伴刀豆球蛋白(Con A)的提取、纯化及其亲和吸附剂的制备

伴刀豆球蛋白A(Concanavalin A,简称Con A)是刀豆(Conavalia gladiata)种子中提取出来的植物凝集素(lectins)。它能与许多多糖形成不溶性的复合物,并能与细胞的多糖、糖蛋白特异结合。它能使血球凝集,凝集性质依赖于同血红细胞表面的多糖相结合的特性。同Con A结合的多糖都含有D-吡喃甘露糖基和D-吡喃葡萄糖基以及类似结构的残基,结合糖以C_3、C_4、C_5的羟基同Con A反应。     

胎儿发育过程中食管上皮糖复合物的改变

采用十种生物素化凝集素 U EA- I、 DBA、 PSA、 BSL、 PNA、 L CA、 RCA- I、SBA、 Con A及 WGA分别对 8W、1 4 W、 2 0 W　 2 8W及 32 W的人胎儿食管上皮进行研究 ,以确定胎儿食管上皮在发育过程中凝集素的结合位点以及结合方式随年龄的变化。结果提示 ,BSL、 RCA- I、 WGA的染色强度及特性不受年龄影响 ,而与之相对应 ,U EA - 1在除 2 0 W以外的食管上皮显色 ;含α- D- Glc NAc的糖复合物 (DBA受体 )只在 2 0 W表达 ;含α- D- man/α- D- Glc的糖复合物 (PSA受体 )则只出现于 32 W;Con A受体虽在胎儿食管上皮各年龄组都表现阳性反应 ,但在 2 8W 时受体在上皮各层细胞中的分布方式及染色强度出现了有意义的变化 ,即腔面细胞染色阴性 ,中层细胞染色强阳性 ,基底细胞弱阳性 ,而在其他时间腔面反应强阳性 ,中层中等阳性 ,基底弱阳性 ;PNA、SBA及 L CA在各年龄组均呈阴性反应。这些结果表明 ,在胎儿食管上皮发育过程中存在着多个凝集素结合位点 ,含 α- L- Fucose、 α- D- Gal NAc、α- D- Man/ α- D- Glc残基的糖复合物在分布方式和 /或量上存在着发育相关性改变 ,即糖复合物的改变具有阶段性。总之 ,本实验资料提示 ,胎儿食管上皮在发育过程中表现出不同的结合位点 ,RCA- 、 BSL、 WGA受体似与年龄变     

N-糖链加工影响里氏木霉形态发生并提高木质纤维素降解能力（英文）

【背景】烟曲霉α-1,2-甘露糖苷酶Msd S在高尔基体中将N-糖链Man8Glc NAc2加工为成熟分泌糖蛋白的糖型Man6Glc NAc2,有研究表明Msd S与烟曲霉的形态发生、细胞壁合成及蛋白质分泌密切相关;与烟曲霉不同的是,里氏木霉的成熟分泌糖蛋白上的N-糖链结构为Man8Glc NAc2,细胞却能正常生长,说明丝状真菌N-糖链的加工具有物种特异性,但其生物学意义不明。【目的】为研究N-糖链加工对里氏木霉细胞生长及蛋白质分泌的影响,本研究将烟曲霉Msd S转入里氏木霉中以改变其成熟分泌糖蛋白的糖型。【方法】构建带有烟曲霉msd S基因的重组质粒并转入里氏木霉中,获得msd S表达菌株Tr-Msd S,分析Tr-Msd S菌株的生长表型、N-糖组、蛋白质分泌途径和纤维素酶活性的变化。【结果】在里氏木霉msd S表达菌株Tr-Msd S中,分泌糖蛋白的主要糖型由出发株的Man8Glc NAc2转变为Man6Glc NAc2,细胞壁组分发生变化,但细胞壁完整性未受影响;与出发株相比,Tr-Msd S菌株产孢、出芽及分枝增多;另外,Msd S的表达还影响蛋白质分泌,在50°C时降解纤维素和β-葡聚糖的能力分别提高9.9%和32.2%。【结论】研究结果表明,N-糖链的加工可影响里氏木霉蛋白质,尤其是纤维素酶的分泌,干扰N-糖链加工可能是提高里氏木酶纤维素酶产量的新策略。     

天花粉凝集素糖结合专一性Ⅱ.

本文报道一些糖类、糖苷、糖蛋白和几种外源凝集素对标记天花粉凝集素和酸处理交联琼脂糖或胎盘细胞膜结合的影响。在低浓度时,所用的糖类中,乳糖是最强的抑制剂,蜜二糖和棉籽糖的抑制能力和乳糖相仿,而纤维二糖、蔗糖、麦芽糖,则无明显影响。三个所用的糖蛋白,它们的抑制活性以下列顺序递减:猪甲状腺球蛋白,人血清转铁蛋白,鸡卵白蛋白。未标记的天花粉凝集素和蓖麻凝集素,两者都专一地和半乳糖结合,它们都能竞争标记天花粉凝集素,而伴刀豆球蛋白A和半夏凝集素则不能竞争。由此,我们推测天花粉凝集素主要是和半乳糖结合,但与乳糖的结合能力最强,故推测其结合部位能容纳半乳糖和另一个单糖。     

天花粉凝集素糖结合专一性Ⅱ.

