A multifunctional protein : Phosphoglucose isomerase / autocrine motility factor / neuroleukin
by
Nathalie Y
B.Sc, Universite de Montreal, 2004
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
MASTER OF SCIENCE
in THE FACULTY OF GRADUATE STUDIES (Anatomy)
THE UNIVERSITY OF BRITISH COLUMBIA April 2007 © Nathalie Y, 2007 II
ABSTRACT
Phosphoglucose isomerase (PGI) is a glycolytic enzyme that moonlights as a cellular
cytokine. The protein is also known as autocrine motility factor (AMF), neuroleukin and
maturation factor. PGI/AMF interaction with its receptor interaction is pH-dependent.
Indeed, at neutral pH, PGI/AMF binds its receptor AMFR at the cell surface and can be
endocytosed via two different pathways: caveolae/raft-dependent endocytosis to the
smooth ER or clathrin-dependent endocytosis to multivesicular bodies (MVBs).
Internalized PGI/AMF can recycle from MVBs to the plasma membrane where it can
undergo further rounds of endocytosis and recycling. Recycling receptor-ligand
complexes can also be sequestered via stable association with FN fibrils. Recent data
show that, at acid pH, endocytosis is inhibited and PGI/AMF binds directly to FN fibrils
or to HS. Heparan sulfate proteoglycans, when expressed on the surface of cells,
modulate the actions of a large number of extracellular ligands while fibronectin is
involved in many cellular processes such as tissue repair and cell migration/adhesion.
However, the mechanisms that regulate PGI/AMF binding to its receptors still remain
unclear.
PGI/AMF cytokine activity, associated with several diseases, has been reported in
rheumatoid synovial fluid and its deposition on synovial surfaces and ability to induce an
autoimmune response in rheumatoid arthritis (RA) identified it as a possible autoantigen
different from normal circulating PGI/AMF. However, more recent manuscripts have
questioned the prevalence of an autoimmune response to PGI in RA. Ill
In this study, recombinant PGI constructs were used to characterize PGI interactions and
functions. We demonstrate that PGI behaves differently after N or C-terminal residue
additions. Our data also suggest that monomerization but not enzymatic activity is
necessary to induce cell motility at neutral pH. The putative function of PGI in RA was
assessed and using the recombinant PGI constructs and PGI autoantibodies was found to be species and conformation-dependant. IV
TABLE OF CONTENTS pages
Abstract II Table of contents IV List of tables VII
List of figures VIII List of symbols and abbreviations X 1. Introduction 1
1.1 Historic 1
1.2 Molecular Biology of PGI 4 Gene structure of PGI 4 Gene 4 Minisatellites 4 Protein structure of PGI 5 Backbone structure 5 Active site 7 Interspecies homology 7 Mutations 7
1.3 Functions 8 Catalytic function of PGI 8 Glycolysis 8 Active sites 9 Moonlighting functions of PGI 13 PGI/AMF/ neuroleukin secretion 13 Neuroleukin 13 Autocrine motility factor and maturation factor 15 Involvement in mineralization during osteoblast differentiation. 19 Embryo implantation 20
1.4 Receptors 21 AMFR / gp78 22 Protein motifs implicated in AMF/PGI cytokine activity and receptor binding 24 Fibronectin 25 Heparan sulfate 26 IGFPB-3 27 Another receptor 27
1.5 PGI/AMF implication in diseases 28 Non-spherocytic haemolytic anaemia 28 Cancer 29 Rheumatoid Arthritis 30 V
2. Hypothesis 32
3. Material and methods 33
3.1 Protein purification 33
3.2 SDS-page and western blots 33
3.3 Enzymatic activity assay 34
3.4 Glutaraldehyde cross-linking assay 35
3.5 Circular dichroism 35
3.6 Fibronectin binding assay..... 35
3.7 Cell motility assay 36
3.8 Sera and synovial fluids 37
3.9 Human RA antisera ELISA screening 37
3.10 Human RA antisera Western Blot screening 37
4. Results 39
4.1 Analysis of recombinant AMF/PGI expression and purification 39
4.2 Enzymatic activity of recombinant PGI/AMF 40
4.3 Recombinant PGI/AMF glutaraldehyde cross-linking 41
4.4 Circular dichroism of recombinant PGI/AMF 42 a. Far UV 42 b. NearUV 43
4.5 Binding to fibronectin 43
4.6 Recombinant cell-induced motility 44
4.7 Implication of AMF/PGI in Rheumatoid Arthritis 45 a. ELISA essay 45 b. Western blot essay 46
5. Discussion 48
5.1 Conformational effects of residue additions to C-terminus and N-terminus. 48
5.2 Cell-induced motility and cell interaction of AMF/PGI 49
5.3 Implications for the Role of PGI in Rheumatoid Arthritis 51
6. Conclusion 53 VI
7. Figures, tables and legends 54
8. Bibliography 79 VII
LIST OF TABLES
Table I Summary: Recombinant AMF/PGI properties 70
Table II Analysis of densitometry 75 VIII
LIST OF FIGURES
1. INTRODUCTION
Figure 1 Structural organization of the human glucose phosphate isomerase
gene 4
Figure 2 Ribbon representation of PGI from different species 6
Figure 3 Glycolysis pathway 8
Figure 4 Interconversion between glucose 6-phosphate and fructose
6-phosphate 9
Figure 5 Proposed catalytic mechanism for PGI 11
Figure 6 Molecular signaling in AMF/PGI motility stimulation 16
Figure 7 Interaction between AMF-AMFR and VEGF-VEGFR signal in
tumor and host endothelial cells 17
Figure 8 Aptosis-related signal pathways induced by AMF/PGI overexpression. 18
Figure 9 The complex biology of PGI/AMF and its receptor 21
Figure 10 Structure of AMFR/gp78 22
Figure 11 Increased association of AMF/PGI to fibronectin at acid pH 26
4. RESULTS
Figure 12 Vector and Constructs 55
Figure 13 Western blot and SDS-page analysis of recombinant AMF/PGI 57
Figure 14 Enzymatic activity of recombinant AMF/PGI 59
Figure 15 Glutaraldehyde cross-linking and semi-log graph analysis 1 61
Figure 15 Glutaraldehyde cross-linking and semi-log graph analysis II 62
Figure 16 Circular dichroism of recombinant AMF/PGI 64
Figure 17 Binding of PGI/AMF to dimeric FN at neutral and acid pH 66 IX
Figure 18 Recombinant AMF/PGI cell-induced motility 68
Figure 19 Human RA antisera ELISA screening 71
Figure 20 Western Blot control 73
Figure 21 Western Blot screening: species-specific recognition of human RA anti-sera to PGI/AMF 75
Figure 22 Western Blot screening: conformation-specific recognition of human RA anti-sera to recombinant PGI/AMF 77 LIST OF SYMBOLS AND ABBREVIATIONS
AMF Autocrine motility factor AMFR Autocrine motility factor receptor ATP Adenosine triphosphate
BSA Bovine Serum Albumine CD Circular Dichroism cDNA Complementary DNA ECM Extracellular matrix ER Endoplasmic reticulum
ERAD Endoplasmic reticulum associated degradation F6P Fructose-6-phosphate FN Fibronectin G6P Glucose-6-phosphate GDI GDP-dissociation inhibitor GTP Guanosine triphosphate HS Heparan sulfate IGF Insulin growth factor
IGFBP Insulin-like growth factor binding protein kDa Kilo Daltons KDR kinase domain region mpCD methyl beta-cyclodextrin mRNA messenger RNA MVB Multivesicular bodies
NAD Nicotamine adenine dinucleotide PGI Phosphoglucose isomerase RA Rheumatoid arthritis RBC Red blood cells SDS Sodium dodecyl sulfate Tfr Transferrin UV Ultraviolet VEGF Vascular endothelial growth factor 1
1. INTRODUCTION
1.1 - HISTORY
Phosphoglucose isomerase is a glycolytic enzyme present in all types of human cells. It had been studied for decades, but its molecular structure and the biological understanding of its numerous functions have emerged only during the past 20 years. Significant technical advances have led to the discovery of its structure, its identity as an extracellular cytokine and its receptor. The human enzyme is of medical interest because
PGI is believed to be involved in several diseases. The interest of PGI/AMF and its receptor is growing not only because of its biological significance, but also because of its practical importance as a major target in the post-genomic era for developing therapeutics for diverse diseases.
Glycolysis
Glycolysis has been studied for over a century. This degradation pathway of sugar molecules leads to the formation of pyruvate releasing energy in the form of ATP and molecules with reduction potential (NAD) [1]. Initially described by Lohman in 1933, phosphoglucose isomerase (PGI) is known to be a ubiquitous cytosolic enzyme that catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate during the second step of glycolysis [2].
Neuroleukin, lymphokin and phosphoglucose isomerase
In response to partial denervation or paralysis, new neurotic processes, called terminal sprouts, can appear from the remaining motor axon terminal,[3]. In their attempt to identify factors produced by denervated or inactive muscle that might be necessary for 2 motor axon terminal sprouting, Gurney and colleagues purified in 1986 a ~56 kD protein, neuroleukin. Neuroleukin was subsequently shown to be capable of increasing survival of cultured sensory neurons [4] and, as they found later that year, to act as a lymphokine.
Following secretion by lectin-stimulated T cells, neuroleukin was able to induce the maturation of B-cells into antibody secreting cells but was not necessary involved in the continued production of immunoglobulin by differentiated antibody-secreting cells [5].
Two years later, in 1988, the mouse phosphoglucose isomerase (PGI) cDNA was isolated
and sequenced. The investigators, a group working on the molecular genetics of
carbohydrate metabolism, surprisingly found a whole sequence identity between the 759 nucleotides at the 3' end of the mouse PGI clone and the sequence of mouse neuroleukin
[6]. Moreover a second group showed that same year a 90% homology between PGI and
neuroleukin [7]. Hence, the question as to how a ubiquitous, cytosolic enzyme could also
function as an extracellular cytokine had been raised. Gurney et al. immediately reacted
to this discovery and subsequently confirmed that both mouse and human neuroleukin
expressed PGI enzymatic activity. However, they found that PGI activity was not
blocked with monoclonal antibodies that were able to block neuroleukin activities. Thus,
they hypothesized for the first time the existence of a PGI/neuroleukin receptor [8].
Autocrine motility factor, maturation factor and phosphoglucose isomerase
Several steps are involved in the progression of a tumor, which include unrestrained
growth and invasive behaviour or active locomotion of tumor cells is also one of its
major properties. Since autocrine growth factors are essential to unrestrained growth of
tumor cells, it was therefore suggested that tumor cell motility might also be regulated by
autocrine mechanisms. This led to the discovery of the autocrine motility in 1986, by 3
Liotta et al. The group showed the presence of a cell motility-stimulating factor present in the serum-free conditioned medium of human melanoma cells. The ~55kDa motility factor, termed 'autocrine motility factor (AMF)', was believed to play a major role in the local invasive potential of tumor cells [9]. The hypothesis was later verified when AMF found in urine from patients with bladder cancer was shown to induce motility of transitional cell carcinoma from urinary bladder in a similar way to that of their own serum-free conditioned medium [10].
