2. 2
Table of contents.
Title page…………………………………………………………………………………1
Table of contents………………………………………………………………………….2
Abstract……………………………………………………………………………………4
List of figures………………………………………………………………………………5
Introduction
1.0 The TGF – β superfamily and interaction with GAGs ………………………………..6
1.1The biological role of Gremlin and its contribution to kidney fibrosis………………...7
1.2 The structure and biosynthesis of heparin and heparan sulfate………………………...8
1.3 Do protein – heparan sulfate interactions exhibit binding specificity?...........................10
1.4 Studies investigating sulfation structures of heparan sulfate………………………......12
1.5 Competitive binding studies of heparan sulfate variants………………………………13
1.6 Homology modelling of Gremlin antagonist…………………………………………..15
1.7 Gremlin mutagenesis…………………………………………………………………...16
1.8 Rationalisation of method………………………………………………………………17
Materials and methods……………………………………………………………………..19
3. 3
Results……………………………………………………………………………………...22
Discussion:
2.0 HSE, HSA and heparin as competitive inhibitors………………………………………32
2.1What gives rise to the distinctive binding nature of KHS?.............................................. 32
2.2 Characterising KHS binding to GAGs, the next steps………………………………… 33
2.3 Comparison with previous studies investigating mutagenized Gremlin binding……....33
2.4 Gremlin mutagenesis and its influence on GAG binding specificity…………………..34
2.5 Heparin binding and its effect on BMP antagonism……………………………………35
2.6 Closing remarks………………………………………………………………………...35
Acknowledgements………………………………………………………………………....37
Reference List ……………………………………………………………………………...38
4. 4
Abstract.
This study has produced strong evidence indicating that the BMP-7 antagonist Gremlin
associates with heparin and heparan sulfate in a specific manner.
Heparin and Heparan sulfate (HS) are linear polysaccharides that belong to a group of
biomolecules called gycosaminoglycans (GAGs). This experiment has employed a selection
of naturally occurring heparan sulfates (HSE, HSA and kidney heparan sulfate) in a
competitive binding assay.
An ELISA technique was used to measure competitive binding activities. A capture plate was
made through coating with covalently coupled heparin-BSA conjugate and each Gremlin
antigen sample was combined with a heparan sulfate to exert binding competition.
The competitive activity of each GAG was compared across 3 different variants of Gremlin.
These variants included the WT form and 2 mutagenised forms (MGR5 and MGR6). For
MGR5 and MGR6, site directed mutagenesis was carried out on 3 basic clusters involved in
heparin binding.
Mutagenesis was shown to result in an alteration in the competitive binding abilities of all
three heparan sulfates. Rather than a quantitative reduction in affinity for all GAGs, there was
a distinct change in binding profile for each mutant. These results indicate that structural
elements of proteins contribute to the interactions made with GAGs. This indicates that
different proteins, exhibiting different surface structures may exhibit unique binding profiles.
It is also clear that a number of different composite features of GAGs can influence their
binding natures. More highly sulfated GAGs may have greater competitive strength whilst
some 3D conformations could reduce steric limitations.
5. 5
List of Figures:
Figure 1: Gremlin signalling and downstream effects in cells………………………………7
Figure 2: Heparan sulfate structure and biosynthesis………………………………………..9
Figure 3: GAG competitive binding experiments for IL-12…………………………………11
Figure 4: Homology modelling of Gremlin with the PRDC monomer………………………15
Figure 5: Ribbon model of Gremlin showing the clusters of 3 basic residues………………16
Figure 6: Graphed absorbance at 405nm wavelength with increasing amounts of WT Gremlin
per well……………………………………………………………………………………….22
Figure 7: Graphed absorbance at 405nm wavelength with increasing amounts of WT Gremlin
per well. (Includes samples coated with x2 immobilised heparin)………………………......23
Figure 8: Graphed absorbance at 405nm wavelength with increasing amounts of
MGR5………………………………………………………………………………………...24
Figure 9: Competitive binding assay for WT and MGR5 with soluble heparin, HSA and
HSE…………………………………………………………………………………………..25
Figure 10: Competitive binding assay for WT, MGR5 and MGR6 with soluble heparin, HSA
and HSE………………………………………………………………………………………26
Figure 11: Competitive binding assay for WT, MGR5 and MGR6 with soluble heparin, HSA,
HSE and kidney heparan sulfate………………………………………………………..........28
Figure 12: Competitive binding assay for WT, MGR5 and MGR6 with increasing amounts of
heparin and soluble kidney heparan sulfate………………………………………………….30
Table 1: Sulfation ratios of HAS and HSE as determined by NMR spectroscopy………….13
6. 6
Introduction.
1.0 The TGF – β superfamily and interaction with GAGs.
The BMPs (bone morphogenic proteins) together with the GDFs (growth and differentiation
factors) constitute one family of cytokine proteins. Both BMP and GDF possess the
characteristic “cysteine knot” fold, placing them within the TGF-β cytokine superfamily
(Rider and Mulloy, 2010). The BMPs, as their name indicates, have a specified role in bone
formation through primary osteoblast differentiation. Besides this, BMPs have additional
developmental roles in other tissues. For example, BMP-7 is essential for kidney
development and BMP-10 has a role in cardiomyocyte proliferation (Rider and Mulloy,
2010).
So far a quarter of cytokines have been shown to bind to the glycosaminoglycans (GAGs)
heparin and heparan sulfate (Rider, 2006). The interactions of BMPs with heparin and
heparan sulfate have been discussed in a wide breadth of literature demonstrating the
influence this has on the protein’s biological effects. A primary role of glycosaminoglycan
binding is to localise the activity of the protein to a desired area (Rider and Mulloy, 2006),
thus aiding paracrine activity. Furthermore, TGF-β has been shown to bind to both heparin
and its receptor simultaneously, resulting in a co-operative potentiating effect (Rider, 2006).
Therefore, in many cases GAG binding acts to enhance the effects of the cytokine activity.
The activity of BMPs is regulated by their antagonists which have also been shown to form
associations with heparin and heparan sulfate. This results in the localisation of BMP and
antagonist in close proximity (Rider and Mulloy, 2010) allowing the antagonist to bind to
BMP; this binding prevents BMP receptor activation (Brazil et al., 2015). Through a finely
controlled balance of BMP/ antagonist activity, cellular processes may be regulated to suit the
developmental context (Brazil et al.,2015). Therefore an imbalance of the two leads to
disease states (see section 1.1).
Interestingly, many cytokine antagonists are also members of the TGF-β superfamily as they
share the same cysteine knot. A particular group of antagonists bearing this domain are
referred to as the CAN (Cerebrus and DAN) family of antagonists (Rider and Mulloy, 2010)
(See section 1.6). This includes the BMP-7 antagonist, Gremlin.
