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Dolichos biflorus agglutinin (DBA) is one of the well known plant lectins that are widely used in clinical serology to differentiate human blood group A1 and A2 erythrocytes and also applied to glycobiology. However, the knowledge of recognition factors of polyvalent (super) glycotopes in glycans and the roles of functional group and epimer in monosaccharide (sub-monosaccharide recognition factor) have not been well established. The size and shape of the recognition (combining) site of DBA has not been clearly defined. In this study, many important recognition factors of DBA-glycan binding were characterized by our established enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results of these assays showed that the intensity profile of the recognition factors for the major combining site of DBA was expressed by Mass relative potency (Mass R.P.) and shown by decreasing order of high density of polyvalent GalNAcα1 → (super glycotopes, 3.7 × 103) >> the corresponding β anomers >> monomeric GalNAcα1 → related glycotopes (GalNAc as 1.0) >> their GalNAc β-anomers >> Gal (absence of NHCH3CO at carbon-2 of GalNAc) and GlcNAc (different epimer of Carbon-4 in GalNAc). From the all data available, it is proposed that the combining site of DBA should consist of a small cavity shape as major site and most complementary to monomeric GalNAcα → located at both terminal reducing end (Tn) and nonreducing end of glycan chains, and with a wide and broad area as subsite to accommodate from mono- to tetra-saccharides (GalNAcβ, Galβ1 → 3/4GlcNAc, lFucα1 → 2Galβ1 → 3/4GlcNAc, GalNAcβ1 → 3Galα1 → 4Galβ1 → 4Glc) at the nonreducing side. This study provides the most (comprehensive) recognition knowledge of DBA-glycan interactions at the factors of glycotope, super glycotope/sub-monosaccharide levels. Thus, it should expand and upgrade the conventional concept of the combining (recognition) site of DBA since 1980s.

The binding property of Con A has been studied intensively and applied widely to glycoconjugates / glycobiology for over 80 years. However, its role and functional relationship of Con A with these mammalian structural units, glycotopes, N-glycan chains, as well as their polyvalent forms in N-glycoproteins involved in the Con A-glycan interactions have not been well defined and organized. In this study, the recognition factors involved in these interactions were analyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all the data obtained, it is concluded that Con A, as previously reported, has a relatively broad and wide recognition ability of the Manα1 → and Glcα1 → related glycans. It reacted not only strongly with yeast mannan and glycogens, but also bound well with a large number of mammalian N-glycans, including the N-glycans of rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobulin and lactoferrin. The recognition specificity of Con A towards ligands, expressed by Molar Relative Potency (Molar R.P.), in a decreasing order is as follows: α1 → 3, α1 → 6 Mannopentaose (M5) and Biantennary N-linked core pentasaccharide (MDi) ≥ α1 → 3, α1 → 6 Mannotriose (M3) > Manα1 → 3Man (α1 → 3Mannobiose), Manα1 → 2Man (α1 → 2Mannobiose), Manα1 → 6Man (α1 → 6Mannobiose), Manα1 → 4Man (α1 → 4Mannobiose) > GlcNAcβ1 → 2Man (β1 → 2 N-Acetyl glucosamine-mannose) > Manα1 → /Glcα1 → > Man > Glc, while Gal / GalNAc were inactive. Furthermore, the Man related code system, in this study, is proposed to express by both numbers of Man and GlcNAcβ1 → branches (M3 to M9 / MMono to Penta etc.) and a table of three Manα1 → and Glcα1 → related biomasses of six recognition factors involved in the Con A-glycan interactions has also been demonstrated. These themes should be one of the most valuable advances since 1980s.

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.

Galα1 → and GalNAcα1 → are the two essential key sugars in human blood group AB active glycotopes, in which GalNAcα1 → related sequences are located at both sides of the nonreducing and the reducing ends of human blood group A active O-glycans. It is also found at the nonreducing ends of GlcNAc N-glycans and glycosphingolipid(GSL) of human blood group A active glycotopes (Ah) and Forssman antigen (Fp). When monosaccharides and their α, β anomers are involved in basic units to express the complex size of the combining sites of the GalNAcα1 → specific lectins, they can be divided into a cavity site to accommodate the GalNAcα → key sugar and a subsite with a wide and broad range of recognition area to adopt the rest part of sugar sequences or glycotopes. The function of the subsite is assumed to act as an enhancement factor to increase its affinity power. The following three points are the theme of this mini review: (1) the loci and distribution of the GalNAcα1 → related glycotopes in mammalian glycoconjugates are illustrated and their chemical structures are advanced by the expression of the disaccharide units and code system; (2) the sizes and motifs of GalNAcα1 → specific lectin-glycan interactions are given and (3) the role of the polyvalent blood group Ah and Bh glycotopes as blood group AB antigens are proposed. These three highlights should provide an essential background required for the advances in this field.