AMF sequence and structure remained unknown until the mid 90's. In 1996, Watanabe et al., working on AMF, microsequenced the tumor-secreted cytokine from a murine fibrosarcoma. They demonstrated that AMF corresponded in fact to the known enzyme and cytokine PGI/Neuroleukin and that AMF exhibited enzymatic/cytokine properties of
PGI/neuroleukin which were inhibited by specific PGI inhibitors [11]. That same year, the identity of a maturation inducer capable of mediating the differentiation of human myeloid leukemic cells to terminal monocytic cells was investigated by Xu et al. They also surprisingly found that the 54.3 kD inducer had PGI enzymatic activity and that its amino acid sequence was 100% homologue to neuroleukin and PGI [12].
Moreover, novel properties of PGI have been discovered more recently and the ubiquitous enzyme has been associated with functions such as involvement in mineralization during osteoblast differentiation [13] or embryo implantation [14], providing a possible hint on the evolutionary development of its original function as a
cytokine. Several glycolytic enzymes serve multiple functions [15] and phosphoglucose
isomerase and its extracellular homologues represent a brilliant example of the evolution
of new functions from existing proteins. 4
1.2 - MOLECULAR BIOLOGY OF PGI
GENE STRUCTURE
Gene
The gene encoding human phosphoglucose isomerase is located on chromosome 19. It is about 50 kb long and contains 18 exons and 17 introns [16].
> Chr.;.19:' Pt3il2 pI3.ii » •.*,«12»:...^.--w ql9Jl^^l3ii2 ^qlJ'.S^ »«13;328«».3:*l3. 4isl3.«t«13;Ml
Size (bp) 122 91 69 120 84 146 72 45 54 61 44 153 130 77 129 76 67 431
Exon 1* 2* 3 4 S 6 7 8 9 10 11 12 13 14 15 1617 18
ItltrOn Size Ikb" MBip 1.6kb ? 1«bp 1.6«> 812bp1.HUi ?
Minisatellites
Minisatellites are tandemly repeated DNA sequences found throughout the genomes of
all eukaryotes. The repeat unit sequence is generally not conserved beyond closely related species [17]. Williams and al. [18] have studied the minisatellite contained in the
intron 9 of the human PGI and have found similar repeats in PGI of other species.
Moreover, these repeat units did not appear in other locus of the genome. Minisatellite
DNA has been reported to be involved in recombination activity, control of gene
expression of nearby gene(s) (transcriptional and translational), whereas others form protein coding regions [19]. Thus, the high level of conservation exhibited by the GPI
minisatellite, coupled with the unique location, strongly suggests a functional role for
these repeated DNA sequences. 5
Protein structure
Backbone structure
While the extracellular cytokine activities have been associated with a 55 kd monomer, the dimeric structure of PGI was found to comprise two identical subunits, each of molecular mass 63 kDa [20]. PGI dimerization is required for its enzymatic activity because the active site of the enzyme is composed of polypeptide chains from both
subunits [21,22]. Although the structural basis for the cytokine activity of the protein is not known yet [21], inhibitors of enzymatic activity are also capable of inhibiting the
extracellular activites of PGI/AMF [11,12].
Human PGI was resolved by Read et al. in 2001. Crystallization of human PGI revealed
that the enzyme is a tight dimer of identical subunits. Each monomer is composed of two
globular domains, and the close association between them is reinforced by the presence
of an 'arm', a 45 residue extension structure at the C terminus which wraps around the
other monomer, while, on the opposite site, another loop termed 'hook' also interacts
with the adjacent monomer. The two domains are historically named large and small,
although they are now known to be pretty similar in size. Both domains consist of an
ahelix-Psheet-ahelix sandwich. The small domain contains a central five stranded
parallel |3-sheet surrounded by helices whereas the large domain has a six-stranded
mixed parallel/antiparallel p-sheet, also packed on both sides by a-helices. The
polypeptide chain begins in the large domain, crosses to the small domain, then returns to
the large domain and finishes at the end of the oc-helical arm [23]. 6
Human PGI monomer L6A Rabbit PGI monomer 2.5A Read et al., 2001 Davies & Muirhead, 2002 ~ 520"
Large Largs ec a21 cfcjffiaih domain I
a15l
«tomai
Small domain
Bacillus stearothermophilus PGI Rabbit PGI dimer monomer 2.3A Davies & Muirhead, 2002 Sun etal., 1999
Figure 2 Ribbon representation of PGI from different species 7
Active sites
For a better understanding of the relationship of PGI enzymatic function and structure, please see section C of the introduction.
Interspecies homology
To date, the pig [24], human [20], rabbit [25] and bacterial (Bacillus stearothermophilus)
[21] PGI have been crystallized and their structure fully characterized. The studies showed that the overall structure of PGI in most species is very similar. Also, the amino acids required in the active site have notably been shown to be conserved in all known
PGI sequences in mammals, plants, flies, bacteria, and yeast [20,22,24].
Mutations in the human PGI
PGI is an essential enzyme and PGI deficiency in humans is an autosomal recessive
genetic disorder resulting in nonspherocytic haemolytic anaemia [26].
Mutations in PGI are homozygotes and can be classified into three groups:
(a) those that impact the precise structure of the enzyme
(b) those that disrupt or alter a dimer-dimer contact, and
(c) those of residues at the active site, which may have a role in catalytic function,
This disease will be covered later on (Section D). 8
1.3 - FUNCTIONS
CATALYTIC FUNCTIONS OF PGI
Glycolysis
Glycolysis is a degradation pathway of sugar molecules that leads to the formation of pyruvate. The breakdown of sugars also releases energy in the form of ATP and some reduction potential molecules [1]. Nine distinct reactions are required to convert glucose
into pyruvate.
Glucose ATP hexokinase glucokinase ADP
Glucose-6-phosphate phosphohexose isomerase
Fructose-6 -phosphate ATP- phospho- ADP' fructokinase-1
Fructose-1,6-bisphosphate
aldolase
Glyceraldehyde-3 -phosphate -Di hydro xyacetone triosephosphate phosphate isomerase M.W. King (1996) [112]
Figure 3 Glycolysis pathway
The second step of glycolysis involves the conversion of glucose-6-phosphate (G6P) to
fructose-6-phosphate (F6P) by phosphoglucose isomerase and as the name suggests,
involves an isomerization reaction, leading to the interconversion between G6P and F6P. 9
The reaction is freely reversible at normal cellular concentrations of the two substrates and PGI is thus also used during gluconeogenesis [27].
The proposed multistep catalytic mechanism for PGI consists in ligand binding, ring opening, isomerization of the substrate, ring closing and ligand release. The isomerization involves an acid/base catalysis, where a rearrangement of the carbon- oxygen bond transforms the six-membered ring (G6P) into a five-membered ring (F6P) or vice-versa [28]. During glycolysis, the rearrangement takes place when the six- membered ring opens, via an enediolate intermediate, and then closes in such a way that the first carbon becomes now external to the ring.
Phosphoglucose isomerase o II 8
-O— P—O—CH2 fJ
Glucose 6-phosphat© Fructose 6-phosphate
AG'0 = 1.7 kJ/mol
Mathews A/an Holde/Ahern, 3rd Ed [27]
Figure 4 Interconversion between glucose 6-phosphate and fructose 6-phosphate
Active sites
The association between the monomers forms the putative active site for PGI's enzyme function in glycolysis and gluconeogenesis. The cleft between the large domain, small domain, and C-terminal arm forms a deep binding pocket in the dimer. This pocket is the 10 postulated binding site for glucose 6-phosphate in glycolysis and fructose 6-phosphate in gluconeogenesis.
The catalytic mechanism of phosphoglucose isomerase should include the following
steps: binding of the cyclic form of the substrate to the enzyme; ring-opening of the
substrate; base-catalyzed isomerization via a cis-endiol intermediate; ring-closure of the product and release of product [29].
The following sketch represents the most recent isomerization mechanism as proposed by
Jeffery and Lee in 2005 [28]. 5 JT V
1 It
LB A f » 11
OUTJ
DOWN Lee and Jeffery, 2005 [28] Figure 5 The proposed catalytic mechanism for PGI as proposed by Lee and Jeffery 12
The proposed multistep catalytic mechanism is shown, including the ligand binding, ring
opening, isomerization, ring closing, and ligand release steps. A helix containing amino
acid residues 512-520 moves between an "in" position, in which it interacts directly with the bound ligand, and an "out" position, in which there is a water molecule located between Lys518 and the ligand. A loop containing amino acid residues 210-214 moves
"up" to interact with the phosphoryl group of the ligand upon ligand binding and "down" upon release of the ligand. The numbering of the steps refers to the F6P to G6P direction
of catalysis. Amino acid residues involved in the ring-opening step are shown in green.
Amino acid residues involved in the isomerization step are shown in blue. The cyclic and
open chain forms of the substrates are shown in red. The dashed lines indicate hydrogen
bond interactions. (1) Ligand binding: The F6P substrate binds in the active site, and the
210-214 loop moves "up." (2) Ring opening: His388 and a water molecule held by
Lys518 and Thr214 catalyze the ring opening step. (3) Conformational changes: The
substrate undergoes rotation about its C3-C4 bond and extends so that C1-C2 approaches
Glu357. The 512-520 helix moves to the "in" position. An ordered water molecule
located between the 512-520 helix and Lys518 is lost. (4) Isomerization: Glu357
abstracts a proton to yield the cis-enediol(ate) intermediate. (5) Glu357 transfers a proton
to the intermediate to yield the open chain form of G6P. (6) Conformational changes: The
512-520 helix moves to the "out" position and an ordered water molecule inserts
between the helix and the open chain form of G6P. The G6P undergoes rotation about its
C3-C4 bond to approach its cyclic conformation. (7) Ring closure: His388, Lys518, and
Thr214 assist in ring closure. (8) Product release: The G6P product is released, and the
210-214 loop moves "down". 13
Moonlighting functions of PGI
PGI/AMF/neuroleukin secretion
One important feature of PGI/AMF/neuroleukin sequence is the lack of a hydrophobic signal sequence suggesting that the protein is not a classical endoplasmic reticulum/Golgi-dependent secretory protein [9,30]. PGI/AMF is predominantly secreted from some types of tumor cells [9,30,31] or T cells stimulated with lectins [5]. It is not clear yet how AMF/neuroleukin is secreted but it apparently follows the non-classical secretory pathway described for other cytosolic proteins such as galecting-3 [113] and
FGF [114,115]. However, overexpression of PGI/AMF in normal or non-AMF-secreting tumor cells was able to induce its secretion [32,33] and that cells secreting PGI/AMF express higher PGI mRNA levels to that of normal cells [34]. Also, some studies suggest that phosphorylation by casein kinase II is associated with the enzyme's secretion [35].
Neuroleukin, and phosphoglucose isomerase
The formation of neuronal sprouts, fine neurotic processes, either from synaptic terminals or nearby nodes of Ranvier, is a widely known form of plasticity of motoneurons. At least four cytokines or growth factors are believed to be involved in motoneuron sprouting, each of which using a distinctive signaling pathway. One of those cytokines is neuroleukin, shown previously to be the ubiquitous enzyme phosphoglucose isomerase
[36].
The original biological role of sprouting was derived from the results of partial muscle denervation experiments. In an attempt to identify factors sufficient to induce motor axon terminal sprouting, Gurney et al. produced a polyclonal antisera [37] and monoclonal antibodies [8] against a 56kD protein. Mono and poly-clonal antibodies to 14 the protein derived from denervated or inactive muscle were capable of certain suppression of botulinum toxin-induced motor axon terminal sprouting. The 56kD protein was named neuroleukin. Gurney remarked that antiserum made against neuroleukin only suppressed about half of the sprouting but did not investigate further.