7. 7
1.1 The biological role of Gremlin and its contribution to kidney fibrosis.
BMP-7 and its antagonist demand attention due their roles in in tissue fibrosis. As shown in
figure 1, Gremlin inhibits the activity of BMP-7 through protein binding thus preventing
BMP-7’s association with its cellular receptor (Ali and Brazil, 2014).
.
Figure 1. Gremlin signalling and downstream effects in cells. (A). Gremlin associates with
BMP in order to prevent its association with receptors type I and II. (B.) Gremlin associates
with VEGFRs to promote angiogenesis. Image is adapted from Brazil et al 2015.
Gremlin has an important role in organogenesis within the embryo and adult levels of this
protein should be far reduced post development. High levels of Gremlin have been found to
be elevated in tissues affected by fibrotic disease (Ali and Brazil 2014). It has been
demonstrated by Carvajal et al (2008) that Gremlin is a mediator in the epidermal to
mesenchymal transition (EMT) signalling pathway which ultimately leads to the generation
of activated fibroblasts. This study evaluated biopsy samples of patients with chronic
8. 8
allograft nephropathy and showed that the expression of Gremlin mRNA had a strong
correlation with tubulointerstitial fibrosis. Biopsies of control patients contained no
expression of Gremlin. Furthermore, BMP-7 has been shown to relieve the state of tissue
fibrosis in an asbestos model of pulmonary fibrosis (Myllarniemi et al., 2008).
Pharmacological agents have already been used to reduce the activity of Gremlin in the form
of anti- Gremlin monoclonal antibodies. These were shown to be effective in ameliorating
hypoxic tissue damage in mouse models (Ciuclan et al, 2013).
Gremlin also has its own cell- signalling role in angiogenesis which requires binding to
vascular endothelial factors (VEGF) as demonstrated in figure 1. This activity can lead to the
generation of highly vascularised tumours. In homeostasis however, these receptors play a
role in blood and lymphatic system development (Chiodelli et al., 2011). This process is
dependent on the presence of heparin and heparan sulfate on endothelial cells (Brazil et al.,
2015)
Due to Gremlin’s role in tissue fibrosis and tumorigenesis it is therefore of great interest to
better define binding characteristics that mediate its association to GAGs.
In order to appreciate this association, the structure of both the protein and
glycosaminoglycans will be explained:
1.2 The structure and biosynthesis of heparin and heparan sulfate.
Glycosaminoglycans (GAGs) are long linear polymers usually attached to a protein core to
constitute ECM proteoglycans. The polymers are constructed from repeating disaccharide
units of a particular nature which give rise to several major forms: heparan sulfate,
chondroitin sulfate, keratin sulfate and dermatan sulfate. Each of the sugar residues may be
sulfated at different positions or epimerised differently at the C5 position, thus giving rise to
distinct disaccharide units. When polymerised, the constituent units provide the molecules
with distinctive functions that provide its identity as one of the 4 above forms (Lodish,
Molecular cell Biology). Unlike most biological macromolecules, the biosynthesis of GAGs
is not underpinned by any genetic instruction or “blueprint”. These molecules are assembled
within the golgi body and the polymer is subject to a number of enzymatic modifications that
sulfate and epimerise the sugar units. The heparan sulfate and heparin polymers are both
largely composed of the same disaccharide unit (see below) and are exposed to the same
9. 9
groups of enzymes for modification. However, it is the overall proportion of highly sulfated
units that distinguishes the two GAGs; heparin is a hypersulfated form of heparan sulfate.
In figure 2, B constitutes 50% of the heparin polymer. By contrast, this is a minor structural
unit in heparan sulfate.
Figure 2. Heparan sulfate structure and biosynthesis. Heparan sulfate and heparin share this
same repeating structural disaccharide unit of glucuronic acid and N- acetyl glucosamine.
Image A represents the initial, unmodified disaccharide unit. B represents a fully sulfated and
epimerised disaccharide unit. Stars denote the points of sulfation before enzymatic
modification (Khan et al., 2013). Image is adapted from Khan et al 2013.
Enzymatic modification occurs in a variable sequential pattern for the generation of heparan
sulfate and heparin. First, a bifunctional enzyme (N- deacetylase/ N- sulfotransferase)
converts GlcNAc to GlcNSO3. Then, followng this sulfation event, an epimerisation enzyme
converts glucuronic acid to iduronic acid. Further sulfation events then follow at the 2-O of
glucuronic acid and at the C6 position of GlcNSO3 (Mulloy et al., 2010). To demonstrate the
extent of variability that can be generated along the polymer, consider: the amino group of
the glucosamine residue may exist in either of the 3 states: acetylated, sulfated or free amino;
the last however occurs rarely in nature. Furthermore, these 3 forms may exist in either of the
4 states: epimerised/ non- epimerised and 2-O sulfated/ 2-O non –sulfated (Mulloy and Rider,
2006). It is now easy to see how within both the heparin and heparan sulfate groups of
polymers, there is extensive heterogeneity that results from this enzymatic modification. As
10. 10
heparin sulfate is subject to lower sulfation events it exhibits more variability along the
polymer chain.
Due to the decoration of sugar units with acidic sulfate groups at conserved positions, this
provides an overall negative charge to the polymeric molecule. Proteins that associate with
GAGs are found to form ionic interactions at physiological PH via basic residues on the
surface of the protein (Rider and Mulloy 2006). For example, Follisatin is known to form a
high affinity association with heparin and heparan sulfate. Crystallographic studies have
revealed that Follistatin’s binding site is rich in basic amino acid residues (Rider and Mulloy,
2006). There is further evidence in the abolishment of protein- GAG association in
transfected cells expressing the sulfyl-transferase competitive inhibitor, chlorate. The ionic
interaction, in this case, is lost as conserved acidic substituent groups are no longer present
(Chiodelli et al., 2011).
Of all the GAGs, heparin and heparan sulfate exhibit the highest level of sulfation. Due to
their high negative charge they have been subject to many studies with an interest in GAG-
cytokine interaction (Mulloy and Rider, 2006). It should be noted that heparin does not bind
to Gremlin in a biological context and it is not expressed on cells or secreted into the ECM
like heparan sulfate. Heparin is released from mast cells but is used experimentally due to its
wide availability.
1.3 Do protein – heparan sulfate interactions exhibit binding specificity?
The extensive variability exhibited between GAG structures, as generated by an organised
array of enzymes, suggests that there could be some intrinsic role in the biosynthetic pathway
to generate unique binding patches. This could confer specificity for particular proteins. It is
also reasonable to suggest that different GAG compositions will vary depending upon the
tissues where they are found, providing specified affinities to particular target molecules. For
example, heparin is produced mostly in mast cells and plays a role in blood clotting through
its binding to the anti – coagulation factor Antithrombin III (Lodish, Molecular Cell
Biology).
