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LECTINS Proteins That Interpret the Sugar Code
GÖTEBORG
Carol L. Nilsson UNIVERSITY (SWEDEN)
Understanding the biochemistry of lectins and the physical nature of the protein–carbohydrate interaction will provide important insights into biological information transfer. With the recent completion of dozens of genomic sequencing projects, including the human genome, the focus of many research groups has shifted to interpreting protein expression to gain new understanding about biological processes. Certainly, proteins are central mediators in cellular processes and perform their duties through interactions with lipids, carbohydrates, small molecules, and other proteins. Therefore, one aspect of functional proteomics is the elucidation of the interactions between proteins and other molecules. Lectins are proteins that recognize and bind to specific carbohydrate structural epitopes. They may be regarded as protein interpreters of the “sugar code” and represent convenient biochemical tools to probe protein–carbohydrate interactions. Understanding the expression patterns of lectins in biological systems and the physical nature of the protein–carbohydrate interaction provides important insights into biological information transfer for such diverse areas as microbiology, oncology, and plant pathology. Proteins that interact with sugars are grouped according to how the interaction takes place. Some proteins transport sugars across cell membranes to provide fuel for the cell. Others modify saccharides in metabolic processes or attach them covalently to lipids or other proteins. Antibodies may be generated by the immune system against carbohydrate epitopes. Lectins, however, are defined as carbohydrate-binding proteins that are not generated in the immune system and lack enzymatic activity (1). Although they were first discovered and characterized in plants (2), lectins are also expressed by microorganisms, fungi, and animals. Some of the functions ascribed to lectins include microbial adhesion, lymphocyte homing, intracellular protein trafficking, cell–cell recognition, fertilization, and cancer metastasis. Secreted lectins may be toxic, as in the case of the ricin toxin, which made newspaper headlines in the 1978 “umbrella homicide” of the exiled Bulgarian Georgi Markov in London and has recently reemerged as a potential terrorist weapon. Lectins were first used as biochemical tools in the 1950s, helping to elucidate the chemical structure of the ABH histo-blood group antigens (3), the A U G U S T 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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FIGURE 1. Human trophoblast adhesion to the uterine wall is mediated by L-selectin. Image shows primary cytotrophoblasts (CTB) in culture stained for L-selectin. The fibroblast, which is a connective tissue cell that does not bind L-selectin, provides a negative control.
system of which is based on differences in terminal saccharides in human glycolipids and glycoproteins. Because lectins recognize saccharide structure motifs, they are invaluable biochemical tools. The coding density of sugars is significantly higher than that of amino acids or nucleotides (4). For a given hexasaccharide, the number of possible structural isomers is 1.44 1015; for a hexapeptide, 6.4 107; and for a hexanucleotide, 4096. The branching nature of oligosaccharides, the substitution of sugars with phosphate or sulfate, and differing anomeric linkages all contribute to the structural diversity of carbohydrates as well as the difficulties associated with developing bioinformatics tools for glycobiologists. In this article, the chemistry and the methods of analysis for lectins are reviewed, and the potential of new analytical techniques is discussed.
Finding old lectins in new places One class of lectins, the selectins, are transmembrane glycoproteins that recognize a subset of sialyl-Lewisx (NeuAc3Gal4[Fuc3]GlcNac)-containing carbohydrate antigens (5). The family is divided into structurally related groups, designated P-, E-, and L-selectins. L-selectins have been extensively studied in the blood and vascular systems because of their ability to mediate leukocyte homing to high endothelial venules under the shear stress of blood flow, an important event in inflammation. The initial tethering of leukocytes to endothelial cells is followed by integrin activation and migration through the blood vessel wall. The role of L-selectins in recognition events occurring outside of the blood–vascular interface has not been studied to any great extent. Recently, Fisher discovered a new role for L-selectin in the establishment of human pregnancy (6). There are morpho350 A
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logical parallels between the processes of white blood cell extravasation and embryonic (trophoblast) attachment to the uterine wall. As in the case of white blood cell attachment, trophoblast adhesion takes place under shear stress caused by fluid flow. Another similarity is that the late stages of embryonic adhesion and implantation are dependent on integrins, which are transmembrane proteins that link the cytoskeleton to the extracellular matrix. Could selectins be expressed at the trophoblastic cell surface during attachment? Immunological methods demonstrated the expression of selectin ligands by human uterine epithelium during the window of receptivity, and human trophoblasts exhibited Lselectin immunoreactivity (Figure 1). The discovery of L-selectin at the fetal–maternal interface is expected to have implications for our understanding of some causes of female infertility and early pregnancy loss.