Acting as a neutrophic factor, neuroleukin promotes survival and sprouting at the neuromuscular junction and also plays a role in motor neuron regeneration in vivo and in the survival of peripheral and central neurons in vitro. In addition to Gurney's studies, more recent studies showed that neuroleukin is upregulated during Huntington's disease, a neurological disorder, and that inhibition of neuroleukin expression potentiates the induction of cell death in PC 12 cells, indicative of its protective role in neuronal cells
[38].
T cells can be induced to secrete several factors. For example, stimulation with lectins stimulates T cell secretion of neuroleukin and increased in neuroleukin mRNA [5]. Also, when added to cultured human peripheral blood mononuclear cells, recombinant neuroleukin induces B cell secretion of antibody. It has not been clearly shown whether neuroleukin acts directly on B lymphocytes, in an autocrine manner to stimulate the T- cell itself or even if it was a T-cell product capable of amplifying monocyte functions.
Unfortunately, due to its identification as the enzyme phosphoglucose isomerase, interest in neuroleukin involvement in neuronal sprouting and B cell antibody secretion diminished, although some papers have recently cited neuroleukin in Huntington's disease [38]. Autocrine motility factor, maturation factor and phosphoglucose isomerase
Tumorigenesis is a multi-step process during which cells acquire a characteristic set of properties which allow them to bypass the normal mechanisms of cellular growth control.
Although the genetic basis of tumorigenesis can vary greatly, the steps required for metastasis are similar for all tumor cells. The various steps in the process of metastasis
are angiogenesis, the attachment of tumor cells to other cells or matrix proteins, the
invasion of the tumor cells and finally, the colonization of the secondary site by tumor
[39].
Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in
tumor metastasis. This requires migration of cancer cells and can be stimulated by
several factors. Through a variety of mechanisms, motility factors may cause one or
more of the following which contribute to motility: changes in cell shape, cytoskeletal
rearrangements, and changes in cell adhesion and/or membrane fluidity [40]. One of
these motility factors is autocrine motility factor, also known as the enzyme
phosphoglucose isomerase.
Unlike associated neuroleukin or T cells activities, the autocrine activations mechanisms
of PGI in cancer cell motility have been assessed in detail. 16
., 2004 [49]
Figure 6 Molecular signaling in AMF/PGI motility stimulation
Members of the Rho family, such as Ras or Rho-like GTPases, induce changes in the actin cytoskeleton, an important step in cell motility. Studies showed that AMF/PGI stimulation of cell motility of some tumor cells via its receptor AMFR activates small
Rho-like GTPase, Racl and RhoA but not Cdc42 in a time- and dose-dependent manner
in human malignant melanoma cells [41].
In response to AMF/PGI, protein levels of JNK1 and JNK2, two MAP kinases working
downstream of Rho-like GTPases, are upregulated, leading to actin fiber rearrangement
and formation of heavy bundles of stress fiber-like structures transversing the cells.
Expression of GDI-p, a Rho GTPase regulatory protein, is enhanced following
stimulation of the tumor cell by AMF/PGI [33]. However, the role of GDI-P in invasion
and metastasis is controversial. Upregulation of the regulatory protein have been
associated with progression of ovarian carcinoma tumors [42] while GDI-p has been
reported as an invasion and metastasis suppressor in bladder cancers [43-45]. 17
Upregulation of GDI-p might be induced as a negative signal in the putative feedback mechanisms against excess signals from AMF/PGI.
Tumor cell Endothelial cell (autocrine) (paracrine)
Angiogenesis Yaganawa et al., 2004 [49] Figure 7 Interaction between AMF-AMFR and VEGF-VEGFR signal in the tumor and host endothelial cells
VEGF is an important signaling protein involved in angiogenesis and affects endothelial cells specifically [46]. VEGF is known to act on endothelial cell mitogenesis and migration via two tyrosine-phosphorylating receptors, fms-like tyrosine kinase (Flt-1), receptor for VEGF-1 [47] and KDR, receptor for VEGF-2 [48]. To better understand the
AMF/AMFR stimulation pathway, crosstalk between AMF-AMFR and VEGF-VEGFR
signals have been assessed. Secreted AMF/PGI was shown to stimulate the host tumor
cell in an autocrine manner and to enhance the production of VEGF. Endothelial cells
exposed to AMF/PGI secreting cells were shown to augment expression of Flt-1 but not
of KDR, suggesting that proliferative signals of VEGF in endothelial cells depend on
KDR while migrational activities depend on Flt-1. PKC and PI3K inhibitors were shown
to inhibit Flt-1 expression induced by AMF/PGI, indicating that Flt-1 expression is
dependent on activation of these two kinases [49]. 18
Yaganawa et al., 2004 [49]
Figure 8 Aptosis-related signal pathways induced by AMF/PGI overexpression
AMF-expressing cells were shown to be resistant to apoptosis induced by serum
deprivation or by mitomycin-C. AMF/PGI signals can activate PI3K which activates
Akt/PKB, which in turn inactivates the proapoptotic protein BAD and caspase-9, leading to suppression of apoptosis induced by serum deprivation [50]. AMF/PGI
overexpression can also suppress the expression of Apaf-1 and caspase-9 which are
important for apoptosis initiation, activating PI3K and MAPK and causing mitomycin-
induced apoptosis resistance [51]. There is a possibility that increased PGI enzymatic
activity may cause hyper-metabolism of glucose, an activity related to malignant tumors,
thereby affecting the apoptosis pathways [49]. 19
Involvement in mineralization during osteoblast differentiation
AMF/PGI has been identified as a key functional molecule in osteoblast differentiation, a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. Zhi et al. showed that AMF/PGI mRNA is temporally expressed during
MC3T3-E1 osteoblast-like cell line cell differentiation and their studies revealed the presence of AMF/PGI in MC3T3-E1 cells as well as in the surrounding matrix,
suggesting secretion of the protein. In addition, AMFR was detected primarily on the cell
membrane. AMF/PGI was expressed at a high level in osteoblasts and superficial
articular chondrocytes of young mice, in fibroblasts and in proliferating chondrocytes.
However, PGI expression was very low in fully differentiated bone cells such as
hypertrophic chondrocytes or osteocytes. Treatment of MC3T3-E1 cells with 6-
phosphogluconic acid, an AMF/PGI inhibitor [11,52], resulted in reduction in alkaline
phosphatase activity and mineralization in MC3T3-E1 cells, especially during the matrix
formation stage of differentiating cells [13]. The investigators showed specific expression
of AMF/PGI in discrete populations of bone and cartilage cells, suggesting a possible role
for this secreted protein in bone development and regeneration. 20
Embryo implantation and phosphoglucose isomerase
Implantation is the first stage in development of the placenta. The role of implantation is to obtain very close apposition between embryonic and maternal tissues. There are
substantial differences among species in the process of implantation, particularly with regard to "invasiveness," or how much the embryo erodes into the maternal tissue.
However, current understanding of embryo implantation in carnivores is limited [14].
A group from Illinois discovered that a 60 kDa protein was necessary in embryo
implantation in the domestic ferret. They later identified this protein as being phosphoglucose isomerase [14]. This discovery demonstrates an uncharacterized
endocrine function of the protein. This role may represent the natural motility-
stimulating activity of PGI, a characteristic later acquired by tumor cells. 21
1.4 - Receptors
AMF/PGI interacts with the cell through several receptors.
Acid PH Neutral pH
• PGI/AMF • AMF-R Mitochondria- Multivesicular associated endosome smooth ER Lagana et al, 2005 Figure 9 The complex biology of PGI/AMF and its receptor. The figure above summarizes AMF/PGI extracellular interactions with the cell. There are 6 proposed pathways. Following AMF/PGI binding, AMFR is internalized via a clathrin-independent pathway to the smooth endoplasmic reticulum tubules (1) or via another route involving a clathrin-dependent pathway to the multivesicular bodies (MVB) or endosomes (2). Following clathrin-mediated uptake to MVBs, AMF/PGI can recycle to fibronectin fibrils (3-5). Under acidic pH conditions, endocytosis of AMF/PGI is inhibited and AMF/PGI binds directly to fibronectin fibrils (7) or to heparan sulfate (8). Finally, at neutral pH, AMF/PGI can also interact with the insulin-like growth factor binding protein 3 (IGFBP-3) (6). 22 AMFR/gp78 In 1990, Nabi et al. identified the receptor for AMF/PGI (AMFR), demonstrating that B16-F1 melanoma cells expressed augmented glycosylation of a 78kDa glycoprotein (gp78) in response to cell shape modulation that correlated with an increased metastatic ability in vivo and motility in vitro [53]. The structure of AMFR was determined in 1999 to be a seven-transmembrane receptor, also called G-protein-coupled receptor, which contains a RING-type zinc finger [54]. MU ,, Extracellular daman NH; 0®©® • Domaina CUE © Domaina da liaison dala CaM O Domaina RING & O Domains Leucine zipper Tran«nambr«i ,=.©< domotnc .®& : 9fi»: O Sites de phosphorylation de CK2 •eta °<&® © Sites de phosphorylation de PKC • Sitesde phosphorylation de TK • Recouvrement des domaines RING et CK2 • Recouvrement des domaines RINGetPKC O Recouvrement desdomaines CaM et CK2 RING ®®©©®(D®®©®aa«®©©!B®©®»® ®€>© CUE ——————uS 99— ##a>«aee@® B® COO H Figure 10 Structure of AMFR/gp78. AMFR/gp78 is a specific receptor for AMF/PGI and is an integral membrane protein that can be found in the endoplasmic reticulum (ER). This receptor can target both itself and other proteins including CD3D and APOB for proteasomal degradation. Interestingly, the receptor possesses ubiquitin ligase activity. As a matter of fact, AMFR/gp78 is a RING finger-dependent ubiquitin protein ligase (E3) of the endoplasmic reticulum, 23 suggesting a potential link between ubiquitylation, ER-associated degradation (ERAD), and metastasis [55]. AMFR/gp78 also contains a Cue domain, another ubiquitin-binding domain, which can interact directly to mono and poly-ubiquitin to promote proteasomal degradation and maybe vesicular trafficking [56]. The AAA ATPase p97/VCP complex dislodges ubiquitin proteins from the ER and chaperones them to cytosol for proteosomal degradation. It has been reported recently that AMFR/gp78 can interact with the AAA ATPase p97/VCP complex and that the interaction was enhancing p97/VCP polyubiquitin-association. The investigators speculated that as a multiple membrane- spanning protein, AMFR/gp78 can form a channel for retrotranslocation [57] which might represent one way of coupling ubiquitination with retro translocation and degradation of ERAD substrates. