Interactions between carbohydrates and proteins underlie key biological processes. Many of these interactions involve carbohydrates on cell surfaces that have essential functions in cell-cell recognition, cellular communication and adhesion.
Moreover, they play crucial pathological roles in host-pathogen interactions, graft rejection and tumor growth. The detailed characterization of protein-carbohydrate interactions forms the basis for defining diagnostic targets and biomarkers, and for glycan-directed drug design. X-ray crystallography, NMR and site-directed mutagenesis are the traditional tools for identifying essential amino acids from protein structures required for carbohydrate recognition; however, their application is limited by large amounts of material required, high purity requirement and by elaborate experimental procedures. Therefore, the development of a robust, straightforward strategy to map carbohydrate- interacting structures in biologically active proteins is of considerable interest. The capability of mass spectrometry (MS) to provide structural characterization for many classes of biomolecules, with high sensitivity, low sample consumption and purity requirement, makes MS a valuable tool for the analytical development of new methods to identify carbohydrate-binding structures in proteins.
The first part of this thesis was focused on the development of novel affinity-mass spectrometric methods for the identification of carbohydrate recognition sites in carbohydrate-binding proteins. The new approaches combine selective proteolytic excision and extraction with mass spectrometry, require only low amounts of material with low purity and can be carried out while maintaining the native structure of the protein. The MS methods were developed, optimized and applied to the identification of the lactose binding peptides in galectin-1, -3, -4, -5 and -8. Galectins are a family of β-galactosides binding proteins that exhibit similar structural characteristics of their carbohydrate recognition domains. Galectins have been shown to be involved in several pathophysiological processes, such as inflammation and cancer, and therefore may represent potential targets for drug development.
For the development of affinity- mass spectrometry methods, columns were prepared by coupling lactose on divinyl sulfone-activated Sepharose. Next, galectins were added over the immobilized ligand and proteolytically digested on column.
Alternatively, the galectins were first digested in solution and the resulting peptide mixture incubated with the immobilized lactose. After washing off unbound galectin fragments, the remaining affinity-bound peptides were eluted from the column using lactose or aqueous-organic mixtures. Careful optimization and standardization were carried out regarding proteolytic conditions, composition of buffers and eluents, and washing protocols. The elution fractions were analyzed by mass spectrometry and the results compared with available crystallographic data. All peptides identified by proteolytic-excision and -extraction MS contain the conserved galactose-binding motifs HxNxR and WGxExR/K, which harbor the essential amino acids required for carbohydrate recognition. Thus, the MS results were in complete agreement with the available X-ray crystallography data on galectins-lactose complexes. For galectins without available crystal structures, the results obtained in this thesis experimentally ascertained the binding sites predicted on the basis of sequence similarities. The newly developed mass spectrometric approaches were also employed for the identification of blood group oligosaccharides recognition sites in human and chicken galectin-3. The identified sequences differed according to the carbohydrate employed, thus demonstrating that the MS methods are capable of discriminating between binding structures of complex oligosaccharides. All experiments were performed under rigorously controlled conditions, optimized to exclude unspecific binding.
The second part of this thesis presents the evaluation of the carbohydrate recognition properties of galectin-derived synthetic peptides. Based on the sequences identified by proteolytic- excision and -extraction MS, corresponding peptides were prepared by solid-phase peptide synthesis. The interaction of the peptides with carbohydrates was first characterized using affinity-mass spectrometry, by incubating the synthetic peptides alone or in mixtures with various immobilized carbohydrates.
After several washing steps, the affinity-retained peptides were eluted and analyzed by mass spectrometry. The results ascertained the specificity of the ligand-contacting synthetic peptides for the individual carbohydrates, thus validating the identifications
made by proteolytic-excision and -extraction mass spectrometry. Moreover, the minimal carbohydrate recognition structures could be defined by comparing shorter synthetic versions of the identified peptides, and the importance of specific amino acids for carbohydrate binding could be assessed by comparing specifically mutated peptides. The specificity of the results was ascertained by control experiments using unmodified matrix, scrambled peptides and carbohydrates lacking the essential galactose subunit.
The binding affinities of the synthetic peptides and intact galectins to lactose were determined quantitatively by surface-acoustic wave (SAW) biosensor analyses.
An affinity probe consisting of a neoglycopeptide was prepared by coupling lactose to a peptide carrier. The probe was immobilized on the SAW gold chip and the equilibrium dissociation constants (KD) determined by analyzing the binding of increasing concentrations of galectins and synthetic peptides. The KD values obtained for the intact galectins were in the micromolar range, in agreement with the results of previous studies employing Isothermal Titration Calorimetry and Surface Plasmon Resonance. The KD values of the ligand-binding peptides were 2-3 orders of magnitude higher, which is consistent with the smaller size and the assembled arrangement in the carbohydrate recognition domain.
The final part of this thesis describes the identification of the galactose binding site in alpha-galactosidase A, and the influence of pharmacological chaperones on galactose recognition by this lysosomal enzyme. Deficiency of α-galactosidase A is the cause of a lysosomal storage disorder, Fabry Disease, and is a recently explored, promising target for new treatment strategies. Binding site identifications were performed using immobilized galactose and the newly developed proteolytic-excision and extraction mass spectrometry methods. The galactose-binding peptides identified contain the key amino acids involved in galactose binding, in agreement with previously reported X-ray crystallography data on the enzyme in complex with galactose. Competition experiments using a galactose structural analogue, 1-deoxygalactonojirimycin (DGJ), revealed that DGJ interferes with the binding of galactosidase-derived peptides to the normal substrate.
In summary, in the present dissertation new affinity-mass spectrometry approaches were developed and shown to be powerful tools for the identification and quantitative characterization of protein-carbohydrate interactions, and for obtaining relevant information on carbohydrate-binding peptide sites. The new methods are fast, require very low amounts of sample and, albeit lower resolution, provide a molecular complement to X-ray and NMR approaches.