Human IgM molecules that bind staphylococcal protein A contain VHIII H chains

Staphylococcal protein A (SPA) is a bacterial membrane protein which has distinct binding sites for Fc gamma and for the Fab region of some IgM, IgG, IgA, and IgE molecules. This study establishes a structure-function correlation responsible for the binding of Ig Fab regions to SPA. Binding of 24 isolated human monoclonal IgM proteins to SPA was measured in a solid phase RIA. VH and V kappa subgroups of each IgM were determined by SDS-PAGE, transfer blotting, and detection with antisera prepared against specific first framework region peptides. Binding to SPA was seen with 10 of 11 VHIII IgM, but none of the 7 VHI or 6 VHII. Similarly, polyclonal IgM fractionated on a SPA-Sepharose CL4B column showed nearly complete partition of VHIII molecules into the SPA-binding fraction, and VHI and VHII subgroup proteins into the fall-through. We conclude that SPA binding is a functional marker for VHIII H chains in human IgM molecules.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

甘露糖结合凝集素基因多态性与结核病的相关性研究进展

结核病的发生是结核分枝杆菌与宿主之问相互作用的结果.并受环境因素影响.人们时结核病的研究重点逐渐转移到其宿主的易感基因上来,其中甘露糖结核凝集素(MBL)基因是当今国内外研究的热点之一.MBL通过结合病原生物表面的甘露糖等糖基受体而直接介导调理吞噬作用和/或通过MBL途径激活补体,在机体的固有性免疫防御中发挥重要作用.本文主要介绍MBL基因多态性与结核病易感性的研究进展.     

Human IgM monoclonal proteins that bind 3-fucosyllactosamine, asialo-GM1, and GM1

Analysis of monoclonal human Ig that occur in association with lymphoproliferative diseases has provided valuable information about antibody structure and idiotypes. We analyzed 940 human sera that contained monoclonal IgM proteins for their ability to bind to four carbohydrate epitopes. Ten sera bound asialo-GM1, five of these sera also bound GM1, 10 bound to 3-fucosyllactosamine (3-FL), and one each bound to levan and galactan. Although the antibody activity in each serum was associated with a single L chain isotype, both kappa and lambda isotypes were represented among the proteins that bound to asialo-GM1 and to 3-FL. Some antibodies against asialo-GM1 were highly specific for this compound, whereas others cross-reacted with the structurally related gangliosides GM1 and GD1b. The antibodies to asialo-GM1 also varied considerably in their ability to lyse liposomes that contain asialo GM1. An association of IgM mAb against gangliosides with peripheral neuropathies has been reported recently, but only one of five patients whose antibodies reacted with GM1 ganglioside had a neuropathy. The antibodies that bound 3-FL exhibited narrower specificity, and less than 10% cross reactivity was noted with structurally related carbohydrates. The frequency of monoclonal proteins that bound 3-FL and asialo-GM1, approximately 1:100 sera for each specificity, was surprisingly high in view of the fact that both of these epitopes are expressed in human tissues. We suggest that these antibodies may be poly-specific and/or that the subset of B lymphocytes that synthesizes these anti-carbohydrate antibodies undergoes malignant transformation more frequently than other B lymphocytes.     

Rapid identification of recombinant Fabs that bind to membrane proteins

Crystallographic studies of membrane proteins have been steadily increasing despite their unique physical properties that hinder crystal formation. Co-crystallization with antibody fragments has emerged as a promising solution to obtain diffraction quality crystals. Antibody binding to the target membrane protein can yield a homogenous population of the protein. Interantibody interactions can also provide additional crystal contacts, which are minimized in membrane proteins due to micelle formation around the transmembrane segments. Rapid identification of antibody fragments that can recognize native protein structure makes phage display a valuable method for crystallographic studies of membrane proteins. Methods that speed the reliable characterization of phage display selected antibody fragments are needed to make the technology more generally applicable. In this report, a phage display biopanning procedure is described to identify Fragments antigen binding (Fabs) for membrane proteins. It is also demonstrated that Fabs can be rapidly grouped based on relative affinities using enzyme linked immunosorbent assay (ELISA) and unpurified Fabs. This procedure greatly speeds the prioritization of candidate binders to membrane proteins and will aid in subsequent structure determinations.     

Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms

Schistosoma mansoni is a blood-dwelling trematode which can persist for several years in the vessels of the human host. The schistosomal surface has been extensively characterized by lectin binding studies, revealing the carbohydrate composition of the worm's tegument. Using fluorescent and scanning electron microscopy we demonstrate that the surface carbohydrates of cercariae and adult worms are the binding ligands for mannanbinding lectin (MBL), a serum protein that is part of the innate immune system. An in vitro complement activation assay with C1q-deficient complement suggests that MBL, in association with the serine proteases MASP-1 and MASP-2, is capable of fixing complement components on the schistosomal tegument and activating the complement cascade via the "MBL pathway." MBL is constitutively expressed by hepatocytes and present in the blood at a stable level. Since it is also a weak acute-phase protein and therefore upregulated in an acute-phase response we investigated the serum MBL levels in patients infected with Schistosoma sp. and in healthy control persons. An enzyme-linked immunosorbent assay indicated no differences between the two groups. Although our results suggest an involvement of MBL activated complement in vitro, its role in vivo remains to be clarified.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Identification and characterization of porcine mannan-binding lectin A (pMBL-A), and determination of serum concentration heritability