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97/Derlin/VFMP complex is unclear. It was reported that p97 interacts directly with several ubiquitin ligases, such as AMFR/gp78, and facilitates their recruitment to Derlin- 1 [58]. Upon binding of its ligand AMF/PGI, AMFR was found to be internalized through two different pathways. Studies showed that AMFR can be localized to a smooth subdomain of the ER [59,60]. AMF-AMFR uptake to the smooth ER tubules in NIH-3T3 fibroblasts is sensitive to the cholesterol extracting reagent methyl-P-cyclodextrin (m(3CD), inhibited by the dynamin-1 K44A mutant and negatively regulated by caveolin-1 [61,62]. Thus, AMF-AMFR uptake to the ER suggests a caveolae or caveolae-like structure-mediated endocytic pathway. Studies describing AMF-AMFR uptake through a second pathway, 24 the clathrin-mediated pathway, have shown that the endocytosis was not totally inhibited by m(3CD, but that the complex receptor-ligand can be internalized to punctate structures in the cells showed by colocalization with LAMP-1 but not TfR to be multivesicular bodies (MVBs) [62]. AMF-AMFR internalization to the MVBs has also been found to be recycled to the fibrils of fibronectin[62]. The nature and the reasons behind AMF-AMFR recycling and binding to fibronectin remain to be elucidated. It has been proposed that AMF activation of the AMFR recycling pathway could be actively involved in the remodeling of the fibronectin ECM of motile cells by regulating fibril formation or turnover [63]. Protein motifs implicated in AMF/PGI cytokine activity and receptor binding In contrast to the well-characterized active site necessary for the catalytic activities of PGI, the sites involved in the cytokine functions of AMF/PGI have not yet been studied in details yet. A recent report provided some insights in AMF/PGI structure needed for cytokine functions [74]. The investigators demonstrated that mutation of residues in the wild-type human AMF/PGI that interact with the phosphate group of PGI substrates and mutations within the C-terminal region significantly reduced cell motility-stimulating activity and AMFR binding than that in the wild-type human AMF/PGI. The report further showed that mutant AMFR lacking the putative N-sugar chain attachment site was expressed on the cell membrane but did not respond to AMF/PGI-stimulation, and that N- glycosidase-treated AMFR did not compete with receptor binding of AMF/PGI. However, there results imply that the unique N-glycosylation site of AMFR is extracellular. Such topology would require an intracellular N-terminus and an extracellular C-terminus of AMFR. This is both inconsistent with the classical topology 25 of seven-transmembrane receptors [116], and would further argue that the long loops forming the C-terminus of AMFR and containing the Cue and Ring domains involved in the E3 ubiquitin ligase function of AMFR is extracellular (Figure 10). Fibronectin The extracellular matrix provides a framework for cell adhesion supports cell movement and serves to compartmentalize tissues into functional units. Fibronectin is an essential component of many extracellular matrices where it regulates a variety of cell activities through direct interactions with cell surface integrin receptors [64]. In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparin [65]. As discussed previously, internalized AMF/PGI can be recycled to the fibrils of fibronectin through the clathrin-mediated pathway [62]. In addition, AMF/PGI can interact directly with fibronectin under acidic conditions, corresponding to a change in PGI tertiary structure [66,67]. AMF/PGI recycling from MVBs did not increase cell motility but AMF/PGI sequestration by fibronectin at acid pH shows an increase in cell motility [67]. Therefore, sequestration of recycling AMF/AMFR complexes by association with fibronectin fibrils may regulate the extent of ligand-dependant AMFR signaling while AMF/PGI interacting directly with fibronectin at acid pH may be used to facilitate internalization after subsequent release upon pH. 26 Control pH 7.0 pH 6.5 I • pH 6.0 jf>R 6.sJf x J / - Amraei et al.. 2003 Figure 11 Stronger association of AMF/PGI to fibronectin at increasingly acid pH. Heparan sulfate Heparan sulfate (HS) is a sulfated polysaccharide that has a very large structural diversity. It is estimated that up to 48 different disaccharides can occur in heparan sulfate, although only 23 have been detected in vivo. Nevertheless, its structural heterogeneity allows HS to interact with a wide range of functionally diverse proteins, such as growth factors, cytokines, chemokines, proteases, lipases and cell-adhesion molecules [68]. Although fibronectin has two defined HS-binding sites [69], presence of HS does not enhance AMF/PGI binding to fibronectin fibrils but selectively increases AMF/PGI cellular binding under acidic conditions. HS therefore mediates fibronectin -independent interaction of AMF/PGI with the cell under acidic conditions [67]. Why AMF/PGI binding to fibronectin but not to HS upon acidic pH increases cell motility is still unclear. 27 IGFBP-3 Insulin-like growth factor binding proteins (IGFBPs) proteins are regulators of insulin growth factors (IGFs). Significant data showed that IGFBPs play important roles in addition to their ability to modulate IGFs. IGFBP-3 is a 266 amino acid protein [70] which can variably be N-glycosylated [71] and is mainly secreted by the liver and to a lesser extent in fibroblasts, ovaries and placenta [72]. It has recently been shown that IGFBP-3 interacts with AMF/PGI, inhibiting AMF/PGI functions [73]. In their studies, Mishra and colleagues showed that IGFBP-3 was able to inhibit catalytic activities of AMF/PGI as well as its ability to induce migration of breast cancer cells. Moreover, AMF/PGI was capable of inhibiting IGFBP-3-induced cell apoptosis. Although AMF/PGI did not bind IGF/IGFBP-3, that binary complex seemed to be a more potent inhibitor of AMF/PGI than IGFBP-3 alone, suggesting a conformation change of IGFBP-3 after its binding to the IGF. Therefore, the results showed suggested a potential ability of IGFBP-3 to disrupt the interaction of AMF/PGI with its receptor AMFR. Another receptor The human acute monocytic leukemia line does not express gp78 and its motile activity is not enhanced by AMF/PGI though it is well differentiated by AMF/PGI exposure. Forced expression of AMFR/gp78 in leukemic cells recovered AMF/PGI motile stimulation with a reduced differentiation ability. Haga et al. detected two unknown proteins by crosslinking between AMF/PGI and leukemic cells, suggesting a new receptor molecule for AMF/PGI in leukemic differentiation [74]. 28 1.5 - AMF/PGI IMPLICATION IN DISEASES Non-spherocytic hemolytic anaemia Aberrations in expression or activity of PGI due to mutations or deletions are of significant clinical importance since in humans they are associated with hereditary non- spherocytic hemolytic anaemia disease. Mutations result in enzyme instability and the defect only affects mature erythrocytes because they are no longer are capable of enzyme synthesis. Since glucose is not digestible by PGI mutated RBCs, it accumulates within the cell to the point where it deforms the cell membrane disrupting the oxygen carrying function of the erythrocytes. The major clinical features of haemolysis include variable degrees of jaundice, slight-to-moderate splenomegaly, an increased incidence of gallstones, and anaemia [75] and severe cases produce mental retardation and can even lead to death. Non-spherocytic hemolytic anaemia was first reported in 1968 [76] and has been found in many patients with PGI mutations [26]. Moreover, this autosomal recessive genetic disorder may be associated in some cases with neurological impairment [77]. Although it is a rare disease, spherocytic hemolytic anaemia is the third most common enzymatic defect resulting in hemolysis. However, it can also be caused by other enzymes deficiencies such as glucose-6-phosphate dehydrogenase, pyruvate kinase and possibly of pyrimidine 5'-nucleotidase [26,78,79]. In contrast to the other red cell enzyme deficiencies, most of the mutations observed in PGI seem to be of independent origin. Therefore, there is no indication of any selection for any particular PGI mutation [26]. 29 Cancer Serum PGI activity has long been reported and is associated with tumor expression indicating that this protein is actively released from both normal and tumor cells [80]. This protein is elevated in the serum or urine of patients with malignant tumors such as gastro-intestinal, kidney, breast, colorectal and lung carcinomas, thereby being useful as a tumor marker [81]. When secreted, AMF/PGI promotes cellular locomotion or invasion [82] and regulates tumor malignancy proliferation [50], secretion [83] or apoptotic resistance [51]. Studies have shown that expression of glycolytic enzymes, including PGI, can modulate cellular life span [84]. Indeed, enhanced glycolysis seems to lead to an uncontrolled proliferation of mouse embryo fibroblasts. The paper correlates an increased glycolysis activity in tumors with their resistance to damaging free radical production. Since overexpression of PGI and phosphoglycerate mutase, an enzyme that catalyzes different steps in glycolysis, were both found to have similar effects on cell proliferation, immortalization seems likely to be caused by enhanced glycolysis rather then by specific properties of a glycolytic enzyme. Because both AMF/PGI and AMFR seem to be very specific to tumor and metastatic cells, interest in both the cytokine and its receptor has grown and the multifunctional enzyme and its receptor are of interest in the development of novel therapeutics against cancer. 30 Rheumatoid arthritis Autoimmunity is the failure of an organism to recognize its own constituent parts as "self, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic disease. RA is strongly hypothesized to be an autoimmune disease. Although a wide range of autoantibodies antibodies can be found in RA, no common antigen has been specifically associated with the disease yet [85] and the true role of these antibodies remains controversial [86]. Studies suggesting implication of AMF/PGI in RA date from the mid 90's, when AMF/PGI was found in the synovial fluid of RA patients [87]. Watanabe et al. suggested that AMF was playing an essential role for communication among leukocytes in rheumatic disease [88]. However, a different role for AMF/PGI implication in RA was suggested later. Using the K/BxN T cell receptor transgenic mouse that spontaneously develops a joint disorder with many of the clinical, histological, and immunological features of human RA, Matsumo and al. identified AMF/PGI this time as a possible autoantigen in RA [89]. The presence of antibodies to AMF/PGI in human RA was assessed and a first study by Schaller et al showed a strong binding of autoantibodies from the sera and synovial fluid of RA patients (64%) to the commercial rabbit AMF/PGI [90]. Two independent groups using recombinant human AMF/PGI contradicted these results, concluding that less than 5% of the sera/synovial fluid contained antibodies to human AMF/PGI [91,92]. Schaller and colleagues responded to these groups and stated that their new study using human 31 AMF/PGI showed that at least 49% of RA patients presented antibodies to both rabbit and human AMF/PGI. They attributed the differences between the studies to parameters such as antibody concentration, blocking solution type and time, incubation time, limitations of certain type of essays and conformation of the commercial and recombinant proteins. Since then, AMF/PGI implication in autoimmune diseases has remained very contradictory. More studies demonstrated no specificity of RA antibodies to AMF/PGI [93,94], while others revealed the importance of AMF/PGI in the disease [95-97]. Recent data have associated AMF/PGI antibodies to extra-articular complication in RA [98] to the pathogenesis of severe forms of arthritis [99] or correlated raised levels of anti- AMF/PGI to different subclasses of IgG [100]. It is unclear why AMF/PGI is secreted in the synovial joints of RA patients. However, AMF/PGI was identified as a hypoxic inducible gene [101] and hypoxia was found to be a characteristic feature of human rheumatoid arthritic joints and animal models of arthritis [102]. Therefore, it has been proposed that hypoxia, within the rheumatoid joints, might lead to upregulation of AMF/PGI which in turn would perpetuate RA [103]. As discussed earlier (section D), upon acidification, AMF/PGI is denatured and can bind directly to the fibrils of fibronectin. It has therefore been proposed that denaturation of PGI at acid pH could lead to increase binding to fibronectin-rich cartilage areas in the synovial joints permitting the generation of a localized autoimmune response against exposed non-native PGI epitopes [66]. 