Many studies have demonstrated that the high affinity binding of Antithrombin III with
heparin is dependent upon a specific pentasaccharide sequence that contains a tri-sulfated
glucosamine. A small deviation in this pentasaccharide sequence has been found to greatly
reduce binding affinity (Rudd et al, 2010). This seems to suggest that the associations made
11. 11
between GAG and proteins may require a higher complexity than ionic interactions alone.
Antithrombin III is however, unique in its possession of such a well- defined binding site, no
such sequence has been found in any other morphogen of the TGF-β family (Mulloy and
Rider 2006). However, it seems likely that certain proteins will have binding preferences
towards particular GAG sequences or “footprints”. Despite there being very few defined
crystallographic structures of GAG- cytokine interactions, it has been calculated that a typical
“binding footprint” is composed of roughly 6 saccharide residues (Mulloy and Rider, 2006).
The sulfation events during biosynthesis do not occur uniformly along the length of the
polymer. Therefore, a pattern emerges where alternating S- domains of high sulfur
concentration and N- domains of low concentration appear (Khan et al, 2013). Heparan
sulfate contains regions of high sulfation interspaced with long regions of lower sulfation.
Heparin however has long regions of high sulfation interspaced with short regions of low
sulfation. As it is the anionic S- regions that interact with the protein, particular arrangements
of these regions may exist which are more permissive for interactions with protein dimeric
units (Mulloy et al., 2010). An example of this can be demonstrated by what are referred to as
“SAS” structures. These are composed of 2 short sulfated regions with an interspacing
acetylated sequence. In the case of platelet factor 4 binding, this intervening sequence allows
for a flexible central portion of the polymer, permitting heparan sulfate to wrap around the
positively charged binding surface (Stringer and Gallagher., 1997). In contrast, a long
sulfation region may not allow for the association with more than one domain on a
multimeric protein (Khan et al., 2013).
Interestingly, these N and S domain regions may further contribute to the 3D conformation of
heparin and heparan sulfate as a degree of “bending” has been shown to result from the
arrangement of N- domains and S- domains. Heparan sulfate has been shown to exhibit a
good degree of bending flexibility due to its more frequent alteration of ionic S- domains
with non-sulfated N domains (Khan et al 2013). In comparison, the heparin molecule is more
rigid. This rigidity is generated by the repulsion exhibited between negatively charged S-
groups. As a result, the occasional occurrence of an unsulfated unit manifests as a “kink”
along the polymer as opposed to the bending seen in heparan sulfate (Khan et al., 2010). X –
ray scattering, carried out by Khan et al (2013) has confirmed that heparan sulfate indeed has
a more bent structure than heparin. This was a highly comprehensive study which utilised
12. 12
constrained modelling in combination with solution scattering techniques in order to increase
the resolution of the structure. This rigidity exhibited by heparin may provide a limitation to
the degree of conformational change the molecule undergoes in order to form associations
with the Gremlin binding motif (Khan et al., 2010).
Here is substantial experimental evidence that a three dimensional level of variability exists
among GAGs. This could bear a strong influence a GAG’s sterical capability to bind to a
particular protein. In many cases the appropriate residues on the protein surface may be
physically inaccessible to the largely rigid polysaccharide (Gahndi et al., 2009). A degree of
flexibility may allow the polymer to reach residues difficult to access on the protein surface.
It therefore seems that the binding affinity of cytokines to heparin and heparan sulfate may be
influenced by the 3D structure and its sulfation pattern. The overall protein interactions are
therefore more complex than simple ionic interactions and charge density. This study aims to
experimentally encompass all the factors that lead to an overall protein interaction with
heparin and heparan sulfate. This shall be referred to as the “totality” of binding.
1.4 Studies investigating sulfation structures of heparan sulfate.
This research project explores the ability of 2 mutagenized forms of Gremlin along with wild
type to associate with a select group of naturally occurring heparan sulfates and heparin.
They are the following: Bovine lung heparin, bovine kidney heparan sulphate, HSE and HSA.
“HSA” and “HSE” are derived from 2 separate fractions isolated from porcine intestinal
mucosa. Their Mrs are 20kDa and 8kDa respectively (Hasan et al., 1999). The structural
details of HSE and HSA have been determined through the use of NMR and the ratios of the
various substituent groups are summarised in the Table 1 (refer to figure 2 for positional
details on the disaccharide):
13. 13
N- acetyl/ N-
sulfate.
2-O-sulfate/ N-sulfate. 6-O-sulfate/N-
sulfate.
HSA 0.9 0.4 0.6
HSE 0.3 1.0 0.5
Heparin 0.2 0.7 0.6
Table 1. Sulfation ratios of HSA and HSE as determined by NMR spectroscopy. The two
heparan sulfates are compared with the highly sulfated heparin, 5th international standard.
This table has been adapted from Rickard et al., 2003
As can be seen in table 1, HSE is highly N- deacetylated and N- sulfated (similar to heparin)
whilst HSA has a much larger proportion of glucosamine residues in its unmodified
acetylated form. This provides the polymer with a lower charge density. Studies have
demonstrated that although removal of all 3 sulfation groups impairs interaction, 2- O
sulfation is crucial for GDNF association with heparin (Rickard et al., 2003, Rider 2003).
This suggests that protein binding can be sensitive to particular GAG sulfation patterns. As
this study is investigating GAG binding in its totality it will be difficult to distinguish the
contribution disaccharide positional sulfation has on protein binding. Nevertheless, a
comparison can be drawn on this basis of overall level of sulfation/ charge density of HSA
and HSE.
1.5 Competitive binding studies of heparan sulfate variants.
This current investigation shall use a method of competitive binding to compare the ability of
different heparan sulfates to associate with Gremlin captured on immobilised heparin.
Previous experiments have also explored the binding abilities of HSE and HSA with the use
of competitive binding. Hasan et al. (1999) demonstrated IL-12’s GAG binding specificity
with the failure of chondroitin sulfate to compete for binding with immobilised heparin. This
same experiment demonstrated that HSA provided no significant inhibition whilst HSE
performed well as a competitive binder (results are shown in figure 3).
14. 14
Figure 3. GAG competitive binding experiments for IL-12. IL-12 has been pre- incubated
with a number of GAG variants before incubation with immobilised heparin. Graph is from a
paper by Hasan et al (1999).
A separate study by Rickard et al (2003) investigated the binding effects of heparan sulfates
to Glial cell derived neurotrophic factor (GDNF) using chemically modified GAGs with
known sulfation patterns. This method was useful in investigating specifically the influence
of sulfation structures on GAG binding and was successful in identifying binding specificity
to O- sulfated groups. This study was compared with the Hasan et al (1999) study with IL-12
and was able to identify that different cytokines have different binding specificities for
glycosaminoglycans.