Probing protein–carbohydrate interactions The field of protein–carbohydrate interactions offers one of the last great frontiers of biology and chemistry. The area presents special analytical challenges because of the weak nature of the biomolecular interactions and the solubility differences between carbohydrates, which are extremely hydrophilic, and proteins, which have a wide range of hydrophobicities. Crystallography has provided the structures of ~30 lectins, which are publicly available on the Web (7 ). However, there is no general lectin structure from which all others can be modeled. Even the binding sites in different lectins lack a general architecture (8). High-resolution crystallography of lectin–saccharide complexes provides evidence that hydrogen bonds, including water-mediated hydrogen bonds, and van der Waals interactions contribute to binding energies. Protein–carbohydrate binding energies are generally lower than those measured for protein–protein complexes. The low affinity of lectins for carbohydrates is, to some extent, compensated for by their multivalency, which increases the avidity of the binding. In addition to crystallography, NMR is a useful technique for studying protein–carbohydrate interactions, but both techniques require relatively larger amounts of analytes. For this reason, MS techniques have been developed for characterizing protein–carbohydrate interactions. MS can be applied as a detector in highthroughput screening schemes to identify lectins or their oligosaccharide ligands, measure the dissociation constants of protein– carbohydrate complexes, and characterize carbohydrate-binding surfaces on proteins. Because the information provided by MS is different from the results of crystallographic and NMR experiments and can be obtained relatively rapidly at a high level of sensitivity, MS is an important complementary technique. In a recent crystallographic study of the lectin of Erythrina cristagalli complexed with two different oligosaccharides, MS mapping of the lectin provided evidence of sequence heterogeneity and protein glycosylation that was not detected by crystal studies (9).
Carbohydrate arrays During 2002, three reports were published that detailed new large-scale methods to study protein–carbohydrate interactions
by microarray techniques (10–12). These reports are based on the discovery that binding partners can be made by either linking the protein or the carbohydrate to a solid substrate. Key issues for developing carbohydrate arrays include chemically binding functional proteins or carbohydrates to a surface while retaining biological activity, minimizing nonspecific binding, and attaining high analytical sensitivity. Although none of the three reports detail the use of a mass spectrometer as a detector, this is expected in the future. Wang and colleagues developed a microarray of ~50 microbial carbohydrate antigens on glass slides, without chemical conjugation (10). On one slide, a pattern of ~20,000 spots can be deposited. A panel of known microbial antigens was tested against murine and human IgM and IgG antibodies using this approach, resulting in the identification of ~40 distinct anticarbohydrate binding specificities. Houseman and Mrksich developed an assay to profile lectin– carbohydrate interactions by immobilizing carbohydrate–cyclopentadiene conjugates to self-assembled monolayers on gold and monitoring lectin binding to the immobilized sugars by surface plasmon resonance (11). The arrays could, in turn, quantitate the inhibitory concentrations of soluble carbohydrates for lectin binding. In another study, Park and Shin designed carbohydrate chips based on lectin multivalency (12). If proper spacing between surface-bound carbohydrates is maintained, each lectin molecule may be able to bind more than one sugar epitope, increasing the avidity of the lectin–carbohydrate interaction. Adding a linker “arm” between the sugar and the surface can modulate the spacing between ligand sugars. For example, maleimide sugars, with linker structures of varying lengths, were attached to a slide displaying thiol groups on its surface using stable thioester linkages. Lectin binding was determined by fluorescence; as expected, longer linker structures attached to the sugars gave the best lectin-binding capacities. Further developments may provide high-throughput methods to profile lectin–carbohydrate interactions and simultaneously characterize inhibitors and native ligand analogues.