32 2. HYPOTHESIS Acid-induced conformational changes in phosphoglucose isomerase (PGI) result in its increased cell surface association and deposition on fibronectin fibrils [66]. We hypothesize that conformational changes of PGI, potentially due to disruption of the monomer-monomer interface under acidic conditions, such as those encountered in the synovial fluid of arthritic joints, could result in its deposition on the surface of joints and the induction of an autoimmune response. In this thesis, the acid-dependent binding of AMF/PGI to FN and conformation-dependent recognition of AMF/PGI by RA antisera are explored. 33 3. MATERIALS AND METHODS 3.1 Protein purification PGI constructs were prepared by Zongjian Jia, a former post-doc. Transformed bacteria were grown in a 37°C shaker overnight in 3 mL Luria broth (LB) medium (Sigma) (Carbenicillin (Sigma) 100 ug/mL; Kanamycin (Invitrogen) 30 ug/mL). 2 mL were used to inoculate 200 mL of LB medium (carbenicillin 100 ug/mL; kanamycine 30 ug/mL) and the culture was incubated in a 37°C shaker. After 2 hours (~0.6 OD600) protein expression was induced by addition of 1 mM of isopropyl-P-D-thiogalactopyranoside (IPTG) (Sigma) and the culture was agitated 4 more hours at room temperature. Afterwards, bacteria were harvested by centrifugation at 3,000g x 15min at 4°C and the pellet was used to purify his-tagged proteins by metal affinity resin following the BD Talon metal affinity resins protocol (Clontech). Proteins were dialyzed against PBS using dialyzing membranes (VWR) overnight at 4°C and concentrations were measured by BCA (Pierce). 3.2 SDS-PAGE and Western blots 5 ug of purified proteins were diluted in loading buffer with or without (3- mercaptoethanol and boiled at 95 °C for 5 minutes. Reduced samples, non-reduced samples and molecular weight markers (Pageruler prestained protein ladder, Fermentas) were loaded on an 8% polyacrylamide gel. Samples were run through the gel for 1 hour 30 minutes and protein bands revealed by Coomassie Blue staining. For western blot analysis, proteins were transferred to a nitrocellulose membrane (Amersham) using a semi-dry unit for 1 hour 30 minutes. Membranes were blocked in PBS containing 2% milk and 0.1% Tween-20 (SIGMA) overnight. Anti-PGI (Raz, KCI, Detroit, USA) was 34 diluted 1:500 in blocking solution and incubated with the nitrocellulose membranes on a rotating support for 1 hour. Membranes were then washed 3 times 20 minutes and incubated with the secondary antibodies, anti-rabbit HRP (Molecular Probes), at a dilution of 1:5000 in blocking solution. Membranes were washed 20 minutes in blocking solution, 20 minutes in PBS-2% milk and twice 20 minutes in PBS. Membranes were revealed in ECL (1:1 solution A : 100 mM Tris pH 8.5, 2.5 mM Luminol, 0.4 mM p- Coumaric acid; solution B : 100 mM Tris pH 8.5, 0.02% H2O2) and exposed to X-Ray films. 3.3 Enzymatic activity assay All reagents were purchased from Sigma. Enzymatic activity of PGI/AMF was carried out using the Robert W. Gracy protocol [52]. Rabbit PGI and human constructs of PGI were prediluted in a 10 mM triethanolamine solution to a final concentration of lug/mL. The reaction solution consisted of a 50mM triethanolamine buffer, 1 mM EDTA pH 8.0, 4 mM fructose-6-phosphate, 0.5 mM NADP and 1 unit of glucose-6-phosphate dehydrogenase. Using a thermostatically regulated spectrophotometer (UNICAM UV visible spectrophotometer equipped with a circulating-water bath, Department of Chemistry, UBC, Vancouver, BC), the reaction solution was incubated for 5 minutes at 30°C in a 1 cm light-path cuvette. 25 uL of the prediluted enzyme was added to the reaction solution and the change in absorbance was read at 340 nm every minute for 5 minutes. The absorbance was then divided by 6.22 (the mM absorbance index for NADPH) to give the uM of glucose-6-phosphate formed per minute per mL of enzyme solution. 35 3.4 Glutaraldehyde cross-linking assay Glutaraldehyde cross-linking of PGI was carried out as previously described [66]. 50 pg PGI was incubated with 0.1% (v/v) glutaraldehyde (SIGMA) in 75 pi PBS for different times at room temperature. The reaction was stopped by addition of SDS sample buffer. Samples were boiled and reduced for 5 minutes, separated in 8% SDS-polyacrylamide gels, and protein bands were revealed by Coomassie Blue staining. 3.5 Circular dichroism Circular dichroism (CD) analysis of PGI and its constructs was performed using a Jasco J-810 spectropolarimeter (UBC Laboratory of Molecular Biophysics). Spectra were recorded in a 2mM quartz cuvette at room temperature in HEPES-based buffers at pH 7.5 and background signal obtained from parallel scans of the buffer alone were subtracted from the measurements. Far UV spectra of lug/ml PGI were recorded from 260 to 190 nm at a speed of 50nm/min in 1 nm steps with a signal averaging time of 4 seconds. Near UV spectra of 1 ug/ml PGI were recorded from 320 to 250 nm at a speed of 50 nm/min in 0.5 nm steps and with a signal averaging time of two seconds. 3.6 Fibronectin binding assay NIH-3T3 cells were plated at a density of 5000 cells/well on 96-well plates and cell containing wells and wells coated with soluble fibronectin at various concentration were fixed with 3% parafomaldehyde, rinsed extensively and labeled with anti-fibronectin and Alexa488 anti-rabbit secondary antibodies in order to determine the concentration (20 ug/ml) of soluble fibronectin that generated an equivalent signal to that of cell associated fibronectin fibrils. Subsequently, empty wells, NIH-3T3 cell containing wells and wells 36 coated with 20 ug/ml fibronectin or BSA were incubated in parallel with 25 ug/ml PGI/AMF-FITC for 30 minutes at 37°C at pH 7.5 or pH 5.0, rinsed, fixed and then labeled with anti-FITC and Alexa488 anti-rabbit secondary antibodies. Fluorescence intensity of the labeled wells was measured using a Bio-Tek FL600 fluorescence plate reader. Fibronectin binding assay of constructs was carried as described above [67]. 25 ug/ml of constructs were added at various pH to 96-well plates coated with soluble 20 ug/mL fibronectin (SIGMA) and fixed with 3% paraformaldehyde, rinsed extensively and labeled with anti-PGI/AMF as previously described [34] and Alexa 488 anti-rabbit (Jackson Laboratory) secondary antibodies, anti-HA (SIGMA) and Alexa 488 anti-rabbit or anti-Flag (SIGMA) and Alexa 488 anti-mouse (Jackson Laboratory). 3.7 Cell motility assay 8 uM pore polycarbonate filters (Falcon) were placed in a 24-well plate and 500 pi AMF/PGI constructs, diluted in serum-free media (25 ug/mL), were added to the lower chamber of the filter. 200 pi of 5X105 MDA-231 cells were added to the upper chamber. After 24 hours of incubation at 37°C in a CO2 incubator, the top of the filters were gently cleaned with a Q-tip, rinsed with PBS and filters were transferred in a well containing methanol-acetone and fixed for 10 minutes at -20°C. Subsequently, filters were washed with PBS and transferred and incubated for 15 minutes in a new well containing 500 uL violet crystal and abundantly washed with water. Pictures of each filter were recorded using a Leica DMRA2 microscope at x40 magnification. 37 3.8 Sera and synovial fluids Sera and synovial fluids samples were obtained from patients with rheumatoid arthritis or normal patients (Pascal Reboul, CHUM, Montreal, Canada; Hideomi Watanabe Gunma University, Japan) 3.9 Human RA antisera ELISA screening 96-well plates were coated with 100 uL of rabbit PGI/AMF at a 5pg/mL concentration diluted in 0.06 M Na-carbonate buffer pH 8.0 overnight. The plates were then blocked with 200 pL PBS-BSA 1% for 30 minutes. Blocking solution was removed and 50 pL of primary antibody (sera or synovial fluid 1:50) and incubated for 1 hour. Plates were washed 3 times with PBS-BSA 0.2% and detection antibody human IgG-HRP was added to the wells and incubated for 30 minutes. Plates were washed 2 times with PBS-BSA 0.2% and 1 time with PBS. lOOpL of ABTS solution (34.5 mg +25 pi of 30% H202 in 100 mL H20) was added to the wells and OD was read at 405 nm on a Bio-Tek FL600 plate reader. 3.10 RA antisera western blots screening 2 ug of PGI, PGI constructs or BSA were diluted in loading buffer with P- mercaptoethanol, boiled at 95°C for 5 minutes. Reduced samples and a molecular weight marker (Pageruler prestained protein ladder, Fermentas) were loaded on an 8% polyacrylamide gel. Samples were run through the gel for 1 hour 30 minutes. Proteins were transferred to a nitrocellulose membrane (Amersham) using a semi-dry unit for lh30min. Membranes were blocked in PBS containing 2% milk and 0.1% Tween-20 overnight. Primary antibodies, normal or RA sera/synovial fluid, were diluted 1:50 in blocking solution and incubated with the nitrocellulose membranes on a rotating support 38 for 1 hour. Membranes were then washed 3 times 20 minutes and incubated with the secondary antibodies, anti-human IgG-HRP (Molecular Probes), at a dilution of 1:5000 in blocking solution. Membranes were washed 1 time 20 minutes in blocking solution, 1 time 20 minutes in PBS- 2% milk and 2 times 20 minutes in PBS. Membranes were revealed in ECL (1:1 solution A : 100 mM Tris pH 8.5, 2.5 mM Luminol, 0.4 mM p- Coumaric acid; solution B : 100 mM Tris pH 8.5, 0.02% H202). 