Currently no such GAG sulfation pattern has been identified which is unique to Gremlin
binding. As unique specificity has been exhibited by these other proteins it can be expected
that Gremlin will also exhibit its own distinct binding pattern.
Unlike the studies by Rickard et al., this current study will investigate the competitive
binding with naturally occurring GAGs, including kidney heparan sulfate, the natural binding
partner for Gremlin. This may provide better insight in a biological context than synthetic
GAG studies.
15. 15
1.6 Homology modelling of Gremlin antagonist.
Unlike the family of TGF- β proteins, their antagonists are highly diverse in their structures.
Some may be multidomained structures while others remain in single domain form (Nolan et
al. 2013). In order to better understand how these proteins form such associations with
heparin sulfates it is necessary to elucidate their structure.
Until recently, the best structural model for Gremlin was based upon homology modelling of
a solution structure of Sclerostin. Since then however, the crystallographic structure of a
more structurally similar protein: Protein Related to DAN and Cererbrus (PRDC) has been
solved in studies by Nolan et al (2013). PRDC is sometimes referred to as Gremlin -2 as it
shares 69% amino acid identity in the cysteine rich motif. This modelling revealed the
characteristic – growth factor like fold that is described as a two- finger- wrist model where a
series of β- strands originate from the N- terminus (See figure 4).
A.
Figure 4. Homology modelling of Gremlin with the PRDC monomer. A. Image is of the
crystallographic monomer of PRDC. Alpha helices are in red and beta strands in blue. Finger
regions are denoted as “F1” and “F2” and the wrist, “W”. B. Figure is of the predicted
Gremlin structure. Structure is based on PRDC X- crystallographic structure. Although this
image is inverted with respect to the PRDC structure, note the structural similarity and the
B.
16. 16
presentation of two- finger wrist structure in each. Modelling of Gremlin was carried out by
Professor Barbara Mulloy using Swiss Model. A is adapted from Nolan et al 2013. B is
Adapted from Tatsinkam 2014 PhD thesis.
1.7 Gremlin mutagenesis.
Utilising this new structure of Gremlin, as shown in figure 4, three clusters of basic residues
were identified on its surface that were thought to contribute to the heparin binding site.
These, like those found in IL-12 in the Hasan et al (1999) study consisted of largely lysine
and arginine basic residues. Using site directed mutagenesis, functional residues were
replaced with alanine; this did not result in misfolding within the protein. Gremlin mutants
were then expressed in Chinese hamster cell ovaries (Tatsinkam 2014, PhD thesis). A number
of different proteins were expressed, but the ones used in this particular study are MGR5 and
MGR6. Their targeted clusters for mutagenesis are as follows:
MGR5: II and IIIb.
MGR6: I and II. (See figure 5).
Figure 5. Ribbon model of Gremlin showing the 3 clusters of basic residues. Green chains
indicate the relevant amino acid residues. Figure is adapted from Tatsinkam 2014, PhD
thesis.
17. 17
Previous studies have also investigated the effects of mutagenizing proteins in order to
investigate their GAG binding. In a study carried out by Severin et al (2010), the researchers
were able to identify a crucial region involved in heparin binding for chemokine CXCL11.
Four individual basic clusters thought to be primarily involved in heparin binding, were
altered by the replacement of Arg and Lys with Ala. The Ec50 value of the one mutant
showed a 10 fold decrease in binding affinity compared with WT. The respective region was
deemed “a key epitope”. This study strongly indicates that proteins will associate with GAGs
using specific surface residues. Again this further supports the idea that associations between
GAGs and their proteins are dependent upon the polyanionic nature of heparin interacting
with cationic patches on the protein.
1.8 Rationalisation of method.
This study utilises a method of competitive binding to demonstrate the ability of heparan
sulfate variants to displace Gremlin binding with an immobilised heparin- BSA complex.
The quantity of Gremlin remaining on the capture plate after washing is measured using an
ELISA technique. This method is useful for measuring the totality of heparin sulfate binding.
As discussed, binding affinity is thought to be determined by a number factors which include
the sulfation pattern, 3D structure and charge density, these combined can be termed the
”totality” of binding. By measuring the affinity of protein binding in its totality this may
provide an idea of how GAGs associate in a biological context. Previous experiments have
investigated Gremlin- heparin binding using column chromatography. In this procedure
however, only the ionic affinity can be measured as elution of Gremlin from the heparin lined
column occurs through salt washing (Tatsinkam et al., 2014).
Alterations have been made to the purification technique of recombinant Gremlin since the
discovery that histidine tags provide artefactual heparin binding (Lacy et al., 2002). The
ELISA method used in this investigation does not require the protein to be covalently bound
to any tags that may present this issue.
Unlike The ELISA method proposed for this investigation, other studies exist where the
protein of interest has been immobilised on the capture plate itself (Marson et al., 2009). This
however may perturb the structure of the protein and influence its binding to the GAG. To
circumvent this issue, this current study shall generate capture plates coated with heparin.
The heparin shall be coupled to BSA to allow for immobilisation on the plate; Gremlin is
then applied to this.
18. 18
As described in section 1.3, heparan sulfates may vary depending upon their tissue of origin.
This study will therefore investigate the binding of Gremlin to heparan sulfates derived from
different tissues. Kidney heparan sulphate (KHS) is of particular interest as Gremlin is known
to provide functional activities for kidney development, also contributing to kidney fibrotic
disease. Therefore, KHS is a GAG that Gremlin binds to in vivo; intestine derived HSA and
HSE are used for comparison.
Aims and Hypotheses.
As demonstrated in the literature, GAGs exhibit highly variable structural and chemical
properties that contribute to their protein binding, therefore, it is expected that they shall each
associate with Gremlin with different affinities. This study aims to demonstrate this using a
competitive binding assay with different variants of heparan sulfates.
This investigation also aims to examine how mutagenesis of surface residues of wild type
Gremlin can affect the strength of its binding to each individual GAG. From this, it can be
elucidated whether perturbation results in a quantitative reduction in binding, equal for all
heparan sulfates or whether there is a qualitative alteration to the specificity of binding to
each. If the latter is true, we can assume that the complexity of binding involves more than
just ionic interactions but involves “key epitopes” and structural features which confer
binding specificity.
This study serves to investigate the hypothesis that Gremlin will bind to heparan sulfates with
structural specificity rather than ionic interaction alone. Thus, further implying that Gremlin
binding is distinctive from other proteins.
19. 19
Materials and Methods.
This experiment uses an ELISA technique to measure competitive binding. Remaining
Gremlin was detected using a specific antibody then followed by an appropriately enzyme
tagged secondary antibody.
Reagents:
Mouse anti- Gremlin Biotinylated Affinity Purified Ab Goat IgG and Streptavidin Alkaline
Phosphatase conjugated Ab were purchased from R&D systems.