本文报道一些糖类、糖苷、糖蛋白和几种外源凝集素对标记天花粉凝集素和酸处理交联琼脂糖或胎盘细胞膜结合的影响。在低浓度时,所用的糖类中,乳糖是最强的抑制剂,蜜二糖和棉籽糖的抑制能力和乳糖相仿,而纤维二糖、蔗糖、麦芽糖,则无明显影响。三个所用的糖蛋白,它们的抑制活性以下列顺序递减:猪甲状腺球蛋白,人血清转铁蛋白,鸡卵白蛋白。未标记的天花粉凝集素和蓖麻凝集素,两者都专一地和半乳糖结合,它们都能竞争标记天花粉凝集素,而伴刀豆球蛋白A和半夏凝集素则不能竞争。由此,我们推测天花粉凝集素主要是和半乳糖结合,但与乳糖的结合能力最强,故推测其结合部位能容纳半乳糖和另一个单糖。     

七种二糖拟糖蛋白的制备及其在肝内源性凝集素组织定位中的应用

各种已知结构的糖蛋白是复合糖和糖结合蛋白研究中必需的工具。由于天然糖蛋白中糖链结构的复杂性和不均一性,不适宜作为研究的工具。人工合成拟糖蛋白(neoglycoproteins)为糖的结构和功能的研究及凝集素和其它糖结合蛋白的研究提供了重要工具,同时为内源性凝集素(endogenous lectin,EL)介导的糖蛋白——药物靶向治疗进一步临床使用提供了物质基础。本文报道以牛血清白蛋白(BSA)和     

一种利用DSA-FACE分析寡糖链的方法

目的：优化糖链结构分析方法,满足高通量、高灵敏度和快速分析糖基结构的要求。方法：基于DNA测序仪的荧光糖电泳（DSA-FACE）,将糖蛋白经过糖苷酶酶切获得糖链,并与荧光标记物8-氨基芘基-1,3,6-三磺酸（APTS）进行衍生化反应,标记样品经过Sephadex-G10、HPLC纯化后,用DNA3100测序仪电泳分离,用Genescan3.7软件进行数据分析。结果：利用DSA-FACE技术分析了标准品Man5GlcNAc2和Gal2GlcNAc2Man3GlcNac2,并得到糖蛋白牛核糖核酸酶B的5种N-糖基结构（Man5～9GlcNAc2）。结论：DSA-FACE是分析糖基结构的有效技术手段,能够实现对糖基结构的高通量、高灵敏度和快速分析。     

人工栽培灵芝中多糖的部分理化性质及免疫调节作用

【背景】灵芝是一种广受关注的药食两用真菌,在中国作为传统药材和健康食品已有几千年历史,近年来中国人工栽培灵芝规模扩展迅速,而多糖被认为是灵芝中最主要的活性成分。【目的】分离和纯化人工栽培灵芝子实体多糖,并对其结构和免疫调节活性进行研究。【方法】采用水提醇沉法提取灵芝子实体多糖(GLP),采用DE-52纤维素柱和Sephadex G-100进行多糖的分离纯化,高效凝胶渗透色谱法测定多糖分子量,1-苯基-3-甲基-5-吡啶啉酮(PMP)柱前衍生法测定单糖组成,红外光谱进行结构分析,噻唑蓝(MTT)比色法测定多糖对小鼠脾细胞增殖的影响,同时评价其对RAW 264.7细胞吞噬能力和细胞因子分泌能力的促进作用。【结果】GLP平均分子量为1.93×10~4 Da,单糖组成包含葡萄糖(Glc)、半乳糖(Gal)及甘露糖(Man),占比为Glc:Gal:Man=3.3:1.3:1.0。红外光谱显示,GLP的异头碳为β构型。GLP能直接促进脾细胞的增殖,且能显著增强刀豆蛋白A(Con A)诱导的T淋巴细胞和脂多糖(LPS)诱导的B淋巴细胞的增殖。此外,对于RAW 264.7细胞的吞噬能力及细胞因子分泌具有一定的促进作用。【结论】灵芝多糖可作为一个潜在的免疫调节药物而开发利用。     