ferent dissociation constants pass through the affinity column at different rates. FAC assays require that the buffer conditions for maximum activity and minimum interference with the desorption/ionization process at the ESI interface be optimized. Recently, a FAC/MS study of a library of 356 compounds targeted to -galactosidase revealed 34 substances with dissociation constant values lower than 10 µM (14). However, not all of the tested compounds could be detected by ESI-MS; therefore, other ionization modes such as atmospheric pressure chemical ionization or MALDI may be needed to complement future assays. Bioaffinity MS. MS is an excellent detector for proteins and oligosaccharides, because structural data can be provided in addition to chemical identity. Derivatization of MALDI probe surfaces represents an important enhancement of selective analyte detection. Lebrilla and co-workers demonstrated that lectins can be immobilized on a MALDI probe and used to capture binding oligosaccharides, after which the carbohydrates can be analyzed by FT-ion cyclotron resonance (FT-ICR) MS and structurally determined (15, 16). The method provided new insights into the fertilization process in the frog Xenopus laevis. The cortical granule lectin (CGL) of X. laevis was immobilized on the MALDI probe surface to capture oligosaccharides released from the frog’s eggs. Researchers studied preferentially binding oligosaccharides using sustained off-resonance irradiation-collision-induced dissociation (17 ), which revealed terminally sulfated structures. Weaker-binding oligosaccharides displayed sulfation at internal residues, indicating that terminal sulfation plays an important role in CGL recognition. Lectin recognition of carbohydrates also plays an important role in microbial adherence to host tissues. Bacteria and viruses possess surface-expressed proteins called adhesins, which recognize glycoconjugates of the host organism and thereby facilitate the colonization and/or invasion of host cells. The first discovered adhesin was the hemagglutinin of the influenza virus (18). Characterization of microbial binding to glycoconjugates and the discovery of the protein ligands are important in microbiology, because they may lead to the development of new vaccines and drugs. Recently, both lectin- and carbohydrate-derivatized MALDI probes were used to study the adhesive properties of pathogenic bacteria (19). Commercially available plant lectins with an affinity for microbial glycoconjugate epitopes were immobilized on an affinity membrane and applied to a MALDI target, then incubated with preparations of pathogenic bacteria or virus-
Because lectins
recognize saccharide structure motifs,
they are invaluable biochemical tools.
Other methods Frontal affinity chromatography. An alternative to carbohydrate chips for high-throughput screening is frontal affinity chromatography (FAC) coupled to online electrospray ionization (ESI)-MS (13, 14). Biotinylated carbohydrate-binding proteins can be immobilized to streptavidin microcolumns. Continuous infusion of ligand libraries over the microcolumn allows measurements of kinetic parameters because compounds with dif-
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es. After washing the binding microorganisms, the researchers lysed them, applied the matrix to the target, and examined them for protein biomarkers by using MS. Likewise, biotinylated carbohydrate polymers (neoglycoconjugates) were immobilized on a membrane coated with streptavidin, and the membrane was attached to a MALDI target and used to capture microorganisms. The carbohydrate-conjugated membranes gave the best sensitivity; protein biomarkers from ~100 cells of the pathogenic E. coli strain O157:H7 could be detected by capture on a Lex-coupled probe. The enhanced sensitivity of the carbohydrate probes can be attributed to the greater number of ligands displayed on the target surface, and hence the higher avidity of the carbohydrate–bacterial lectin interactions. Glass slides are typically used for high-throughput analysis and can also be used as sample supports for MALDI MS. In a recent study, biotinylated lectins immobilized on streptavidincoated glass slides captured agglutinated Bacillus spores, which were analyzed by TOF and FT-ICR MS (20). Interestingly, less postsource fragmentation occurred in samples deposited on glass slides, which improved the quality of the mass spectra. Unfortunately, a lower ion current than that seen with metal MALDI targets was also observed, but instruments equipped with highvoltage extraction MALDI sources may ameliorate this problem. The ability to couple activated glass slides to MALDI analysis is an important development, because this technique offers
relative ease of designing high-throughput assays and highly specific MS detection. Affinity proteomics. Lectin–oligosaccharide interactions can be exploited to capture cells for microbe identification by MS and identification of specific interacting membrane lectins on microbial surfaces. Identifying interacting partners is a necessary step before determining the protein structure. In many cases, microbial lectins are challenging to analyze because of their hydrophobicity and low copy number per cell. Numerous carbohydrate-binding affinities for different strains of the gastric pathogen Helicobacter pylori have been described, including Leb, sialyl-Lex, and sulfated oligosaccharides. A strategy for assigning the identity of the membrane-bound microbial adhesin to its oligosaccharide affinity through a proteomics approach has been described for the Leb-binding adhesin (21). Live bacteria are incubated with a heterotrifunctional neoglycoconjugate probe containing an oligosaccharide, a biotin, and a light-reactive cross-linker (Figure 2). After incubation, the probe is cross-linked to the binding adhesin by UV irradiation. Subsequent reduction of the disulfide bond yields adhesin molecules containing a minimal cross-linker with biotin. The successful identification of the sialyl-Lex-binding adhesin of H. pylori by selective enrichment of the biotinylated proteins from lysed bacteria on streptavidin-coated magnetic beads, followed by electrophoresis, MS, and database searches, allows identifi-
Neoglycoprotein/cross-linker conjugate Cross-linker NHS Disulfide bond
Albumin
Aryl azide Oligosaccharide
Biotin
Carbohydrate-binding activity Albumin A
UV cross-linking Reducing conditions
Albumin
Albumin
A
A
FIGURE 2. Adhesin affinity tagging procedure for selective protein enrichment and identification. The use of a multivalent neoglycoconjugate probe that displays multiple copies of carbohydrate epitopes increases the avidity of adhesin binding to the probe. A, adhesin; NHS, N-hydroxysuccinimido.
