16 research outputs found
Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen
12/IA/1398
16/IA/4419
GOIPG/2016/858
IF/00780/2015
PTDC/BIA-MIB/31028/2017
UIDB/04378/2020
PD/BD/142847/2018
RTI2018-094751-B-C22
RTI2018-099592-B-C2.The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisX and GalNAc, respectively.publishersversionpublishe
Influence of protein (human galectin-3) design on aspects of lectin activity
The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms
Synthesis and evaluation of multivalent ligands for macrophage galactose C-type lectin. Development of new chemical tools for immunological research and structural investigation of the lectin with GalNAc and derivatives
Synthetic chemistry can provide powerful tools to explore the correlation between an antigen’s
chemical structure and the type of immune response triggered. This thesis work has focused
on the design, synthesis and evaluation of multivalent molecules which target a relevant protein
of the immune system, the human macrophage galactose C-type lectin (hMGL).
In chapter 2 the synthesis of multivalent glycomimetics is described as well as measurements
of their affinity for hMGL. The main objective was
to learn more about the structure activity relationship
with regard to ligands based on tetraphenyl ethylene
(TPE) for hMGL and other lectins. To achieve this
divalent, trivalent and tetravalent ligand, based TPE
geometry as well as trans-stilbene presenting α-
mannopyranoside, β-lactose and α-thio-GalNAc as
headgroups, were synthesised. The identified
synthetic route to such compounds is reproducible and scalable. The thermodynamics of the
interaction of α-thio-GalNAc derivatives were studied using the extracellular domain of hMGL
and hMGL CRD, revealing insights into the possible interaction of these compounds with the
lectins.
In chapter 3 the synthesis of an extensive anti-cancer synthetic vaccine candidate library based
on MUC1/MUC4 peptide backbone is described. The main objective of this work was to
enhance antigen presentation by hMGL via the major histocompatibility complex II. To
achieve this objective mucin epitopes
were decorated with a trivalent
glycocluster selective for hMGL.
Through solid phase synthesis synthetic
peptides, including those presenting the
glycocluster were obtained. These were
found to bind hMGL from 10 to 200-
fold better than the natural peptides in an ELISA performed in the S. van Vliet laboratory in
Amsterdam. The next step of this project will be to identify peptides which induce the desired
signalling cascade and test them on mouse model for the production of long-lasting antibodies
IgG. This is the first reported attempt to target a receptor with neoglycosylated peptides baring
a fragment specifically designed to selectively target hMGL.In chapter 4 the synthesis of a (neo)peptide containing a glycocluster with potential to give a trimeric coiled-coil sequence is described. The glycocluster is based on the tetraphenylethene residue. The peptide was successfully synthesised and purified. While some evidence for helix formation was obtained by CD spectroscopy, the refolding of the peptide after heating/denaturing was not observed and may have occurred partially.
In chapter 5 the first structural elucidation of hMGL carbohydrate binding mode is reported using X-ray crystallography. hMGL is the only lectin on human DCs that selectively recognise carbohydrate antigens present on many cancer cell lines and was proven to have the ability to lead the immune system to produce permanent antibody against cancer. The aim of this work was to understand the structural basis of the hMGL/carbohydrate interaction and to allow a more reliable in silico ligand design. To achieve this the protein was expressed in bacterial cell lines. As the expression resulted in insoluble protein, a refolding screen optimisation was carried out, which facilitated recovery of the protein in high purity and good yield for crystallisation screening. As a result of screening and optimisation, suitable crystals for diffraction were obtained. This led to the elucidation of the first crystal structure of hMGL carbohydrate recognition domain (CRD), binding with several monovalent GalNAc derivatives. The structure offers a possible explanation of hMGL binding mode and selectivity.2024-02-1
Synthesis and evaluation of multivalent ligands for macrophage galactose C-type lectin. Development of new chemical tools for immunological research and structural investigation of the lectin with GalNAc and derivatives
Synthetic chemistry can provide powerful tools to explore the correlation between an antigen’s
chemical structure and the type of immune response triggered. This thesis work has focused
on the design, synthesis and evaluation of multivalent molecules which target a relevant protein
of the immune system, the human macrophage galactose C-type lectin (hMGL).
In chapter 2 the synthesis of multivalent glycomimetics is described as well as measurements
of their affinity for hMGL. The main objective was
to learn more about the structure activity relationship
with regard to ligands based on tetraphenyl ethylene
(TPE) for hMGL and other lectins. To achieve this
divalent, trivalent and tetravalent ligand, based TPE
geometry as well as trans-stilbene presenting α-
mannopyranoside, β-lactose and α-thio-GalNAc as
headgroups, were synthesised. The identified
synthetic route to such compounds is reproducible and scalable. The thermodynamics of the
interaction of α-thio-GalNAc derivatives were studied using the extracellular domain of hMGL
and hMGL CRD, revealing insights into the possible interaction of these compounds with the
lectins.
In chapter 3 the synthesis of an extensive anti-cancer synthetic vaccine candidate library based
on MUC1/MUC4 peptide backbone is described. The main objective of this work was to
enhance antigen presentation by hMGL via the major histocompatibility complex II. To
achieve this objective mucin epitopes
were decorated with a trivalent
glycocluster selective for hMGL.