Heparin BSA conjugate was generated previously in the lab by treatment with
cyanoborohydride which covalently couples the reducing end of heparin to BSA.
Bovine lung heparin and kidney HS were both purchased from Sigma – Aldrich.
Two HS’s isolated from porcine intestinal mucosa were previously isolated in the lab by
fractionation (HSA and HSE) (indicated in Table 1.)
For recombinant Gremlin synthesis, see section 1.7.
All above reagents were kept below 4 ̊ C.
Stock of 10 x PBS (PH 6.87) buffer and stock of TRIS- EDTA (PH 7.4) were both previously
made in the lab and kept at room temperature. (In an effort to generate conditions similar to
nature, buffer PH has been matched to biological PH.)
96 well plates are purchased from Sigma – Aldrich.
Method
5ng of heparin –BSA conjugate, was diluted in TRIS- EDTA (PH 7.4) buffer and 100μl
added to each well of a 96 well plate. The plate was left to incubate at 4 ̊ C overnight on a
rotating platform. An additional set of 4 “blank” wells which was not exposed to any reagents
was measured for background absorbance.
Following incubation the plate was washed three times with 250μl 1 x PBS. Wells were then
blocked with 2% BSA- PBS, 200μl per well. Plates were covered and left for an hour at room
temperature on a rotating platform.
20. 20
Blocker was then removed and wells were washed three times with 250μl of 0.05% BSA 1x
PBS solution. 100μl of Gremlin antigen was added to each well. Plates were covered and left
at room temperature for 90 minutes on a rotating platform.
Gremlin was prepared in 0.05% BSA- 1x PBS to the desired concentration, in 100μl volume.
(The exact protein weight is unknown as Gremlin samples are suspended in the supernatant
so samples are given volume units.)
Gremlin was then removed and wells were washed three times with 250μl of 0.05% BSA-1x
PBS solution. 100μl of Biotinylated anti – gremlin antibody was added to each well. Plates
were covered and left at room temperature for 90 minutes on a rotating platform.
Biotinylated primary antibody was diluted to 1:250 with 0.05% - BSA 1X PBS before adding
to plate.
Primary antibody was removed and wells were washed three times with 250 μl of 0.05%
Tween 20 -1 x PBS. 100μl of streptavidin alkaline phosphatase conjugated antibody was
added to each well. Plates were covered and left at room temperature for 30 minutes on a
rotating platform.
Streptavidin Alkaline Phosphatase conjugated secondary antibody was diluted to 1:100 in
0.05% -BSA 1x PBS before adding to plate.
Secondary antibody was removed and wells were washed with 250μl of 0.05% Tween 20- 1x
PBS. Then washed twice with 1x PBS. (This second set of washes is important to remove any
bubbles that may have formed in the detergent wash.)
100μl of P- nitrolphenol substrate was added to each well. Plates were covered and left to
incubate at 37 ̊ C on a rotating platform for 25-30 minutes.
Substrate was prepared as directed by manufacturer.
Plates were read at 405nm wavelength using an ELISA plate reader. The absorbance value
indicates the remaining quantity of Gremlin on the capture plate.
This experiment used 4 repeat wells for each Gremlin sample and the negative control.
Negative control consisted of wells coated with BSA prepared without heparin conjugation.
21. 21
Statistical Analysis.
Data typical of figures 9 and 10 was analysed using student’s t-test. The comparison was
made between the “no GAG” condition and each GAG competitor individually.
For data typical of figure 11, statistical analysis was completed using the mean values of 3
identical competitive binding experiments individually, then an analysis was carried out using
the combined mean values of the 3 experiments: Data was analysed using ANOVA or
Kruskal- Wallis testing (Levene’s test was used to indicate which test was suitable based
upon variance.) This was followed by post- hoc Bonferronni test. This testing allowed for the
determination of the performance of each GAG as a competitive inhibitor, relative to one
other.
Both ANOVA and Kruskall- Wallis tests have more power than the T- test when the number
of conditions compared is greater than 2.
22. 22
Results
This experiment has aimed to recreate, as best possible, the process of heparan sulfate –
Gremlin binding as it occurs in a biological system. However, it must be kept in mind, the
conditions that influence binding can vary greatly in a biological system. Therefore, the data
represented in this report only accounts for experimental conditions.
Before competitive binding assays are carried out, a suitable fixed concentration of Gremlin
antigen to use must first be determined. A significant portion of antigen may be removed
during competitive assays so ideally, a high initial absorbance should be present before
addition of competing GAG.
A titration of increasing amounts of WT Gremlin was carried out to find a suitable value:
Gremlin was prepared in 0.05% BSA- 1x PBS to achieve 10μl, 15μl, 20μl and 25μl in each
well in 100μl volume.
Figure 6. Absorbance at 405nm wavelength with increasing amounts of WT
Gremlin per well (Mean+- SEM values are plotted, although bars are smaller than
symbol diameter). Each data point is the mean of 4 repeat wells. Red points
denote negative control, blue points denote immobilised heparin binding.
0
0.5
1
1.5
2
2.5
3
3.5
0 5 10 15 20 25 30
Absorbance(405nm).
Gremlinper well (µl).
23. 23
As may be seen in figure 6, the negative control values indicate that Gremlin is only
associating with the immobilised heparin and only a low background level of binding is
observable. These negative control wells were made using 5 ng per well of mock- BSA
conjugate. This has been treated with the same chemicals for heparin conjugation but in the
absence of heparin.
In figure 6, a plateau is reached at 20µl per well of Wild type Gremlin. This indicates that the
immobilised heparin binding sites have become saturated. In order to determine if this is true,
the experiment was repeated with an additional set of wells with twice as much immobilised
heparin (10ng per well) for Gremlin concentrations 0μl and 25μl.
Figure 7. Absorbance at 405nm wavelength with increasing amounts of WT Gremlin per
well (Mean+- SEM values are plotted, although are smaller than symbol diameter). Each
data point is the mean of 4 repeat wells. Red points denote negative control, blue points
denote immobilised heparin binding. Grey points denote Gremlin samples in wells coated
with twice the heparin (10ng) as other samples. Data labels are included to distinguish
between overlapping points.
0.1295
0.13
0.5995
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 5 10 15 20 25 30
Absorbance(405nm).
Gremlinper well (μl)
24. 24
As can be seen in figure 7, the same maximal absorbance is obtained for 25μl Gremlin in
wells coated with twice as much heparin (10ng) as the standard wells (5ng). Therefore, it is
unlikely that immobilised heparin has become saturated and more likely that “levelling off”
in figure 6 is due to the detection limit having been reached on the reader or because all
substrate has been consumed.
As the WT curve in figure 7 is irregular, a 3rd titration experiment of this sort was carried out
which was similar to of that in figure 6. From this data a 10µl per well was selected as the
most appropriate quantity of Gremlin to be used in competitive binding assays. This value
provides a high enough absorbance to allow for significant competitive binding without
existing in the “plateau” region in figure 6.