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cation of an adhesin against known genomic sequences (22). Both the Leb- and sialyl-Lex-binding adhesins are putative vaccine candidates. Blackbody IR radiative dissociation. A novel application of the thermal dissociation technique blackbody IR radiative dissociation (BIRD) to gaseous protein–carbohydrate complexes provided measurements of dissociation activation energies (23) and a map of the intermolecular hydrogen bonds in one of the complexes. The measurements were performed by gently desorbing protein–carbohydrate complexes by ESI and transferring the ions to a heated ICR cell. Thermal (IR) emission from the heated chamber is absorbed by the ions, causing the complexes to dissociate. By measuring dissociation rates as a function of temperature, researchers can accurately determine Arrhenius activation energies and pre-exponential factors. The retention of biospecific interactions in multiply protonated supramolecular complexes has been reported, but nonspecific interactions may also occur (24). The charge state of the complex may influence its conformation as well. For example, mapping a single-chain variable fragment (scFv) of an antibody with four different trisaccharide ligands at various charge states provides evidence that the native conformation of the scFv with its natural ligand is retained in the +7 to +10 charge states (25). At charge states >+10, charge-induced structural changes in the complexes are observed. Although few studies have been published, BIRD applied to protein– carbohydrate complexes holds promise for the quantitation of these complexes in the gas phase. H/D exchange MS. This technique provides information on protein conformation and identifies protein surfaces that interact with ligands. Amide hydrogens are exchangeable with hydrogen or deuterium in the solvent. When proteins are dissolved in D2O, protein backbone hydrogens that are solventexposed will rapidly exchange with deuterium atoms. The resulting 1 Da/deuteron increase in mass can be measured by MS, and the peptide segments protected from amide hydrogen exchange by protein folds or ligand interactions can be identified. H/D exchange MS was applied to the protein–carbohydrate complex endopolygalacturonase II (EPG-II) from Aspergillus niger and an octamer of galacturonic acid (26). The location of the binding cleft on the protein surface could be deduced by monitoring the differences in deuterium incorporation of EPGII in the presence and absence of the carbohydrate. The data obtained agreed with those obtained by site-specific mutagenesis. A second study of EPG-II, the carbohydrate substrate, and the polygalacturonase inhibitor protein (PGIP) provided new information on the nature of the enzyme inhibition (27 ). PGIP contacts EPG-II at a site remote from the carbohydrate-binding cleft. New methods are still emerging that will expedite the identification and characterization of lectins and their binding partners. Chip-based methods should be highly useful to rapidly profile environmental, medical, and forensic samples. Novel MSbased structural techniques such as BIRD and H/D exchange MS have been applied to protein–carbohydrate complexes and
also are likely to increase our understanding of lectin–oligosaccharide interactions in the future. I thank Susan Fisher (who also provided Figure 1) and John Klassen for helpful comments on the text. This work was funded by the National Medical Research Council of Sweden. Carol L. Nilsson is an associate professor at the Institute of Medical Biochemistry at Göteborg University (Sweden). Her research interests include applications of MS to functional and structural proteomics. Address correspondence about this article to Nilsson at carol.nilsson@ medkem.gu.se.
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