Through solid phase synthesis synthetic
peptides, including those presenting the
glycocluster were obtained. These were
found to bind hMGL from 10 to 200-
fold better than the natural peptides in an ELISA performed in the S. van Vliet laboratory in
Amsterdam. The next step of this project will be to identify peptides which induce the desired
signalling cascade and test them on mouse model for the production of long-lasting antibodies
IgG. This is the first reported attempt to target a receptor with neoglycosylated peptides baring
a fragment specifically designed to selectively target hMGL.In chapter 4 the synthesis of a (neo)peptide containing a glycocluster with potential to give a trimeric coiled-coil sequence is described. The glycocluster is based on the tetraphenylethene residue. The peptide was successfully synthesised and purified. While some evidence for helix formation was obtained by CD spectroscopy, the refolding of the peptide after heating/denaturing was not observed and may have occurred partially.
In chapter 5 the first structural elucidation of hMGL carbohydrate binding mode is reported using X-ray crystallography. hMGL is the only lectin on human DCs that selectively recognise carbohydrate antigens present on many cancer cell lines and was proven to have the ability to lead the immune system to produce permanent antibody against cancer. The aim of this work was to understand the structural basis of the hMGL/carbohydrate interaction and to allow a more reliable in silico ligand design. To achieve this the protein was expressed in bacterial cell lines. As the expression resulted in insoluble protein, a refolding screen optimisation was carried out, which facilitated recovery of the protein in high purity and good yield for crystallisation screening. As a result of screening and optimisation, suitable crystals for diffraction were obtained. This led to the elucidation of the first crystal structure of hMGL carbohydrate recognition domain (CRD), binding with several monovalent GalNAc derivatives. The structure offers a possible explanation of hMGL binding mode and selectivity.2024-02-1
Synthesis and evaluation of multivalent ligands for macrophage galactose C-type lectin. Development of new chemical tools for immunological research and structural investigation of the lectin with GalNAc and derivatives
Synthetic chemistry can provide powerful tools to explore the correlation between an antigen’s
chemical structure and the type of immune response triggered. This thesis work has focused
on the design, synthesis and evaluation of multivalent molecules which target a relevant protein
of the immune system, the human macrophage galactose C-type lectin (hMGL).
In chapter 2 the synthesis of multivalent glycomimetics is described as well as measurements
of their affinity for hMGL. The main objective was
to learn more about the structure activity relationship
with regard to ligands based on tetraphenyl ethylene
(TPE) for hMGL and other lectins. To achieve this
divalent, trivalent and tetravalent ligand, based TPE
geometry as well as trans-stilbene presenting α-
mannopyranoside, β-lactose and α-thio-GalNAc as
headgroups, were synthesised. The identified
synthetic route to such compounds is reproducible and scalable. The thermodynamics of the
interaction of α-thio-GalNAc derivatives were studied using the extracellular domain of hMGL
and hMGL CRD, revealing insights into the possible interaction of these compounds with the
lectins.
In chapter 3 the synthesis of an extensive anti-cancer synthetic vaccine candidate library based
on MUC1/MUC4 peptide backbone is described. The main objective of this work was to
enhance antigen presentation by hMGL via the major histocompatibility complex II. To
achieve this objective mucin epitopes
were decorated with a trivalent
glycocluster selective for hMGL.
Through solid phase synthesis synthetic
peptides, including those presenting the
glycocluster were obtained. These were
found to bind hMGL from 10 to 200-
fold better than the natural peptides in an ELISA performed in the S. van Vliet laboratory in
Amsterdam. The next step of this project will be to identify peptides which induce the desired
signalling cascade and test them on mouse model for the production of long-lasting antibodies
IgG. This is the first reported attempt to target a receptor with neoglycosylated peptides baring
a fragment specifically designed to selectively target hMGL.In chapter 4 the synthesis of a (neo)peptide containing a glycocluster with potential to give a trimeric coiled-coil sequence is described. The glycocluster is based on the tetraphenylethene residue. The peptide was successfully synthesised and purified. While some evidence for helix formation was obtained by CD spectroscopy, the refolding of the peptide after heating/denaturing was not observed and may have occurred partially.
