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 α-anomer of galactose (Gal) at the non-reducing terminal than the corresponding β-anomer; (ii) Galα1-3Galα1- (B_α), Galα1-4Gal (E), Galβ1-3GalNAc (T) and Galβ1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II_β, prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I_β/II_β and E_β 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 α1-3/4/6Gal, β1-3GalNAc, β1-3/4/6GlcNAc, β1-4/6Glc, β1-3DAra and β1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galα1-3Galβ1-4GlcNAc β1-3Galβ1-4Glc (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 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.     

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

以合成多肽为抗原的戊肝抗体检测试剂的研制

用戊肝病毒（ＨＥＶ）基因组编码氨基酸序列１－９０１－９１４／２－５１５－５３０、３－９１－１２３、２－６１３－６５４相应的三段合成多肽为抗原、研制出一种检测抗－ＨＥＶＩｇＧ的ＥＬＩＳＡ试剂。以该试剂检测中国、缅甸、印度和前苏联肠道传播非乙型肝炎（ＥＴ－ＮＡＮＢＨ）病人血清１０５份,仅３份中国病人血清阴性,阳性率为９７．１％;检查实验感染ＨＥＶＬ赤猩猩血清,感染前阴性,感染后阳性;检查正常人血清９９     

Macoma birmanica agglutinin recognizes glycoside clusters of β-GlcNAc/Glc and α-Man

Macoma birmanica agglutinin (MBA) that seems to play crucial roles in the innate immunity of marine bivalve, M. birmanica has been earlier defined as GlcNAc/Man specific. However, most complementary carbohydrate structures to its binding domain and ligand clustering in its recognition profile have not been established. In this study, the complete recognition profile of MBA was examined by enzyme-linked lectin-sorbent assay and inhibition assay. Among the monosaccharides tested, GlcNAc was more reactive followed by Man and Glc, others were non-reactive; revealing the importance of equatorial -NAc group at C-2, -OH group at C-4 and C-6, and pyranose conformation of hexose. Moreover, β-glycosides of GlcNAc and Glc were more potent whereas for Man it was α-glycoside. MBA recognized both exposed and internal α-Man and β-GlcNAc/Glc residues well with most linkages except (β1-4). This binding pattern was further extended and confirmed by polyvalent glycoside clusters of GlcNAc(β1-2)Man(α1-, which was a better inhibitor than Man(α1-2/3/6)Man(α1- or Man(α1-3/6)Man(β1- present in well-defined naturally occurring glycoproteins. This broad range specificity explains the importance of MBA as an important pattern recognition molecule that provides more realistic picture of carbohydrate-based immune response triggering.     

生物技术在食品检测方面的应用

综述了DNA探针、PCR、生物芯片、胶体金免疫层析及ELLSA等生物技术的基本原理及其在食品检测方面的应用。