Glycosides are formed when carbohydrates are bound to non-sugar molecules such as proteins by specific enzymes (glycosylation). Glycosylation is extremely common, and over half of all proteins are glycosylated. Protein-carbohydrate interactions are among the most important biological interactions involved in recognition and signal transmission on cell surfaces, in cell membrane transport processes, in infections, and in tumor cell metastasis. A number of carbohydrate-binding proteins act as starter molecules for investigating and designing peptides as new active ingredients, such as antitumor agents. The team of experts at the Steinbeis Transfer Center for Biopolymer Analysis and Biomolecular Mass Spectrometry at the University of Constance has developed a new method called CREDEX-MS (“Carbohydrate REcognition Domain EXcision - Mass Spectrometry”) to identify these kinds of biologically inspired biomolecules.
CREDEX-MS combines enzymatic proteolysis with mass-spectrometric identification of the specific affinity-bound peptide fragments of a carbohydrate-binding protein. The Steinbeis team used it to identify the exact carbohydrate-specific binding epitope peptides in galectins, which have an antitumoral effect. This procedure makes it possible to determine peptide biomarkers of lectins at the level of molecular binding sites from very small protein quantities, as a basis for developing new antitumoral peptide agents.
The wide-ranging biological functions of protein-carbohydrate recognition processes harbor significant potential for medical applications [1]. One example of this: the interactions of galactosides – glycosides that contain galactose – which are important binding partners for endogenous lectins due to their strategic location on cell surfaces and their major role in cell-cell recognition. Beyond their function as glycol-biomarkers, galactosides are also important bioactive ligands for the development of lead compounds for new lectin-based carbohydrate active ingredients. Galectin-1 and galectin-3 (Gal-1, Gal-3) are two of the best-known human galectins, and their functions include acting as cell effectors in cell cycle control and as prognostic factors for the spread of tumor cells in tissue. As such, the identification and availability of peptides that imitate the specificities of galectins could enable the development of diagnostic tests to prove the presence of malignant processes. Consequently, designing biologically-inspired peptides that imitate the binding site(s) of a receptor is an area of growing focus.
How does the CREDEX-MS procedure identify carbohydrate-binding peptides? In an experiment (called “excision”), the Steinbeis experts
immobilized lactose (a disaccharide comprised of galactose and glucose) on a gelatinous solid carrier (Sepharose 4B). 50 µg of galectin-3, which regulates human growth, was then bound with 200 µL of the resulting affinity matrix. The lectin recognizes and binds the lactose to form a complex. Excess lectin and non-binding material (impurities) were removed by washing. This was followed by proteolytic degradation of the affinity-bound Gal-3 lactose complex by trypsin. The parts of the lectin bound to the sugar are protected from the degradation by trypsin and remain bound, including after washing. The non-binding trypsinized peptides were completely removed by washing. The remaining affinity-bound peptide fragments were detached with lactose or a suitable organic-water mixture and analyzed via MALDI mass spectrometry. The MALDI mass spectrometry showed the presence of two specific peptides, hG3.1 (Gal-3(152-162)) and hG3.2 (Gal-3(177-183)), which contain all of galectin-3’s main lactose binding sites and thus represent the biologically active sequences of lectin, in full agreement with the X-ray crystallography structure of the Gal-3 lactose complex (see Fig. 1 B, C) [3]. Lactose binding experiments with the synthetic peptides hG3.1 and hG3.2 fully confirmed the binding specificity [3]. It is of particular interest that the synthetic peptide ligands are shown to specifically inhibit binding of galectin-3 to tumor cell surfaces. In recent experiments, the CREDEX-MS method has already been successfully applied in a series of lectin carbohydrate complexes: in all cases, specific bioactive peptide ligands were identified, in most lectins without the structural data of the carbohydrate complexes being known.
The results so far clearly demonstrate the potential of the CREDEX-MS method to directly identify bioactive peptides through proteolytic excision with minimal quantities and only low purity requirements for carbohydrate complexes. The peptide sequences identified always contain the main interactions of the intact lectin. This combination of proteolytic excision and mass spectrometry establishes an important basis for investigating and optimizing bioactive peptides from lectins and other carbohydrate-binding proteins. It harbors a range of promising opportunities such as the development of specific peptides that regulate tumor growth, the development of peptides for T cell communication, and the identification of secondary or extended carbohydrate binding sites.
[1] Gabius, HJ.; André, S.; Jimenez-Barbero, SS.; Romero, A.; Solís, D. From lectin structure to functional glycomics: principles of the sugar code. Trends in Biol. Sciences 2011; 36 (6), pp. 298-313.
[2] Przybylski, M.; Moise A. Method for the identification of ligand-contacting peptides. EU patent application (2011).
[3] Moise, A.; André, S.; Eggers, F.; Krzeminski, M.; Przybylski, M.; Gabius, HJ. (2011) Toward Bioinspired Galectin Mimetics: Identification of Ligand-Contacting Peptides by Proteolytic-Excision Mass Spectrometry. J. Am. Chem. Soc. 133, 14844-14847.
Frederike Eggers | Adrian Moise | Prof. Dr. Michael Przybylski
SteinbeisTransfer Center for Biopolymer Analysis and Biomolecular Mass Spectrometry, University of Constance (Constance)
su0723@stw.de