In chapter 5 the first structural elucidation of hMGL carbohydrate binding mode is reported using X-ray crystallography. hMGL is the only lectin on human DCs that selectively recognise carbohydrate antigens present on many cancer cell lines and was proven to have the ability to lead the immune system to produce permanent antibody against cancer. The aim of this work was to understand the structural basis of the hMGL/carbohydrate interaction and to allow a more reliable in silico ligand design. To achieve this the protein was expressed in bacterial cell lines. As the expression resulted in insoluble protein, a refolding screen optimisation was carried out, which facilitated recovery of the protein in high purity and good yield for crystallisation screening. As a result of screening and optimisation, suitable crystals for diffraction were obtained. This led to the elucidation of the first crystal structure of hMGL carbohydrate recognition domain (CRD), binding with several monovalent GalNAc derivatives. The structure offers a possible explanation of hMGL binding mode and selectivity.2024-02-1
Anomer preferences for glucuronic and galacturonic acid and derivatives and influence of electron-withdrawing substituents
Equilibrium anomeric ratios are reported for pyranoses (hemiacetals) of glucuronic and galacturonic acid and their derivatives. These are compared to related gluco- and galactopyranoses and to deoxyfluorogluco- and deoxyfluorogalactopyranoses. An association between axial anomer stability and the sum of H-1 NMR downfield chemical shifts for protons H-3 and H-5 was observed in D2O with gluco- and galactopyranoses as reference compounds. When compared to 2-hydroxytetrahydropyran in water, introduction of three OAc substituents and one carboxylic acid substituent leads to an increase in stability of the axial anomer by 0.89-1.05 kcal/mol. This is interpreted as the electron-withdrawing substituents causing a reduction in the steric (gauche) interaction and an increase in favourable Coulombic interaction between CH groups of the pyranose and the anomeric group through substituent deshielding effects. Anomer preferences for galacturonic acid and its derivatives were more sensitive to solvent polarity compared to other pyranoses, and this may be linked to the electrostatic potential and reduced stabilization of the equatorial anomeric OH group due to reduced hydrogen bonding. The latter is more notable in nonpolar chloroform. Analysis of crystal structures combined with molecular dynamics indicated there are conformational distinctions between galacturonic acid and glucuronic acid that could influence properties.This publication has emanated from research supported by Science Foundation Ireland (SFI, grant number 12/IA/1398) and is co-funded under the European Regional Development Fund under Grant Number 14/SP/27102019-07-0
Anomer Preferences for Glucuronic and Galacturonic Acid and Derivatives and Influence of Electron-Withdrawing Substituents
Equilibrium
anomeric ratios are reported for pyranoses (hemiacetals)
of glucuronic and galacturonic acid and their derivatives. These are
compared to related gluco- and galactopyranoses and to deoxyfluorogluco-
and deoxyfluorogalactopyranoses. An association between axial anomer
stability and the sum of <sup>1</sup>H NMR downfield chemical shifts
for protons H-3 and H-5 was observed in D<sub>2</sub>O with gluco-
and galactopyranoses as reference compounds. When compared to 2-hydroxytetrahydropyran
in water, introduction of three OAc substituents and one carboxylic
acid substituent leads to an increase in stability of the axial anomer
by 0.89–1.05 kcal/mol. This is interpreted as the electron-withdrawing
substituents causing a reduction in the steric (gauche) interaction
and an increase in favourable Coulombic interaction between CH groups
of the pyranose and the anomeric group through substituent deshielding
effects. Anomer preferences for galacturonic acid and its derivatives
were more sensitive to solvent polarity compared to other pyranoses,
and this may be linked to the electrostatic potential and reduced
stabilization of the equatorial anomeric OH group due to reduced hydrogen
bonding. The latter is more notable in nonpolar chloroform. Analysis
of crystal structures combined with molecular dynamics indicated there
are conformational distinctions between galacturonic acid and glucuronic
acid that could influence properties
Synthesis and biological evaluation of migrastatin macrotriazoles
The synthesis of three macrotriazoles that are analogues of migrastatin is reported. The synthesis is based on copper(I)- and ruthenium-catalyzed azide-alkyne cycloaddition reactions. The enantiopure terminal alkyne derivatives were prepared by using the Trost desymmetrization, Brown alkoxy-allylation and an efficient Colvin reaction. Biological evaluation of the products revealed a promising efficiency in reducing the ability of MDA-MB-361 cell lines to migrate
Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform
The increasing realization of the involvement of lectin-glycan recognition in (patho)physiological processes inspires envisioning therapeutic intervention by high-avidity/specificity blocking reagents. Synthetic glycoclusters are proving to have potential for becoming such inhibitors but the commonly used assays have their drawbacks to predict in vivo efficacy. They do not represent the natural complexity of (i) cell types and (ii) spatial and structural complexity of glycoconjugate representation. Moreover, testing lectins in mixtures, as present in situ, remains a major challenge, giving direction to this work. Using a toolbox with four lectins and six bi- to tetravalent glycoclusters bearing the cognate sugar in a model study, we here document the efficient and versatile application of tissue sections (from murine jejunum as the model) as a platform for routine and systematic glycocluster testing without commonly encountered limitations. The nature of glycocluster structure, especially core and valency, and of protein features, i.e. architecture, fine-specificity and valency, are shown to have an influence, as cell types can differ in response profiles. Proceeding from light microscopy to monitoring by fluorescence microscopy enables grading of glycocluster activity on individual lectins tested in mixtures. This work provides a robust tool for testing glycoclusters prior to considering in vivo experiments.Inspiring discussions with Drs B. Friday and A. Leddoz are gratefully acknowledged. Research in this paper was funded in part by Science Foundation Ireland (Grant Number 12/IA/1398) and the Irish Research Council (Grant Number GOIPG/2016/858). L. L. R.-H. thanks CONACYT-MĂ©xico (Grant 290936) for financial support.peer-reviewe