39 4. RESULTS 4.1 Analysis of recombinant AMF/PGI expression and purification Denaturation of PGI is believed to lead to autoimmune disease [66,67]. To disrupt PGI/AMF structure, N- and C-terminal tags were added to generate chimeric forms of the protein. Six constructs using human PGI/AMF constructs made by Zongjian Jia, a former post-doc in the lab, were used to study conformational effects of tag additions to the C or N-terminus. The PGI cDNA was tagged with HA or Flag epitope tags at the N or the C-terminus (His-HA-AMF, AMF-HA-His, His-Flag-AMF and AMF-His-Flag). In order to study whether dimerization was able to restore AMF/PGI properties, a cysteine residue was added to the C-terminus or within the N-terminal tag, His-Flag-AMF-cys and His-cys-Flag-AMF, respectively. The six constructs, AMF-His-Flag, His-Flag-AMF, AMF-HA-His, His-HA-AMF, His- cys-Flag-AMF, His-Flag-AMF-cys (Fig. 12b), were transformed in E. coli and purified, yielding concentrations between 200 pg/mL and 800 pg/mL. Under similar conditions, AMF-His-Flag and AMF-HA-His, were purified at lower concentrations than His-Flag-AMF and His-HA-AMF. His-Flag-AMF-cys showed the highest protein expression/purification but His-cys-Flag-AMF showed low protein concentration after purification. Some amino acids can resist degradation more than others and their presence at the beginning of proteins can be used to protect proteins from degradation. For example, proteasome is known to have a preference for hydrophobic residues [110] and RGS (arginine, glycine, serine) residues found in the pQE-31 vector are highly polar (arginine, 40 serine) or neutral (glycine), therefore making the site more hydrophilic. Because the pQE-31 RGS residues were kept intact while cloning His-Flag-AMF and His-HA-AMF but removed from the AMF-His-Flag and AMF-HA-His constructs, this probably explains why AMF-His-Flag and, to a lesser extent, AMF-HA-His showed expression levels lower than His-Flag-AMF and His-HA-AMF. The addition of a cysteine at the C-terminus of AMF/PGI (His-Flag-AMF-cys) increased levels of expression (800 ug/mL) but expression level remained low (200 pg/mL) when a cysteine was added to the N-terminus (His-cys-Flag-AMF). AMF/PGI samples were subjected to 8% SDS polyacrylamide gel. A plot of log molecular weight versus the migration, measured from the top of the gel, was prepared to calculate the molecular weight of each band. The actual molecular weights are shown in place of their corresponding log values. Analyzes showed that under reducing conditions, rabbit PGI migrated as a band of about 60 kDa while recombinant human AMF/PGI consistently migrated a little bit slower, as a band of about 62 kDa (Fig. 13a). Under non-reducing conditions, His-Flag-AMF-cys presented an additional higher band. That higher molecular band of 123 kDa corresponds to the molecular weight of a His-Flag- AMF-cys dimer (Fig. 13b). 4.2 Enzymatic activity Because AMF/PGI is an enzyme that mediates interconversion between glucoses- phosphate and fructose-6-phosphate, the different recombinant human AMF/PGI were tested for their enzymatic abilities, using Gracy's PGI enzymatic assay [52]. His-Flag- AMF, His-HA-AMF, and His-cys-Flag-AMF exhibited complete loss of enzymatic 41 activity. However, AMF-HA-His remained 50% active relative to the positive control rabbit PGI, and His-Flag-AMF-cys showed enzymatic activity recovery of about 55% to that of rabbit PGI (Fig. 14). 4.3 Glutaraldehyde cross-linking Cross-linking of recombinant AMF/PGI with glutaraldehyde for different times prior to SDS-PAGE was used to further characterize their ability to form larger complexes. It was previously demonstrated that in the absence of cross-linking, PGI was detected exclusively as a monomer of ~60 kDa but with increasing time of cross-linking distinct protein bands appeared progressively at ~120 kDa, corresponding to PGI dimers, and at 240 kDa, which was suggested to be a tetrameric form of PGI [66]. Recombinant AMF/PGI that did not show dimerization on non-reducing gels (AMF-His- Flag, His-Flag-AMF, AMF-HA-His, His-HA-AMF, His-cys-Flag-AMF) were assessed for they ability to form multimers with 0.1 % glutaraldehyde for different time and then analyzed by reducing SDS-polyacrylamide (8%) gel electrophoresis. Coomassie Blue staining revealed the progressive transition of monomeric PGI to dimeric and then to tetrameric forms of the protein with increasing times of cross-linking. A plot of log molecular weight versus the migration, measured from the top of the gel, was prepared to calculate the molecular weight of each band. The molecular weights were applied to their corresponding log values. Cross-linking of His-Flag-AMF, AMF-His-Flag, AMF-HA-His and His-HA-AMF were similar to that of rabbit PGI (Fig. 15a,b,c,e,f). At time 0 of incubation with glutaraldehyde, rabbit PGI and the different recombinant human PGI/AMF were detected 42 only as a monomer, and as time increased, bands at ~ 120 kDa and -240 kDa appeared. His-Cys-Flag-AMF exhibited a monomeric band at time 0 of cross-linking. However, with increasing time of cross-linking, higher molecular weight bands did not appear, indicating that His-cys-Flag-AMF was incapable of multimerization (Fig. 15d). Other bands visible on the gels are due to the staining of contaminating proteins. 4.4 Circular Dichroism 4.4a Far UV Circular dichroism is a form of spectroscopy that measures the differential absorption of left- and right-handed circularly polarized light. The six recombinant forms of human AMF/PGI were assessed by circular dichroism in both the far UV and near UV to identify loss or change of secondary and tertiary structure, respectively (Fig. 16). Far UV (190-260 nm) CD spectrum of recombinant human AMF/PGI show a slight difference from the rabbit enzyme and is sensitive to the presence of additional amino acids located at the N and C terminal of the enzyme. There is a decrease of signal in the negative bands at 208 and 222 nm and an increase in the amplitude of the positive band at 198 nm. The presence of a Flag tag (Fig. 16a AMF-His-Flag and His-Flag-AMF) seems to be more disruptive than that of a Ha tag (Fig. 16a AMF-HA-His and His-HA-AMF). However, loss of secondary structure is increased when either tag is located at the N terminus (Fig. 16a His-Flag-AMF and His-HA-AMF). Therefore, the addition of a Flag at the N-terminus (Fig. 16a His-Flag-AMF) seems to show the most severe effects on the secondary structure. The presence of a cysteine at the C-terminus (Fig. 16a His-Flag- 43 AMF-cys) stabilized the secondary structure, but not at the N-terminus (Fig. 16a His-cys- Flag-AMF). 4.4b Near UV Information regarding tertiary structure can be obtained from the near UV CD spectra (250 to 320 nm). All recombinant human PGI/AMF (AMF-His-Flag, His-Flag-AMF, AMF-HA-His, His-HA-AMF, His-cys-Flag-AMF, His-Flag-AMF-cys) presented a change in ellipticity of the bands, reflecting an altered conformational state (Fig. 16b). AMF-HA-His seems to retain a near UV spectrum more comparable to rabbit AMF/PGI. It is interesting to point out the apparent contribution of the disulfide bond in the near UV CD spectrum His-Flag-AMF-cys. It can be observed that between 250 and 270 nm, the intensity of the CD spectrum is larger when the Cys residues are forming a disulfide bond. This is the region where the CD absorption bands of the disulfide bond are expected [111]. Although the amplitude of the bands showed some variation, the structure and shape of the spectrum was retained across the different forms of recombinant AMF/PGI. 4.5 Binding to fibronectin Recombinant human AMF/PGI were subsequently tested for its ability to interact with cell receptors. AMF/PGI is known to bind in an increasing manner to fibronectin (FN) at acid pH. A fibronectin binding assay was carried out to determine whether PGI/AMF was able to bind to the soluble form of fibronectin. PGI/AMF binding to soluble FN was assessed using a fluorescent plate reader assay. To compare PGI/AMF binding to soluble FN relative to cell associated FN, a soluble FN 44 concentration (20 ug/mL) that generated an equivalent fluorescent signal using anti-FN antibody relative to plated NIH-3T3 cells was determined. At pH 7.5, PGI/AMF-FITC did not exhibit detectably increased binding to soluble FN or to NIH-3T3 cells relative to control wells left empty or coated with an equivalent concentration of BSA. Binding to FN at pH 5 was detectable and was essentially equivalent to PGI/AMF binding to NIH- 3T3 cells indicating that at acid pH, PGI/AMF binds to soluble FN (Fig. 17a). Recombinant human AMF/PGI binding to FN was assessed using the previously described assay (Fig. 17b). The control rabbit AMF/PGI strongly bound at pH 5.0 and binding to FN decreased as pH increased. AMF-His-Flag, His-Flag-AMF, AMF-HA- His, His-HA-AMF and His-Flag-AMF-cys showed an increased binding to FN at acid pH although to a lesser extent compared to that of rabbit AMF/PGI. His-cys-Flag-AMF showed low binding to FN. 4.6 Recombinant cell-induced motility AMF/PGI is known to induce cell motility. Therefore, recombinant human AMF/PGIs were assessed for their ability to promote migration of MDA-231 cells through 8pM pore polycarbonate filters (Fig. 18). After 14 hours of treatment with rabbit AMF/PGI, MDA- 231 cell migration through the filters was quantified, exhibiting a 2.6-fold increase above negative control (p<0.01) . Under similar conditions, AMF-His-Flag and AMF-HA-His increased cell motility by a 1.6-fold while cell-induced migration by His-HA-AMF had a 2-fold increase compared to the control with no sera (p<0.01). However, His-Flag-AMF, His-cys-Flag-AMF and His-Flag-AMF-cys did not significantly increased cell motility (p<0.01). 45 4.7 Implication of AMF/PGI in rheumatoid arthritis 4.7a ELISA PGI has been postulated to play a role as an autoantigen in RA, although this has been controversial. Some studies reported a high prevalence of PGI autoantibodies [90,95,96] while other reports showed no specificity of RA antibodies against PGI [91,92,93]. Previous findings in our lab demonstrated denaturation of PGI/AMF under an acidic environment and binding to fibronectin following conformational changes of PGI/AMF [66,67]. The synovial fluids of arthritic joints are often found to be acidic [108], therefore we attempted to demonstrate that conformational changes of PGI might be responsible for the autoimmune response in RA [66]. An ELISA essay against rabbit PGI was first used to determine the existence of autoantibodies in the sera of patients with RA (Fig. 19). 49 sera and 14 synovial fluids from patients with RA and 10 sera from 'normal' patients were tested for their ability to bind rabbit PGI, using BSA as a negative control. The results showed reproducible variations between each sera or synovial fluids, with a small increase of binding at pH 5.5 compared to the binding at pH 7.5. RA sera binding to rabbit PGI did not show more specificity than 'normal' sera (Fig. 19), More importantly, binding of antibodies to control BSA showed a very similar binding pattern to the control BSA. As a matter of fact, differences between PGI binding compared to BSA binding were specific at 8% of the time at pH 7.5 and 5% of the time at pH 5.5, showing that the assay had no or very low specificity to rabbit PGI (Fig. 19) (p<0.05). 46 4.7b Western blot The presence of autoantibodies to AMF/PGI in the sera of RA patients was then assessed by western blot. Control showed that rabbit PGI, recombinant proteins His-Flag-AMF, His-Flag-AMF-cys and AMF-HA-His, but not BSA could be recognized by polyclonal rabbit anti-PGI by western blot (Fig. 20). 20 RA sera, 7 RA synovial fluids and 7 sera from 'normal' patients were tested for their ability to bind rabbit PGI, His-Flag-AMF and BSA (Fig. 21). Western blots were quantified (Fig. 21b) and ratios are shown in Table II (Table II). From the results obtained, it can be seen that, among the samples tested, only one RA serum showed binding to rabbit PGI (Fig. 21 #002) (p<0.05), while other samples presented no significant binding to rabbit PGI. Comparatively, binding to the recombinant human PGI His-Flag-AMF was detected. Hence, about 70% of RA sera significantly bound His-Flag-AMF (p<0.05). Binding to His-Flag-AMF between the samples showed high variations, some exhibiting an increase of 4 or 5 times above background while others showed an increase of up to 15 times above background. Synovial fluids from RA patients demonstrated strong binding (Fig. 21 #554sf, 584sf) (p<0.05) to His-Flag-AMF, which correlated with the presence of PGI autoantibodies in RA sera from the same patients (Fig. 21 RA sera #554, #584). One 'normal' serum showed strong binding to His-Flag-AMF (Fig. 21#573) (p<).05). Among the 34 samples used, 3 RA sera exhibited significant binding to BSA (Fig. 21 #502,512,554) (p<0.05) and none of the other samples significantly recognized the BSA control. 