3.1 Human embryonic kidney 293 cell line culturing ................................................................................ 76
2.3.2 Transport experiments with Chinese hamster ovary K1 cells expressing SLC6A19 and collectrin ..... 77
2.4 Molecular Genetics ........................................................................................................................... 78
2.4.1 Molecular cloning ................................................................................................................................ 78
2.4.2 Site-directed mutagenesis................................................................................................................... 81
2.4.3 RNAi knockdown and reverse transcription PCR ............................................................................... 82
2.5 Protein Biochemistry ......................................................................................................................... 82
2.5.1 Enzymatic assays ................................................................................................................................. 82
2.5.2 Bradford assay ..................................................................................................................................... 83
2.5.3 SDS PAGE and western blotting .......................................................................................................... 83
2.5.4 Blue-native PAGE ................................................................................................................................ 86
2.5.5 Preparation of detergent-resistant membranes ................................................................................. 87
2.5.6 Co-immunoprecipitation of membrane proteins ................................................................................ 87
2.5.7 Tissue homogenate preparation ......................................................................................................... 88
2.5.8 Co-localisation of membrane proteins using the Venus fly trap ......................................................... 88
2.6 Radiolabelled Tracer Experiments and Electrophysiology ................................................................. 89
2.6.1 Radiolabelled uptake........................................................................................................................... 89
2.6.2 Electrophysiology: the two-voltage clamp configuration ................................................................... 90
2.6.3 Steady-state Michaelis-Menten kinetics ............................................................................................. 94
2.6.4 Recordings of APN induced tetrapeptide currents and inhibitors ...................................................... 95
2.6.5 Simultaneous current and radiolabelled uptake recordings ............................................................... 95
2.7 In Silico Computation Methods ......................................................................................................... 96
2.7.1 Genomic and protein sequence databases and tools ......................................................................... 96
2.7.2 Protein model construction and verification ...................................................................................... 96
2.7.3 Hidden Markov analysis and multiple sequence alignment of collectrin ........................................... 98
2.7.4 Putative motif and PTM analysis of collectrin ..................................................................................... 99
2.7.5 Protein sequence alignments and phylogenetic trees ...................................................................... 100
2.8 Data Analysis and Statistics ............................................................................................................. 101
2.8.1 Data analysis, graphical representations, and nomenclature ........................................................... 101
2.8.2 Equations and curve fitting ............................................................................................................... 101
2.3.8 Statistical analysis ............................................................................................................................. 102
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