Mannan-binding lectin (MBL) is an innate immune collectin present in the serum of humans and many farm animals. This oligomeric pattern-recognition protein effectively binds to the glycoconjugate arrays present on the surfaces of microorganisms and activates the complement system to enhance pathogen killing and clearance. MBL deficiency is often associated with immunodeficiency in humans. Although two MBLs (MBL-A and MBL-C) have been characterized in various species, the identity of porcine MBL (pMBL) was not clearly defined. In this study, we purified an MBL from porcine serum by mannose affinity, ion exchange, and size exclusion chromatography and determined many of its characteristics. Based on the N-terminal sequence, multiple sequence alignment, and relative affinities to various carbohydrate ligands, we propose that the MBL purified in this study is pMBL-A. We have generated antibodies to this protein and established an immunoassay to quantify pMBL-A in serum. Using this assay, we found breed differences in pMBL-A concentration distributions and heritability estimates. In the Duroc breed (n=588), pMBL-A concentrations show a unimodal distribution with a mean of 9,125 ng/ml. In contrast, the pMBL-A concentration distributions in the Landrace breed (n=533) show three distinct mean values: 301, 2,385, and 11,507 ng/ml. Furthermore, heritability calculations based on an additive genetic variance model with no fixed effects indicate that serum pMBL-A concentration is highly heritable in the Landrace (h ²=0.8) but not in the Duroc breed (h ²=0.15). These genetic differences may be useful in selecting breeding pigs for improved disease resistance.     

Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores

Here we show that human Zeste White 10 (Zw10) and Rough deal (Rod) are new components of the mitotic checkpoint, as cells lacking these proteins at kinetochores fail to arrest in mitosis when exposed to microtubule inhibitors. Checkpoint failure and premature mitotic exit may explain why cells defective for hZw10 and hRod divide with lagging chromosomes. As Zw10 and Rod are not conserved in yeast, our data, combined with an accompanying study of Drosophila Zw10 and Rod, indicate that metazoans may require an elaborate spindle checkpoint to monitor complex kinetochore functions.     

Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)

Background

Lectin immunosorbant assays (LISAs) have been widely used for analyzing protein glycosylation. However, the analysis of serum samples by LISAs could suffer from high sample-dependent background noise. The aim of this study is to develop a differential lectin immunosorbant assay (dLISA) with reduced background interferences.

Methods

For the analysis of protein glycosylation, dLISA establishes a dose–response curve for every serum sample. The sample is split into five aliquots. Four aliquots undergo differential removal of the glycoprotein of interest by immunoprecipitation. Then, all five aliquots are subject to two measurements: protein by immunoassay and protein glycans by LISA. A dose–response curve is established by plotting glycans signals on the y-axis and protein levels on the x-axis for all the aliquots. Slope of the curve, calculated by linear progression analysis and expressed as fluorescence per concentration of protein, is used for the measurement of protein glycosylation in the serum sample.

Results/conclusions

To demonstrate the feasibility of the dLISA approach, we used recombinant, fucosylated tissue inhibitor of metallopeptidase 1 (TIMP-1) as the target glycoprotein. Magnetic beads based TIMP1 immunoassay and TIMP-1 UEA LISA were developed for the measurement of TIMP1 protein and terminal α1, 2 fucosylated glycans on TIMP1, respectively. Serum samples supplemented with differentially fucosylated recombinant TIMP-1 were used to demonstrate that the slopes measured the TIMP-1 fucosylation, and were less prone to background interference.     

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.     

The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.     

Analysis of plasma proteins that bind to glycosaminoglycans

Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-alpha-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties.     

NPC2, the protein deficient in Niemann-Pick C2 disease, consists of multiple glycoforms that bind a variety of sterols

Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.     

Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14-20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k(on)) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (DeltaCp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.     

Evidence that lithium ions can modulate lectin stimulation of lymphoid cells by multiple mechanisms

Inhibition of PHA stimulation of hamster lymph node cells by theophylline, DBcAMP, or indomethacin or PHA stimulation of thymocytes by theophylline or DBcAMP was partially reversed by addition of 10 mM LiCl to the cultures. Addition of LiCl to Con A-stimulated lymphoid cells treated with the same reagents did not alter the inhibition. In contrast, addition of 10 mM LiCl to Con A-stimulated cultures enhanced the inhibition induced by the Na,K ATPase inhibitor, ouabain. Like LiCl, this latter inhibitor was found to be effective in modulating stimulation only if added early in the culture.These data support the hypothesis that LiCl can modulate lymphocyte responsiveness at the level of cyclic nucleotide metabolism, as exemplified by PHA stimulation, or at the level of the Na,K ATPase, exemplified by Con A stimulation. The site of involvement of Li⁺ ion would appear to be dependent on the biochemical nature of the stimulating signal.