47 To determine whether binding to human PGI was conformation specific (Fig. 22), His- Flag-AMF-cys and AMF-HA-His were chosen because the recombinant proteins retained enzymatic activity (Fig. 14), suggesting that their conformation is less altered, also confirmed by circular dichroism spectra (Fig. 16). Hence, in addition to rabbit PGI and His-Flag-AMF, RA sera were tested for their ability to bind to His-Flag-AMF-cys and AMF-HA-His by western blot. Sera that did not significantly bind His-Flag-AMF did not show binding to any other recombinant AMF/PGI (Fig. 22 sera #009, 552, 556, 560, 572). When autoantibodies in RA sera showed binding to His-Flag-AMF, they exhibited binding to His-Flag-AMF-cys (73%) and to AMF-HA-His (60%) as well. In many cases, autoantibodies bound His-Flag-AMF more significantly than His-Flag-AMF-cys or AMF-HA-His (66%). Several proteins can renature on a membrane following western transfer. Hence, PGI might renature following the transfer step. Therefore, stronger antibody binding to His-Flag-AMF than to His-Flag-AMF-cys and AMF-HA-His might be caused by the more aberrant conformation of His-Flag-AMF. 48 5. Discussion 5.1 Conformational effects of residue additions to C-terminus and N-terminus PGI is a globular enzyme active as a dimer. The association between the monomers is reinforced by a C-terminus "arm-like" tail that will embrace the other subunit when PGI forms a dimer. On the other hand, the N-terminus of PGI is found within the large domain [104]. When Flag and Ha were added to the C-terminus of PGI, only the Ha tag showed enzymatic activity (Table I AMF-His-Flag, AMF-HA-His). However, both recombinant protein forms showed low structural alterations by CD (Fig. 16) and increased cell motility (Table I). When Flag and Ha tags were added to its N-terminus, PGI showed loss of enzymatic activity (Table I His-Flag-AMF, His-HA-AMF). However, His-HA-AMF showed less structural changes (Fig. 16) and increased cell motility while His-Flag-AMF, which showed weak secondary structure CD spectra (Fig. 16), did not increase cell motility (Table I). There are some suggestions that not all residues make equal contributions to protein stability. In fact, it makes sense that amino acids located inside the protein, which become inaccessible to the solvent in the native state, would have a much greater effect than those on the surface [111]. These characteristics confer greater flexibility to the C-terminus tail but also cause the introduction of N-terminus residues to induce destabilization. Hence, because N- terminus is located between the dimer subunits, additions would be more disruptive than C-terminus additions, and interactions leading to dimerization would be less likely to happen, which explain loss of activity when tags are added to the N-terminus of PGI. 49 Disulfide bonds are believed to increase stability of the native state by decreasing the conformational entropy of the unfolded state due to the conformational constraints imposed by the cross-link [105]. C-terminal addition of a Cys residue stabilized PGI, showed by high levels of protein expression (800ug/mL), by restoration of enzymatic activity (Table I His-Flag-AMF-cys) and by restoration of the Far UV spectra measured by CD (Fig. 16a). On the other hand, the addition of the cysteine to the N-terminus did not restore expression levels, enzymatic activity (Table I His-cys-Flag-AMF), the overall CD spectra showed altered conformation (Fig. 16a His-cys-Flag-AMF). The N-terminal Cys residue destabilized PGI even more, showed by loss of multimerization and of fibronectin binding (Summary Table I). Thus, cysteine increased PGI stability when added to the C-terminus but not to the N-terminus. One possible reason is that the cysteine residues introduced to the N-terminus would themselves lead to some destabilization. On the other hand, N-terminal cysteines may create a disulfide bond not strong enough to offset the destabilizing effects of the N-terminal Flag. A more simple explanation is that the added N-terminal cysteines can not interact with each other for steric reasons and therefore have no effect. 5.2 Cell-induced motility and cell interaction of AMF/PGI AMF was shown to be capable of glycolytic activities but AMF/PGI cell-induced motility does not correlate with enzymatic activity, although active sites from both enzymatic activity and motility overlap. AMF exhibited enzymatic/cytokine properties of PGI/neuroleukin which were inhibited by specific PGI inhibitors [11]. Site-directed mutagenesis of two residues involved in binding erythrose-4-phosphate results in 50 impaired autocrine motility factor activity, suggesting that the active site is involved in binding to the autocrine motility factor receptor [11]. We showed (Fig. 14,18) that loss of enzymatic activity does not correlate with induction of cell motility, hence our results suggest that not all domains involved in the enzymatic active site are required for cytokine activity. It should also be noted that recombinant PGI/AMF were purified from bacterial culture and we cannot exclude the possibility that bacterial contaminants may contribute to the motile response observed. Indeed, AMF/PGI cell-induced motility requires a certain degree of structure: recombinant AMF/PGI showing loss of enzymatic activity (Table I His-Flag-AMF and His-cys-Flag-AMF), a strong change in structure (Fig. 16 His-Flag-AMF and His-cys- Flag-AMF) and low binding to fibronectin at acid pH (Table I His-cys-Flag-AMF) did not increase cell motility (Table I His-Flag-AMF and His-cys-Flag-AMF) while proteins showing no enzymatic activity (Table I His-HA-AMF and AMF-HA-His) but less changes in secondary structure (Fig. 5a His-HA-AMF and AMF-HA-His) induced cell motility (Table I His-HA-AMF and AMF-HA-His). Therefore, alteration to a certain extent of AMF/PGI can disrupt cell-induced motility but enzymatic activity is not required, confirming that glycolytic and motility active sites are not totally identical. Thus interpretation is consistent with previous reports showing that enzymatic activity of phosphoglucose isomerase is not required for its cytokine function [61,62] PGI dimers are responsible for enzymatic activity but the monomer has been proposed to be responsible for neurotrophic activity of neuroleukin. In the presence of monomeric PGI, neuroblastoma cells have enhanced neurite extension and a reduced proliferation 51 rate [106]. Sites required to activate cell motility are not well-defined and it is still unclear whether dimerization is required for cell-induced motility [21]. The C-terminal covalently bound dimer (His-Flag-AMF-cys), showed restoration of enzymatic activity (Table I His-Flag-AMF-cys) and secondary structure (Fig. 5a His-Flag-AMF-cys) but was not able to induce cell motility (Table I His-Flag-AMF-cys). Thus, the inability of induced dimerization of His-Flag-AMF-cys to restore motility stimulation argues that dimerization is not sufficient for the cytokine function of AMF/PGI and suggests that monomerization of AMF/PGI might be required to activate cell motility pathway. Cross- linked AMF/PGI has been shown to endocytose at neutral pH [66]. Therefore, several reasons might explain the lack of cytokine activity of dimerized His-Flag-AMF-cys. For example, it might suggest that N-terminal modification will prevent receptor activation or that structural changes or dimerization of His-Flag-AMF-cys lead to AMF/PGI inability to react with the molecules involved in the cell motility pathway. On the other hand, dimerized His-Flag-AMF-cys might bind different receptor or be endocytosed through a different pathway, explaining why cell motility did not occur. 5.3 Implications for the Role of PGI in Rheumatoid Arthritis Direct binding in large amounts of monomeric PGI to cell surface extracellular matrix fibrils at acid pH provides one possible explanation for the postulated role of PGI in RA. PGI cytokine activity has been found in rheumatoid synovial fluid [87] which has been reported to be acidic, as low as pH 6.0, particularly in rheumatoid patients [108]. Also, PGI increased expression has been associated with hypoxia. In response to hypoxia, normal cells will increase gene expression of glycolytic enzymes to adapt environmental 52 stress through activation of hypoxic-inducible transcription factor [102]. At lower pH, AMF/PGI undergoes partial denaturation. Therefore, acid pH-association of PGI with the joint surface would result in exposure of epitopes different from the native form of the enzyme leading to an immune response and destruction of the cells through direct or indirect antibody mediated cytotoxicity [67]. Similarly, addition of tags to PGI results in a loss of structure and could mimic acid pH denaturation of PGI. Moreover, non- recognition of rabbit PGI by RA antibodies demonstrates a high specificity to human PGI of RA autoantibodies (Fig. 21) Differences between the ability of RA antisera to recognize different forms of recombinant human AMF/PGI at different levels (Fig. 22 His-Flag-AMF, AMF-HA-His and His-Flag-AMF-cys) may be due to the degree of structural alteration of recombinant AMF/PGI. Recent studies have shown that RA autoantibodies to PGI are associated with the occurrence of extraarticular complications [98] and that elevated levels of circulating anti-PGI antibodies in serum may be masked by non-specific binding [100]. However, patient information was unavailable for the current study and further research is required to determine whether high titers of PGI antibodies correlate with more severe forms of arthritis. It is interesting to note that the only normal sera that showed elevated anti-PGI antibodies was diagnosed with Sarcoidosis, an immune disorder which has also been correlated with high levels of secreted AMF [117] 53 6. Conclusion The prevalence of autoantibodies in RA sera was found in this study to be high (70%) (Fig. 21,Table II), consistent with original reports claiming elevated levels of antibodies to PGI in the sera/synovial fluids of RA patients (64%) [90]. Increased binding to His- Flag-AMF compared to other forms of recombinant AMF/PGI may indicate more specific recognition by autoimmune Abs of conformationally modified AMF/PGI, confirming our original hypothesis that antisera in RA may be due to conformational changes in AMF/PGI. Although they have conserved a very similar structure throughout evolution, only mammalian but not bacterial or yeast forms of AMF/PGI acquired cytokine functions [109]. PGI extracellular functions, such as osteoblast differentiation [13] or embryo implantation [14], may have evolved in order to fill other functions. Bacteria and yeast do not have that complex multicellular system, which might explain that lack of PGI cytokine activity. Protein multifunctionalism is a clever mechanism for generation of complexity using existing proteins without requiring expansion of the genome [104]. However, loss of functional specificity may leave openings and opportunities for diseases. For instance, AMF/PGI structure may not have been primarily built to be secreted or to resist an acidic environment, resulting in the exposure of distinct motifs uncovered by the unfolding process, susceptible to autoantibody recognition. Therefore, AMF/PGI denaturation in the synovial fluid of RA patients might be responsible for RA autoantibody recognition and our data might provide new insights into the detection of epitopes involved.in autoimmune diseases. 