Formaldehyde (FA) is frequently used in sterilizing surgical instruments and materials. Exposure to FA is highly concerned for eye tissues. Rabbit corneal epithelial cells were examined for changes after FA exposure. Our results showed that cell survival decreased 7 days after transient 3 min exposure to more than 100 ppm FA by trypan blue staining while MTT assay detected significant decrease at 20 ppm at 24 hours observation. The decrease of cell survival rate was concentration (up to 600 ppm)- and observation time (1-7 day)- dependent. The cell number decreased after 100 ppm FA exposure for more than 10 min at 7-day observation. The FA treated cells showed increased apoptosis/necrosis and cell cycle accumulation at sub G1 phase as well as mitochondria clustering around nucleus. The in vivo rabbit eye exposure for tear production by Schirmer's test revealed that the FA-induced overproduction of tear also exhibited observation time (1-10 day)- and FA concentration (20-300 ppm for 5 min exposure)-dependent. Activated extracellular signal-regulated kinase (pERK2) in cornea explants by western blotting was reduced and increased c-Jun amino - terminal kinase (JNK) activation (pJNK) in cornea and conjunctiva was evident at 2 month after exposure to 50-200 ppm FA for 5 min. In conclusion, injury to the eye with transient exposure of up to 100 ppm FA for 3 min decreased corneal cell survival while a more sensitive MTT test detected the cell decrease at 20 ppm FA exposure. Morphology changes can be observed even at 5 ppm FA exposure for 3 min at 7 days after. The FA exposure also increased apoptotic/necrotic cells and sub-G1 phase in cell cycle. Long term effect (2 months after exposure) on the eye tissues even after the removal of FA can be observed with persistent JNK activation in cornea and conjunctiva.

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate–protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le a ) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.

Human ovarian cyst glycoproteins (HOC, cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the richest and pioneer sources for studying biosynthesis, structures and functional roles of blood group ABH, Lea,b,x,y, sLea and sLex active glycoproteins. After 70+ years of exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Lex and Ley; (iii) recognition profiles of cyst glycans and a sialic acid-rich (18%) glycan with lectins and antibodies were shown. (iv) Co-expression of Blood Group A/ A-Leb/y and B/B-Leb/y active Glycotopes in the same glycan chains were isolated and illustrated. These are the most advanced achievements since 1980.

Lectins, in combination with our established enzyme-linked lectin sorbent assay (ELLSA) and inhibition study, have been used as powerful tools in many glycoconjugate recognition studies. In this short review, we highlight the following: (i) The recognition profiles of Gal/GalNAc-specific lectins were updated and upgraded. (ii) Based on the cross-specificities of applied lectins, a new classification system was introduced. (iii) Applications of lectins for the detection and identification of N-glycan and/or Tn glycotope in glycoconjugates were intergraded. (iv) The polyvalency of the glycotopes in glycans was found to play a critical role in glycan–lectin recognition. This is an unexplored area of glycobiology and one of the most promising directions toward the coming glycoscience transformation.

Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4-GlcNAc-capped terminal epitope functionally implicated in protection against Helicobacter pylori infection. Being more readily available and reasonably well characterized, it serves as a good reagent for immunobiological studies, as well as a standard for analytical methodology developments. Current approaches in mass spectrometry (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) mapping of permethylated glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode. This was facilitated by introducing a mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/MS fragmentation patterns, all previously known structural features of porcine gastric mucin including the terminal epitopes and location of sulfates could be readily defined. We additionally showed that both arms of the core 2 structures could be extended via 6-O-sulfated GlcNAc to yield a series of disulfated O-glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MS n analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y epitope implicated by positive antibody binding.