亲和层析法分离大鼠脂肪细胞膜上腺苷A_1受体

本文采用腺苷亲和层析法从大鼠脂肪细胞膜上分离出了一种亚基分子量为38kD的腺苷结合蛋白质。此蛋白在SDS-聚丙烯酰胺凝胶电泳上显示单一带,糖蛋白染色阳性;能与[8-~3H]腺苷特异结合(Kd=0.269nmol/L,Bmax=6.05pmol/mg.Pr);结合抑制实验表明它与腺苷A_1受体激动剂R-PIA、A_2受体激动剂NECA和腺苷的亲和力大小顺序为:R-PIA>腺苷>NECA。这表明所分离出的38kD蛋白是大鼠脂肪细胞膜上的腺苷A_1受体。     

地衣霉素对细胞膜表面运铁蛋白受体功能的影响

应用抑制糖蛋白Ｎ－糖链合成的地衣霉素处理ＳＭＭＣ－７７２１人肝癌细胞,３Ｈ甘露糖掺入实验显示细胞膜表面糖蛋白Ｎ－糖链的合成受到显著抑制,但细胞膜表面运铁蛋白受体内吞再循环的过程无显明变化,进一步的研究表明受体与运铁蛋白的亲和力亦无改变,但细胞膜表面运铁蛋白受体数减少。结果提示用地衣霉素处理细胞后,在内质网合成的无Ｎ－糖链的运铁蛋白受体影响其运输到细胞膜表面表达。     

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.     

Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.     

The binding of fucose-containing glycoproteins by hepatic lectins. Re-examination of the clearance from blood and the binding to membrane receptors and pure lectins

The nature of the hepatic receptors that bind glycoproteins through fucose at the non-reducing termini of oligosaccharides in glycoproteins has been examined by three different approaches. First, the clearance from blood of intravenously injected glycoproteins was examined in mice with the aid of neoglycoproteins of bovine serum albumin (BSA). The clearance of fucosyl-BSA was rapid and was not strongly inhibited by glycoproteins that inhibit clearance mediated by the galactose or the mannose/N-acetylglucosamine receptors of liver. The clearance of Fuc alpha 1,3(Gal beta 1,4)GlcNAc-BSA (where Fuc is fucose) was inhibited weakly by either Fuc-BSA or Gal beta 1,4GlcNAc-BSA but strongly by a mixture of the two neoglycoproteins, suggesting that its clearance was mediated by hepatic galactose receptors as well as by a fucose-binding receptor. Second, the binding of neoglycoproteins to a membrane fraction of mouse liver was examined. Fuc-BSA binding to membranes was Ca2+ dependent but was not inhibited by glycoproteins that would inhibit the galactose or the mannose/N-acetylglucosamine receptors. In addition, the binding of Fuc-BSA and Gal beta 1,4GlcNAc-BSA differed as a function of pH, in accord with binding of Fuc-BSA through fucose-specific hepatic receptors. Finally, the binding of neoglycoproteins to the pure galactose lectin from rat liver was examined. Neither Fuc-BSA nor Fuc alpha 1,2Gal beta 1,4GlcNAc-BSA bound the galactose lectin, although Fuc alpha 1,3(Gal beta-1,4) GlcNAc-BSA bound avidly. Taken together, these studies suggest that a fucose-binding receptor that differs from the galactose and the mannose/N-acetylglucosamine receptors may exist in rat and mouse liver.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean)

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I > Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.     

The Conformational Properties of the Glc3Man Unit Suggest Conformational Biasing within the Chaperone-assisted Glycoprotein Folding Pathway

A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcα1-3Manα linkage, formed by trimming of the Glcα1-2Glcα1-3Glcα1-3Manα (Glc₃Man) unit in Glc₃Man₉GlcNAc₂. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc₃ManOMe, Glc₃Man₄,₅,₇GlcNAc₂ and Glc₁Man₉GlcNAc₂ using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc₃ManOMe. Our results show a single conformation for the Glcα1-2Glcα and Glcα1-3Glcα linkages, and a major (65%) and a minor (30%) conformer for the Glcα1-3Manα linkage. Modeling of the binding of Glc₁Man₉GlcNAc₂ to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.     

Interactions of substrate with calreticulin,an endoplasmic reticulum chaperone

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K(b)) and changes in enthalpy of binding (Delta H(b) degrees ) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed.     

小刺猴头菌丝体多糖的结构研究

对小刺猴头过滤掉发酵液的发酵菌丝体,水提和碱提后获得的均一组分多糖HMP-w1.1和HMP-a1.1进行结构性质的研究。结果表明:HMP-w1.1是分子量为36.3 kD的α型吡喃糖,单糖组成为甘露糖(Man),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc);HMP-a1.1是分子量为42.8 kD的β型吡喃糖,单糖组成为甘露糖(Man),半乳糖醛酸(GalUA),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc)。综合高碘酸氧化和Smith降解的试验结果,推断HMP-w1.1的糖苷键构型可能为1→、1→4、1→4,6、1→6、1→2、1→2,6;HMP-a1.1的糖苷键构型可能为1→6、1→2、1→2,6。     

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Le(x) trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Le(x) less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Le(x) at all.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].