7. FIGURES AND LEGENDS 55 Vector and Constructs Sma I EcoRI/RBS 6xH" Bom HI Sph I Soc I Kpn I Xmo I Sol I Pstl Hind III fa I ATGTAGAGGATCI BMAC |GGATCCGCATGCGAGLTCGGTACCCCGGGTCGACCTGCAGCCAAGCTT]AATTAGCTGAG I 1 RGS His epitope B AMF-HA-His M - AMF - YPYDVPDYALHHHHHHK L Q stop... siartlHHB AMHHHHHHHHBBHtHHIH— stop AMF-His-Flag M - AMF - H H H H H H DYKDDDDK A stop start mmm: AMFflaHMi^i^HHHB^PM-st^ His-HA-AMF DPHASSVPY st P MRGSKH ^ HiHiBH^HBBB AMlvilHKHHr^ ° His-Flag-AMF M RG Sj^^^^T DPHASSVPR VD^y His-cys-Flag-AMF MRGSH hJH T DPHASSVPRV D^^^^)K - AMF - C stop start-—•••• Flag AMF cysstop His-Flag-AMF-cys MRGSH HCTDPHASSVPRVDYKDDDD^^^ AMF -,^^^_^_stop start —-IHHHbys Flag AMF stop Figure 12 56 Figure 12 Vector and Constructs (A) Map of the expression vector pQE-31. (B) Maps of recombinant human PGI/AMF. PGI/AMF was tagged with HA or Flag epitope tags at the C and N-termini as well as 6XHis residues for purification (AMF-HA-His, AMF-His-Flag, His-HA-AMF, His-Flag- AMF). His-Flag-AMF was flanked with a cysteine residue at the C-terminus (His-Flag- AMF-cys) and N-terminus (His-cys-Flag-AMF). 57 Western Blot and SDS-PAGE analysis of recombinant AMF/PGI Western blot SDS-PAGE Reducing Dimer Non-reducing Monomer c Semi-log graph 0 20 40 60 80 Relative migration Figure 13 58 Figure 13 Western blot and SDS-page analysis of recombinant AMF/PGI. Western blot (A) and SDS-PAGE (8%) (B) analysis of rabbit PGI and of the purified recombinant human AMF/PGI with (Reducing) or without (3-mercaptoefhanol (Non- reducing). Lane 1, molecular weight marker (Fermentas); lane 2, rabbit PGI (not shown on SDS-PAGE); lane 3-8, purified AMF-His-Flag, His-Flag-AMF, AMF-HA-His, His-HA-AMF, His-Flag-AMF-cys , and His-cys-Flag-AMF Ni-NTA affinity chromatography. (C) A plot of log molecular weight, measured from the top of the gel, was prepared to calculate the molecular weight of each band. The actual molecular weights are shown next to their corresponding log values. 59 Enzymatic activity of recombinant AMF/PGI Control AMF/PGI Rabbit i AMF-His-Flag His-Flag-AMF AMF-HA-His en His-HA-AMF His-cys-Flag-AMF His-Flag-AMF-cys 0.02 0.04 0.06 0.08 0.1 pM glucose 6-phosphate / min / mL Figure 14 60 Figure 14 Enzymatic activity of recombinant AMF/PGI. An enzymatic activity assay was used to determine the abilities of the purified constructs to convert fructose-6-phosphate to glucose-6-phosphate. The reaction was initiated by the addition of rabbit AMF/PGI or recombinant AMF/PGI (0.1 unit/mL) to lmL of reaction mixture [50 mM triethanolamine buffer (pH 8.3), 1 mM EDTA, 4 mM fructose 6-phosphate as a substrate, 0.5 mM NADP, and 1 unit of glucose 6-phosphate dehydrogenase]. The graph shows activity for the construct AMF-HA-His and restoration of activity to the inactive His-Flag-AMF by insertion of a cysteine residue at the C-terminus of the protein. Glutaraldehyde cross-linking of 61 recombinant AMF/PGI A Rabbit AMF/PGI Semi-Log Graphs Min 0 1 5 10 15 30 60 (T)~240--> •Jretamer (~240 kDa) (D)~120-> (M)~60--> 20 40 60 Relative migration B AMF-His-Flag Min 0 1 5 10 15 30 60 (T)~240~> f Jretamer (~-240_kQa) (D)~120-> (M)~60--> Relative migration C His-Flag-AMF Min 0 1 5 10 15 30 60 (T)~240~> (D)~120--> (M)~60~> Monomer (-62 kDa) 10 20 30 43 50 Relative migration Figure 15 Glutaraldehyde cross-linking of 62 recombinant AMF/PGI (continuation) D His-cys-Flag-AMF Semi-Log Graphs Min 0 1 5 15 30 60 (M)~60- Monomer (-62 klpa) 10 20 30 40 50 Relative migration AMF-HA-His Tretamer (~ 240 kDa) Monomer (-62 kba) 10 2 0 30 40 50 Relative migration His-HA-AMF Min 0 1 5 15 (T)~240~> Tretamer (~ 240 kDa) (D)~120--> (M)~60»> kDa) 10 20 30 40 50 Relative migration Figure 15 63 Figure 15 Glutaraldehyde cross-linking and semi-log graph analysis. (A) Rabbit PGI and (B-F) recombinant human PGI were cross-linked with 0.1% glurataldehyde for 0, 1, 5, 10, 20, 30 and 60 min, as indicated, and then analyzed by reducing SDS-polyacrylamide (8%) gel electrophoresis. (A-C, E-F) Coomassie Blue staining reveals the progressive transition of monomeric PGI to dimeric and then to tretrameric forms of the protein with increasing times of cross-linking. (D) Coomassie Blue staining reveals no multimerization of the purified recombinant protein His-cys- Flag-AMF. (A-F) Plots of log molecular weight versus distance (in mm), measured from the top of the gel, were used to calculate the molecular weight of each band. The calculated molecular weights are shown next to their corresponding log values. Circular Dichroism of recombinant AMF/PGI B Far UV Near UV AMF-His-Flag His-Flag-AMF 0 His-cys-Flag-AMF a His-Flag-AMF-cys AMF-HA-His • His-HA-AMF • rabbit PGI CD 65 Figure 16 Circular dichroism spectra of recombinant PGI/AMF. Circular dichroism (CD) analysis performed by recording spectra in a 2mM quartz cuvette at room temperature in HEPES-based buffers at pH 7.5. Far UV spectra of recombinant PGI/AMF were recorded from 260 to 190 nm and near UV spectra of recombinant PGI/AMF were recorded from 320 to 250 nm. (A) Effects of tag additions upon the far UV CD spectra of PGI/AMF. The far UV CD spectrum of AMF-HA-His ()IOIOK) shows less structural alteration than that of the other recombinant PGI/AMF. His-Flag-AMF-cys (eee), relatively to His-Flag-AMF (AAA), shows restoration of its far UV CD spectra, to an extent comparable to that of AMF-HA-His (HOIQH). (B) Effects of tag additions upon the near UV CD spectra of PGI/AMF. The near UV CD spectrum of AMF-HA-His ()ipipi() show less structural alteration than that of other recombinant PGI/AMFs. The addition of a cysteine at the C-terminal of His-Flag-AMF (AAA) leads to the apparition of a strong band between 250 and 270 nm. 66 Binding of PGI/AMF to dimeric FN at neutral and acid pH Labeling Figure 17 67 Figure 17 Binding of PGI/AMF to dimeric FN at neutral and acid pH. (A) The fluorescent signal due to binding of PGI/AMF-FITC to uncoated wells of a 96- well plate, to wells coated with 20ug/mL BSA or FN, or to wells plated with NIH-3T3 cells was amplified by anti-FITC and Alexa488 anti-rabbit secondary antibodies and measured with a fluorescence plate reader. Absolute relative fluorescence values were normalized to maximal values and binding at pH 5.0 and pH 7.5 in the presence or absence of PGI/AMF-FITC was determined. To assess relative FN levels in the wells containing soluble FN or NIH-3T3 cells, parallel wells were labeled with anti-FN and Alexa 488 anti-rabbit secondary antibodies. (B) Recombinant PGI/AMF binding to fibronectin was assessed using the same assay and showed increased binding at acid pH except for His-cys-Flag-AMF. 68 Recombinant AMF/PGI cell-induced motility Figure 18 69 Figure 18 Recombinant AMF/PGI cell-induced motility. MDA-231 cells plated on 8uM pore polycarbonate filters were treated with 25 ug/mL of each form of recombinant human AMF/PGI or rabbit PGI each construct or rabbit PGI. After 24 hours, the cells were fixed with methanol/acetone, dyed with crystal violet and pictures of each filter taken. The number of migrating cells was quantified. Rabbit PGI, AMF-HA-His, AMF-His-Flag and His-HA-AMF significantly (*) stimulated cell motility, and His-Flag-AMF, His-cys-Flag-AMF and His-Flag-AMF-cys did not. Table I Summary of recombinant AMF/PGI properties Non- Enzymatic Glutaraldehyde Cell Construct Tag Caractenstics FN binding reducing gel activity cross-link motility Flag-Hi s at Monomer, AMF-His-Flag ** C-terminal 0 Dimer, Tetramer His-Flag at Monomer, * His-Flag-AMF 0 N-terminal Dimer, Tetramer His-Flag at Monomer, no His-cys-Flag- Cysteine at N 0 AMF N-terminal terminal multimerization IC Monomer His-Flag at Cysteine at C Presence of His-Flag-AMF- Dimer * cys N-terminal terminal dimer Tetramer Monomer Ha-His at AMF-Ha-His *** Dimer ** ^terminal Tetramer Monomer His-Ha at *** His-Ha-AMF 0 Dimer N-terminal Tetramer 71 Aijsueiuj JO|OO aAi}B|9u Ajisuejui JO|OO 8Ai}B|ey Figure 19 .72 Figure 19 Human RA antisera ELISA screening. The binding of anti-sera to wells coated with 5ug/mL of rabbit PGI or BSA at pH 7.5 (A) or pH 5.5 (B) was detected using anti-human-HRP, visualized with a chromogenic substrate and quantified using a plate reader. (A) At pH 7.5, 8% of the sera/synovial fluid showed significant (*) differences of binding to rabbit PGI and BSA and (B) at pH 5.5, 5% of the sera/synovial fluid showed significant (*) differences of binding to rabbit PGI and BSA. 73 RA antisera Western Blot screening Western Blot control Figure 20 74 Figure 20 Western Blot control Rabit PGI, His-Flag-AMF, His-Flag-AMF-cys and AMF-HA-His were run through a polyacrylamide gel, transferred to a nitrocellulose membrane and botted for anto-PGI. Polyclonal rabbit anti-PGI is able to specifically recognize the -60 kDa rabbit PGI, His- Flag-AMF, His-Flag-AMF-cys and AMF-HA-His monomeric bands but not 66.4 kDa BSA band. Western Blot screening : species-specific 75 recognition of RA anti-sera to AMF/PGI RA sera RA synovial fluid Normal sera 554 552sf Pa 554sf Jo 555sf Al 560sf 558 n-ra 570sf 559 n-ra 572sf 573 n-ra 584sf 583 n-ra Densitometry ll Audi iniAi T-NOJt-NNOfNIO^UlNOT-M'tlOOD^ M V) CA H OT OT a ;= o co co TO O O O O O in in ll) if. in m Hi t'.i I I' I-- 1^- |- Qj 1(1 — *— i— i— oooininininmmmminmmmmininmm Ra sera I rahhit PGI • his-flag-Ah Dvial fluid 'Normal' sera Figure 21 Binding to (p<0.05) RA sera Ra synovial fluids 'normal' sera Rabbit PGI 5% (1/20) 0% 0% His-Flag-AMF 70% (14/20) 30% (2/7) 15% (1/7) BSA 15% (3/20) 0% 0% 76 Figure 21 Western Blot screening : species-specific recognition of human RA anti-sera to PGI/AMF Table II Analysis of densitometry (A) RA sera and synovial fluids and normal sera were assessed for the presence of autoantibodies against rabbit PGI, His-Flag-AMF and BSA by western blot. (B) The bands were quantified. (See Table II) Quantification showed high prevalence of antibodies to His-Flag-AMF in RA sera (70%). RA synovial fluids that presented antibodies to His-Flag-AMF correlated to patient sera that showed elevated levels of antibodies to His-Flag-AMF (#554, #584). 77 Western Blot screening : conformation-specific recognition of RA anti-sera to recombinant AMF/PGI Figure 22 78 Figure 22 Western Blot screening : conformation-specific recognition of human RA anti-sera to recombinant PGI/AMF (A) RA sera and synovial fluids and normal sera were assessed for the presence of autoantibodies against rabbit PGI, His-Flag-AMF and BSA by western blot. (B) The bands were quantified (see table II). Quantification showed a high prevalence of antibodies to His-Flag-AMF in RA sera (70%). RA synovial fluids that presented antibodies to His-Flag-AMF correlated to patient sera that showed high levels of antibodies to His-Flag-AMF (#554, #584). 79 8. Bibliography 1. 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