Next, the same procedure was carried out for mutagenized MGR5 in place of WT in order to
determine a suitable quantity of Gremlin for competitive binding assays.
These results in figure 8 are typical of 2 similar experiments that show a plateau reached at
25µl per well of MGR5. This is likely to occur for the same reasons proposed for WT. Note
Figure 8. Absorbance value at 405nm wavelength with increasing amounts of MGR5
per well (plotted as Mean+- SEM). Each data point is the mean of 4 repeat wells. Red
points denote negative control, blue pints denote immobilised heparin binding.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 10 20 30 40 50 60
Absorbance(405nm).
Gremlinper well (µl)
25. 25
that a range of higher concentrations was used than for WT, as binding strength is expected to
be reduced for this mutant.
From this data 10µl per well was selected as the most appropriate quantity of Gremlin to be
used in MGR5 competitive binding assays.
Competitive binding assays.
In order to investigate how the structure of Gremlin contributes to its association with
different GAGs, the recombinant form of Gremlin (MGR5) was compared with WT in a
competitive binding assay
Both WT and MGR5 were combined with soluble forms of either 0µg/ml GAG, 50µg/ml
heparin, 50µg/ml HSA or 50µg/ml HSE. This concentration was selected as previous
experiments indicated that 50μg/ ml of soluble heparin exerted maximum inhibition on these
capture plates.
As indicated by the “No GAG” value in the right panel of figure 9, MGR5 has an absorbance
approximately a third of WT. In the left panel, where the data has been normalised, the
Figure 9. Absorbance values at 405nm wavelength. Left panel: Normalised data.
Right panel: Raw data. Data plotted as mean +/- SEM. Each data point is the mean
of 4 repeats. Blue represents WT and red is MGR5. A t- test was carried out on the
data:
*** -.significant at p< 0.001, **** - significant at p< 0.0001
****
***
****
****
****
0
0.5
1
1.5
2
2.5
No GAG Heparin HSA HSE
Absorbance(405nm).
SolubleGAG.
0
0.2
0.4
0.6
0.8
1
1.2
No GAG Heparin HSA HSE
Normalisedabsorbance.
SolubleGAG.
26. 26
competitive binding of each GAG can be better compared. Here it can be seen that HSA has a
poor competitive ability whilst HSE competes for Gremlin binding well.
It should be kept in mind that HSE, HSA and heparin are not natural binders for Gremlin as
these are not found in tissues where Gremlin is expressed. However, HSE and heparin still
exhibit strong binding and are useful in observing variation in GAG structure derived from
different tissues.
Some caution should be taken when interpreting the results as the MGR5 “No GAG”
absorbance values appear unusually low, compared with previous experiments and
conversely WT is unusually high. This may indicate some discrepancy which may present
after normalisation of the results.
It was decided that the experiment should repeated to obtain a more meaningful result. The
following experiment also investigates competitive binding of MGR6.
This set of data shown in figure 10 is typical of 1 other similar experiment. This is also in
accordance with the data in figure 9. The right panel “No GAG” value shows that both
MGR5 and MGR6 have an overall reduced affinity to immobilised heparin. As can be seen in
the normalised data (left panel) all competing GAGs are able to successfully compete for
Figure 10. Absorbance at 405nm wavelength. Left panel: Normalised data. Right
panel: Raw data. Data plotted as mean +/- SEM. Each mean value is calculated
from 4 repeats. Blue represents WT,red is MGR5 and grey is MGR6.
A two tailed, paired t- test was carried out on raw data.
*** -.significant at p< 0.001, **** - significant at p< 0.0001
****
****
****
***
********
***
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
No GAG Heparin HSA HSE
Absorbance(405nm)
SolubleGAG.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
No GAG Heparin HSA HSE
Remainingabsorbance(405nm)
Solube GAG.
27. 27
MGR5 binding to immobilised heparin. Interestingly, MGR6 plate binding is poorly
competed for by the soluble GAGs.
The left panel shows HSA is only able to notably influence MGR5’s binding to immobilised
heparin. In contrast, HSE offers considerably more competition than HSA for both mutants;
MGR5 showing particular sensitivity to HSE competitive binding. As expected, soluble
heparin is able to eliminate immobilised heparin binding to background absorbance levels
due to its high charge/ sulfation density.
The following experiment explores the competitive binding ability of kidney heparin sulfate.
This should broaden the analysis of Gremlin binding across the HS variants. This may help to
further indicate whether mutagenesis of Gremlin has resulted in a qualitative reduction in
affinity or whether any distinct binding profile is exhibited by the protein variants.
28. 28
Figure 11 is typical of 3 similar experiments where a consistent binding pattern is
demonstrated for the 3 Gremlin variants. As can be seen in the normalised data (upper panel),
kidney heparan sulfate (KHS) has a similar competitive binding activity to HSE and serves as
a good competitor. Statistical analysis of the 3 combined experiments (as explained in
Figure 11.Absorbance at 405nm wavelength. Upper panel: Normalised data. Lower panel:
Raw data. Data plotted as mean +/- SEM. Each mean value is calculated from 4 repeats. Blue
represents wildtype, red is MGR5 and grey is MGR6. Stars indicate the significance of
inhibition P< 0.001 as calculated by ANOVA or Kruskal- Wallis testing (Variance was
measured using Levene’s test.) This was followed by post- hoc Bonferronni test.
*
* *
*
*
*
*
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
No GAG Heparin HSA HSE Kidney HS
Absorbance(405nm).
SolubleGAG.
0
0.2
0.4
0.6
0.8
1
1.2
No GAG Heparin HSA HSE Kidney HS
Remainingabsorbance(405nm).
SolubleGAG.
29. 29
methods) demonstrated that the inhibition observed in the data, typical of figure 11, was
significant for all the soluble GAGs except for HSA. HSA may therefore be considered a
non- competitor. Heparin however, always reduces the absorbance value to nearly
background levels. It should be kept in mind, HSA’s low sulfation density, this data indicates
that ionic charge density contributes strongly to binding.
Interestingly, the normalised data (upper panel) reveals KHS has a poorer competitive
binding ability for MGR5 than HSE does; this was not the case for WT or MGR6. As shown
in the lower panel, for all three conditions of competitive binding: (No GAG, HSA and
KHS), MGR5’s absorbance has remained around a half of WT’s, however, the remaining
absorbance was lower in comparison, following HSE competitive binding. Therefore, the
reduction in affinity exhibited by MGR5, relative to WT has not been equal for all GAGs; a
new binding pattern is exhibited.
