Zhangung Yang

Lectins as tools in glycoconjugate research

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate–protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin–carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin–glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, β-elimination), which extend the applicability of these methods, are also described.

Differential affinities of Erythrina cristagalli lectin (ECL) toward monosaccharides and polyvalent mammalian structural units

Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galβ1→4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 × 104, 3.9 × 103 and 2.4 × 103 more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galβ1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galβ1→4GlcNAc (II) and some Galβ1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with Galβ1→4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galβ1→4GlcNAcβ1→6Gal. These analyses should facilitate the understanding of the binding function of ECL.

Glycomic mapping of O- and N-linked glycans from major rat sublingual mucin

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galβ1→4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.

Differential contributions of recognition factors of two plant lectins – Amaranthus caudatus lectin and Arachis hypogea agglutinin, reacting with Thomsen-Friedenreich disaccharide (Galβ1–3GalNAcα1–Ser/Thr)

Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for the Thomsen-Friedenreich (T(alpha), Galbeta1-3GalNAcalpha1-Ser/Thr) glycotope, but differ in monosaccharide binding--GalNAc>>Gal (inactive) for ACL; Gal>>GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was based on studies with a small number monosaccharides and T-related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that the main recognition factors of ACL, GalNAc was the only monosaccharide recognized by ACL as such, its polyvalent forms (poly GalNAcalpha1-Ser/Thr, Tn in asialo OSM) were not recognized much better. Human blood group precursor disaccharides Galbeta1-3/4GlcNAcbeta (I(beta)/II(beta)) were weak ligands, while their clusters (multiantennary II(beta)) and polyvalent forms were active. The major recognition factors of PNA were a combination of alpha or beta anomers of T disaccharide and their polyvalent complexes. Although I(beta)/II(beta) were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galbeta1-3GalNAc (T), while the PNA binding domain is proposed to be Galbeta1-3GalNAcalpha or beta1--as the major combining site with an adjoining subsite (partial cavity type) for a disaccharide, and most complementary to the linear tetrasaccharide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glc (T(beta)1-4L, asialo GM(1) sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes for the interaction with these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions.

Expression of sialyl Lex, sialyl Lea, Lex and Ley glycotopes in secreted human ovarian cyst glycoproteins

Human blood group A, B, H, Ii, Le(a) and Le(b) antigens and their determinants expressed on ovarian cyst glycoproteins have been studied for over five decades. However, little is known about sialyl Le(x) and sialyl Le(a) glycotopes, which play essential roles in normal immunity, inflammation, and cancer cell metastasis. Furthermore, Le(x) and Le(y) were classified as glycotopes of unknown genes. Identification of these Lewis epitopes was hampered by the lack of specific antibodies. In this study, the occurrence of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) reactivities in cyst glycoproteins was characterized by enzyme-linked immunosorbent assays. The results indicated that most human ovarian cyst glycoproteins carried Le(x) (8/25) and/or Le(y) (17/25) glycotopes. The expression (epitopes) of the new genes described in previous reports are Le(x) and Le(y) glycotopes; the reactivities of sialyl Le(x) and sialyl Le(a) glycotopes in secreted cyst glycoproteins may be affected by the conditions of purification; the relationship between Le(y) and human blood group ABH was confirmed; recognition profiles of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) present in the carbohydrate chains of water-soluble cyst glycoproteins were illustrated; possible attachments of glycotopes to the internal carbohydrate complex of cyst glycoproteins have been reconstructed; proposed biosynthetic pathways for the formation of sialyl Le(a), sialyl Le(x), Le(x), Le(y), ALe(y) and BLe(y) determinant structures on Type I and Type II core structures of human ovarian cyst glycoproteins are also included in this study.

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

BACKGROUND Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed. METHODS The roles of human and bovine lactoferrins involved in lectin-N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins. RESULTS AND CONCLUSIONS Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins - Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA. GENERAL SIGNIFICANCE This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin-N-glycan recognition processes.

Identification of blood group A/A‐Leb/y and B/B‐Leb/y active glycotopes co‐expressed on the O‐glycans isolated from two distinct human ovarian cyst fluids

Although the individual human blood group A and B determinants are well defined, their co‐expression pattern on a particular glycan carrier in individuals of blood group AB status has not been delineated. To address this issue, complex O‐glycans were isolated from two distinct sources of human ovarian cyst glycoproteins (HOC 89 and Cyst 19) and profiled by advanced MS analyses, in conjunction with defining their binding characteristics against a panel of lectins and monoclonal antibodies. The major O‐glycans of HOC 89 were found to correspond to sialyl Tn, mono‐ and di‐sialyl T structures, whereas those of Cyst 19 were apparently more heterogeneous and extended to larger sizes. A minimal structure that carries both A and B determinants on the same molecule was identified, in which the A epitope is attached directly to the core GalNAc, whereas the B epitope is preferentially located on the six arms of a core 2 structure. Both arms can be further extended with internal fucosylation that appears to be restricted to those non‐sialylated chains already carrying the terminal ABH determinants, thus giving rise to rather prominent A/B‐Leb/y glycotopes on larger O‐glycans.

Recognition intensities of submolecular structures, mammalian glyco-structural units, ligand cluster and polyvalency in abrin-a-carbohydrate interactions

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having alpha-anomer of galactose (Gal) at the non-reducing terminal than the corresponding beta-anomer; (ii) Galalpha1-3Galalpha1- (B(alpha)), Galalpha1-4Gal (E), Galbeta1-3GalNAc (T) and Galbeta1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II(beta), prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I(beta)/II(beta) and E(beta) glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of alpha1-3/4/6Gal, beta1-3GalNAc, beta1-3/4/6GlcNAc, beta1-4/6Glc, beta1-3DAra and beta1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs.

Recognition roles of mammalian structural units and polyvalency in lectin--glycan interactions.

Lectins are an important class of proteins or glycoproteins of nonimmune origin that specifically or selectively bind carbohydrate moieties of complex carbohydrates. They play many critical roles in life processes, such as fertilization, embryogenesis, cell migration, organ formation, inflammation, immune defense, and microbial infection [8, 22, 40]. Specificity of lectins toward particular carbohydrate structures allows them to be used for characterization of unknown structures and identification and fractionation of glycoconjugates [23]. Moreover, this unique group of proteins has provided researchers with powerful tools to explore many biological processes in which lectins are involved. Considering the role of lectins in vivo, we have to realize that lectins in living organisms interact with multivalent macromolecules (glycoproteins) or clusters of oligosaccharide ligands on the cell surface.