In the lower panel of figure 11, note the “No GAG” raw data value for MGR6 which
indicates that it has around 60% of WT absorbance. Therefore a significant amount of affinity
for immobilised heparin has been lost. However, MGR6 appears to bind more strongly to
immobilised heparin than any soluble heparan sulfate, all of which displace this binding
poorly (shown in the upper panel). Compared with the wild type condition, the range of GAG
binding partners that MGR6 will associate with successfully, appears to be limited. Thus, like
MGR5 there has not been an equal reduction in GAG binding affinity for all GAG variants as
a result of mutagenesis. This was also evident in figure 10.
Some caution should be taken when interpreting the data in figure 11. As can be seen in the
lower panel, the value for MGR6 with soluble HSA exceeds that of the MGR6 with no GAG.
This may indicate that the uninhibited value is anomalously low which may influence the
normalised data. This however, seems to have minimal effect as data is still consistent with
the other experiments
The experiment to follow this examines more closely the ability of kidney heparan sulfate as
a competitive binder.
30. 30
Titration experiments for KHS and WT.
A titration experiment was conducted for MGR5, MGR6 and WT individually where soluble
heparin and kidney heparan sulfate were added in the following concentrations:
0 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 100 μg/ml. (MGR5 only.)
0 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml. (with WT and MGR6).
This range of concentrations was selected based on the knowledge that 50μg/ ml of soluble
heparin has previously shown to provide maximum binding inhibition.
WT and MGR6 titrations were carried out over a smaller range as inhibition shown in the
MGR5 experiment indicated that inhibition was substantial at lower concentrations.
It should be kept in mind that previous competitive assays used a fixed concentration of
50μg/ml soluble GAG. As shown in figure 12, KHS is a much poorer competitive binder than
heparin for all Gremlin mutants. Maximal inhibition for heparin is reached at around 10μg/ml
for all Gremlin variants whilst KHS reaches maximal inhibition between 50 μg /ml and 100
Figure 12. Normalised absorbance at 405nm wavelength. Data points are the mean of 4 repeat
values. Please refer to the legend above.
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100 120
Remainingabsorbance.
GAG Concentration (μg/ml)
WT Heparin
WT KHS
MGR5 Heparin
MGR5 KHS
MGR6 Heparin
MGR6 KHS
31. 31
μg /ml for all Gremlin variants. It appears from the slope of the curve that further increase in
KHS concentration would not reduce bound Gremlin levels to those seen with soluble
heparin.
Soluble heparin has displaced the binding of all 3 Gremlins quite equally. Take note that
MGR6 binding to immobilised heparin is poorly competed for by soluble KHS when
compared with WT and MGR5. Interestingly, MGR5 and WT appear to behave quite
similarly with competitive inhibition from KHS. Similar behaviour was previously observed
in experiments that looked at competitive binding at fixed concentrations (See figure 11,
upper panel).
Following examination of all the data, of particular interest is the observable change in
binding profile for both MGR5 and MGR6 from the wild type condition. In figure 11, upper
panel, the data shows MGR5 and WT to bare a similar resemblance in competitive behaviour
with all GAGs except for HSE; here there is a significant difference. Furthermore, MGR6 is
shown to have a limited preference towards heparin over all other GAGs.
Mutagenesis has not only resulted in a quantitative reduction in binding affinity to all GAGs
but the binding behaviour has changed relative to the WT. This observation was consistent
over many experiments.
32. 32
Discussion
2.0 HSE, HSA and Heparin as competitive inhibitors.
As supported by statistical analysis, all the GAGs except HSA provide a significant
competition for Gremlin binding with immobilised heparin. HSE was a strong competitor for
all 3 Gremlins and heparin was able to reduce absorbance to background levels. As HSE and
heparin are more densely sulfated whereas HSA has lower N and O sulfation (Rickard et al.,
2003) (See table 1) this indicates that the ionic interactions made between ionic sulfate
groups on GAGs and basic protein residues bear a strong contribution to binding. This
however, is contrary to the binding of HSA with human Betacellulin where significant
activity is shown (Mummery et al., 2007). This indicates that the binding nature observed in
our experiment is unique to Gremlin.
Furthermore, as heparin is hypersulfated, this allows for an improved “furnishing” of the
polymer thus increasing the chances of presenting the appropriate binding motif to the
protein; this occurs in addition to increased ionic interactions. Despite the molecule having
increased overall rigidity compared with heparan sulfate, the ability to present more moieties
means that bending to accommodate sterically is unnecessary.
2.1 What gives rise to the distinctive binding nature of KHS?
KHS was shown to demonstrate binding activity that was distinct from HSA and HSE. As
can be seen in the results, KHS exhibits similar binding activity to HSE for WT and MGR6
however, it competes more poorly for MGR5 binding than HSE does. It must now be
considered what provides this distinct binding nature that distinguishes KHS from HSA and
HSE and does it possess structural features that are unique to KHS? As mentioned earlier,
heparan sulfates derived from different tissues may be subject to different enzymatic
synthesis and as a result are composed of different disaccharide units. Therefore, it may be
the case that a structural feature, for which MGR5 is dependent, is less frequently expressed
on KHS than the intestinal mucosa derived HSE. A concept similar to that of the
pentasaccharide motif in heparin –Antithrombin binding. Furthermore, perhaps the regulation
of spacing of the S – N domains varies in KHS from that of HSE, making it less flexible to
shape to MGR5’s surface.
33. 33
2.2 Characterising KHS binding to GAGs, the next steps.
As HSE and KHS have similar binding activities for all except for MGR5 it would be of
interest to examine the binding features of these two GAGs more closely. In order to
elucidate what distinguishes HSE binding to MGR5 from KHS it would be of benefit to
examine the structural features of KHS in more detail. This may include, investigation of
sulfation patterning along the polymer through spectroscopy and x-ray scattering to
determine conformation and “bending” of the polymer. These details could then be compared
with HSE.
Elucidation of KHS binding features would be of particular interest as KHS is what Gremlin
proteins would associate with in vivo.
2.3 Comparison with Previous Studies investigating mutagenized Gremlin binding.
Previous experiments exploring the binding affinities of mutagenized Gremlin against wild
type were in agreement with the results of this investigation. Most of these studies
demonstrated an overall reduction in binding affinity to heparin with mutagenesis. An
example of this are the heparin affinity column experiments where mutagenized Gremlin was
observed to elute sooner than wild type (mentioned in section 1.8). A novel experiment
demonstrating the reduction in KHS affinity for Gremlin mutants was carried out by Joy
Askew (MSc) at Royal Holloway, University of London. This investigation utilised KHS
present within kidney tissue: Tagged WT Gremlin was incubated with the C57B1/6 derived
renal tissue overnight. Incubation with secondary antibody then revealed WT Gremlin indeed
binds to specific cell types within the glomerulus. This binding was able to be blocked by
heparinise digestion. The same experiment was repeated with 3 mutants: MGR3, MGR5 and
MGR6 (generated using the same method as described in section 1.7). This investigation
demonstrated that Gremlin does indeed localise in kidney with kidney HS but loses this
natural ability in mutants. A competitive binding assay with soluble heparin was also carried
out.
Askew’s investigation was useful in observing the biological activity of KHS in situ and the
competitive binding results were in agreement with the immobilised heparin method used in
this experiment. This should affirm that the results obtained from the immobilised heparin
method have biological credibility. However what has been possible with the immobilised
heparin method is to cross compare the mutants in their ability to bind to a range of GAGs as
34. 34
opposed to just KHS. This has been more revealing of how their binding specificities to
different GAG structures have altered.
2.4 Gremlin mutagenesis and its influence on GAG binding specificity.
As established from normalising the data, although MGR5 has experienced an overall
reduction in affinity to immobilised heparin, the lower sulfated GAGs can still compete for
its binding. Although the lower sulfation ratios of HSs result in a less frequent presentation
of binding motif on the molecule, each of the soluble heparan sulfate samples is composed of
a heterogenous mix of molecules. Therefore, a portion of these molecules will present to
MGR5 an appropriate binding site. Furthermore, heparan sulfate may be able to bend to
accommodate binding whereas the immobilised heparin is too rigid to permit this (See
section 1.3). It may be the case that mutagenesis has altered the surface structure of Gremlin
in such a way that necessitates the respective GAG have a degree of flexibility to
accommodate binding to a particular patch. Thus, some of the Gremlin will be able to
preferably bind to the competing heparan sulfate and is removed from the immobilised
heparin. Now, it must be considered whether MGR5 mutagenesis has altered binding in such
a way that changes the nature of specificity towards certain GAGs:
It is evident that MGR5’s overall lower affinity for GAGs has not resulted in an equal
reduction for all. Whilst the competitive nature for KHS remains virtually the same as WT,
the competitive nature with HSE is comparatively greater with respect to the WT. This
indicates that mutagenesis has resulted in an alteration in the specificity of the protein to bind
to particular GAGs. It may be of interest to perform a titration experiment comparing HSE
competitive binding for WT and MGR5 to investigate further whether this behaviour is true.
If this is the case, it would be expected that HSE more successfully competes for MGR5
binding than it does for WT on a heparin capture plate.
As mentioned earlier, MGR6 has also exhibits an overall reduction in affinity to immobilised
heparin. Despite this, the data indicates that soluble GAGs have very poor competitive
activity for MGR6’s binding to the immobilised heparin. As a result, MGR6 is retained well
on the plate. It seems that mutagenesis at the binding clusters for MGR6 has limited the
selectivity of protein binding to a particular structural feature which may be presented in
more abundance on a highly sulfated heparin. It may also be the case that mutagenesis has
reduced the surface ionic nature of Gremlin and so binding is more preferential towards the
35. 35
more highly anionic charge of heparin. The result is that MGR6 will bind to fewer GAG
molecules overall and will be more likely to bind to heparin molecules.
It can therefore be confirmed that surface residues on Gremlin proteins contribute to their
ability to associate with particular GAGs. This concept could potentially be extrapolated to
the binding activity of other proteins. As each protein exhibits different surface structures,
they will exhibit different binding strengths for different GAGs. This further supports the
concept that Gremlin exhibits binding specificity distinct from other proteins.
2.5 Heparin binding and its effect on BMP antagonism.
It would now be of interest to determine the biological importance of heparin binding and
what effect this has on BMP activity. Experiments have already been conducted that explore
this in vivo however, in a cellular context Gremlin’s activity is highly complex. Although
glycosaminoglycans may localise Gremlin to bind to BMPs closer to cell receptors, making
inhibition more effective, this also brings Gremlin closer to recycling entities present on the
cell. This may increase endocytosis of Gremlin, removing its activity. What can be inferred
from GAG affinity binding as to how this affects biological states must therefore be used
with caution.
2.6 Closing remarks.
The results are in support of the hypothesis that heparan sulfates associate with proteins with
specificity rather than simple ionic interaction alone. This has been demonstrated as
mutagenesis results in a different GAG binding profile to WT.
There seems to be no discrepancies in the results of this investigation with the literature so
there can be confidence in these findings. Furthermore, previously proposed mechanisms of
GAG-protein associations can be successfully used to infer why these results are observed.
Structural GAG elements such as flexibility and sulfation pattern of the molecule may well
underlie the binding events that have been observed in this investigation. Therefore binding
interactions between GAGs and proteins encompass far more than ionic interactions alone.
Instead all these elements summit to a “totality” that determines the binding strength.As
GAGs exist as highly polymeric molecules, each molecule will possess, to different degrees,
these composite elements. Therefore, Gremlin interactions will vary widely in nature for all
36. 36
GAGs which may give rise to this specificity demonstrated in this investigation. As this
investigation only observed GAG binding in its totality, further investigations should now be
carried out which isolate factors that give rise to Gremlin – GAG binding. This may include,
comparing GAGs with different sulfation patterns or GAGs with different degrees of
“flexibility”. Currently, it is unclear the degree of contribution each of these features
provided to binding. From further studies, more conclusive evidence of GAG binding
behaviour may be drawn.
As predicted, both composite elements of GAGs and surface residues on Gremlin contribute
towards a unique binding activity. This concept may be applied to other protein- GAG
interactions and therefore it is reasonable to suggest that the unique surface structure of
Gremlin gives rise to a unique binding activity which is distinct from other proteins.
Furthermore, some GAG interactions have reported different binding profiles with other
cytokines such as Betacellulin (See section 2.0).
This investigation has been the first to examine the effects of perturbation towards Gremlin
surface residues in binding activity for a number of heparan sulfate variants. Therefore, this
investigation has presented new findings in the way in which Gremlin associates with
different GAGS.
37. 37
Acknowledgements.
I would like to thank first and foremost Nkola Tatsinkam, or as I have come to know as
“Arnold Junior”. Without whom, I would have struggled endlessly with calculations. Your
explanations were always so clear and I feel that I have mastered a skill that I never thought I
could. Despite having a mountain of work to complete for your own PhD you always had
time to help me out when I hit a problem or to just have a chat about life! It was a pleasure
sharing the lab with and I wish you all the best with your work in Switzerland. Thank you.
I would also like to thank Dr Rider for his constant support. I felt very privileged to be so
included in the larger research project. This really helped me to see the bigger picture and
value my work beyond just my own project. Thank you for time spent explaining where my
small contribution stood in the wider world of research and thank you for helping me learn
that project handbooks are not to be scrutinised to the last detail.
Also thank you to the kind masters student that worked in the lab next to me for your patience
when I urgently needed a plate reader and for the time when I needed a calibrated pipette. I
hope I didn’t cause too much disruption!
38. 38
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Tatsinkam 2014, PhD thesis. Royal Holloway, University of London.
