Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220- and 180-kD components by means of lectins

Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA₁ reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction. © 1995 Wiley-Liss, Inc.     

Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane

When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin-conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC-PNA-labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA-labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC-PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.     

Gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk,Unio elongatulus,binds to and induces acrosome reaction in human spermatozoa through a protein kinase C-dependent pathway

In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewisa-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

Biochemical maturation of Spam1 (PH‐20) during epididymal transit of mouse sperm involves modifications of N‐linked oligosaccharides

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spam1 protein, which is associated with structural and functional differences of the molecule. Spam1 is uniformly distributed over the surface of the head of caput sperm while in caudal sperm, light and confocal microscopy demonstrate that it is localized to the anterior and posterior regions. The hyaluronidase activity of Spam1 in acrosome‐intact caput sperm was significantly lower (4.3‐fold; P < 0.0001) than that of caudal sperm. The increase in enzymatic activity in caudal sperm is accompanied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at ∼74 kDa and a minor band at ∼67 kDa; while for the cauda there was a major band at ∼67 kDa and minor bands at ∼70 and ∼56 kDa. Additionally, the bands from caput sperm were 4.9 to 7.7‐fold less intense than those from caudal sperm. This decreased affinity for the polyclonal anti‐Spam1 suggests the presence of different surface characteristics of the molecule from the two epididymal regions. Computer analysis of the protein structure from Spam1 cDNA sequence reveals four putative N‐linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After endoglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of ∼56 kDa, the size of the membrane‐anchored polypeptide backbone. Based on the difference in size and intensity of the Spam1 bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spam1 during epididymal maturation is regulated by deglycosylation. Mol. Reprod. Dev. 52:196–206, 1999. © 1999 Wiley‐Liss, Inc.     

Isolation and Partial Characterization of a Glycoprotein Complex with Sperm-Receptor Activity from Ciona intestinalis Ovary1

Sperm-egg interaction in the ascidian Ciana intestinalis is mediated by a fucosyl-glycoprotein (FP) component of the egg vitelline coat. FP are responsible for sperm binding, sperm activation and the acrosome reaction. In this paper we report a detailed biochemical and functional characterization of FP purified from the ovaries by affinity chromatography. thic component with sperm receptor activity is a high molecular weight glycoprotein complex (>10⁷) with a protein-carbohydrate ratio of 2:1, which inhibits the binding of the spermatozoa to the vitelline coat and induces sperm activation and the acrosome reaction. Exhaustive proteolytic digestion of FP yields high molecular weight glycopeptides (> 4×10⁵), which contain N-acetylgalactosamine, fucose, galactose and rhamnose. These glycopeptides retain some receptor activity, thus raising the question of the involvement of the polypeptide backbone in the sperm-egg binding process. However, the glycopeptide fraction fails to induce the acrosome reaction: we suggest that the polypeptide fraction plays a role in the induction of sperm activation and the acrosome reaction.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

FLOW‐CYTOMETRIC CHARACTERIZATION OF THE CELL‐SURFACE GLYCANS OF SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM SPP.)1

Symbiodinium spp. dinoflagellates are common symbionts of marine invertebrates. The cell‐surface glycan profile may determine whether a particular Symbiodinium is able to establish and maintain a stable symbiotic relationship. To characterize this profile, eight Symbiodinium cultures were examined using eight glycan‐specific fluorescent lectin probes. Confocal imaging and flow‐cytometric analysis were used to determine significant levels of binding of each probe to the cell surface. No significant variation in glycan profile was seen within each Symbiodinium culture, either over time or over growth phase. No cladal trends in glycan profile were found, but of note, two different Symbiodinium cultures (from clades A and B) isolated from one host species had very similar profiles, and two other cultures (from clades B and F) from different host species had identical profiles. Two lectin probes were particularly interesting: concanavalin A (ConA) and Griffonia simplicifolia‐II (GS‐II). The ConA probe showed significant binding to all Symbiodinium cultures, suggesting the widespread presence of cell‐surface mannose residues, while the GS‐II probe, which is specific for glycans possessing N‐acetyl groups, showed significant binding to six of eight Symbiodinium cultures. Other probes showed significant binding to the following percentage of Symbiodinium cultures examined: wheat germ agglutinin (WGA), 37.5%; peanut agglutinin (PNA), 50%; Helix pomatia agglutinin (HPA), 50%; phytohemagglutinin‐L (PHA‐L), 62.5%; soybean agglutinin (SBA), 50%; and Griffonia simplicifolia‐IB₄ (GS‐IB₄), 12.5%. This study highlights the complexity of cell‐surface glycan assemblages and their potential role in the discrimination of different dinoflagellate symbionts by cnidarian hosts.     

Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (>10⁶), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.     

A proof‐of‐concept study in engineering synthetic protein for selective recognition of substrate‐free polyubiquitin

Similar to substrate‐conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961–1969]. They designed a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnFCUBP domain, and a linkage‐selective UBA domain, to specifically recognize unanchored Lys48‐linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate‐free Lys48‐linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48‐polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.     

Proteomic profiling of N‐linked glycoproteins identifies ConA‐binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N‐linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty‐eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high‐levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA‐binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, “ConA‐binding proCD” (ConA‐pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5–5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA‐pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA‐pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Multiple and multivalent interactions of novel anti-AIDS drug candidates, sulfated polymannuronate (SPMG)-derived oligosaccharides, with gp120 and their anti-HIV activities

Sulfated polymannuronate (SPMG), a novel anti-AIDS drug candidate, combats HIV-1 infection mainly by binding to gp120 protein with high affinity. To explore the structural basis of this anti-HIV-1 action, size-defined oligosaccharides were prepared by semi-synthesis or separated from native SPMG. In this study, a series of homogeneously sized SPMG fragments are evaluated for their capacity to bind rgp120 using surface plasmon resonance (SPR) analysis. The minimum SPMG fragment size that interacts with rgp120 is a hexasaccharide. Additionally, binding capacity increases with the molecular size of oligosaccharides, with the affinity of large fragments (> or = 15-16 saccharides) approaching that of full-sized SPMG. Competitive inhibition and stoichiometric analyses disclose that SPMG oligos bind to multiple binding sites on gp120. Sugar chains longer than 15-16 saccharide residues (SPMG) display multivalent interactions, with one sugar chain binding to two or three gp120 molecules. Consistent with binding data, a positive correlation exists between the size of SPMG oligosaccharides and their anti-HIV activity. The octasaccharide is established to be the minimal active fragment inhibiting syncytium formation and lowering the P24 core antigen level in HIV-IIIB-infected CEM cells. Alternatively, about 50% anti-HIV activity was observed for 15-16 saccharides, whereas a 19-20-saccharide fragment displayed anti-HIV activity equivalent to native SPMG. The structures of the unique minimum hexasaccharide specifically recognized by gp120 and the minimum octasaccharide combating HIV-IIIB infection were representatively structured as [ManA (2s)beta1-4 ManA(2s/3s)]n.     

Recognition of defined epitopes by affinity‐purified anti‐immunoglobulin Fab autoantibodies isolated from HIV‐infected humans

Infection of humans with HIV‐1 has previously been independently shown to result in the generation of autoantibodies (AAbs) reactive with immunoglobulin Fab fragments (Heidelberg), and with autoantibodies to T‐cell receptors (TCRs) (Tucson). Here, we carry out epitope mapping studies of affinity‐purified AAbs to Fab fragments prepared from individual HIV‐positive patients for their capacity to bind recombinant constructs and peptide‐defined epitopes modeling TCR and Ig light chains. Some affinity‐purified autoantibodies reacted strongly with TCRs expressed by intact T‐cells, and recombinant V_α/V_β constructs as well as with certain synthetic peptide epitopes. The binding reactions of affinity‐purified AAbs of individual patients were distinct, and the AAb preparations consisted of populations of polyclonal lgs as reflected in specificity and isotype. AAb pools from individual patients all bound particular regions of TCR and Ig chains defined by comprehensive peptide synthesis including the CDR1 and Fr3 segments of the variable domains and the joining segment/switch peptide. In addition, other reactivities to restricted regions of α, β and λ light chains were documented. These results substantiate the cross‐reactivity of TCR and Ig–Fab determinants, and are consistent with the hypothesis that autoantibodies arising as a consequence of HIV infection can have an immunomodulatory role. Copyright © 1999 John Wiley & Sons, Ltd.     

Characterization of a pectic polysaccharide from the leaves of Diospyros kaki and its modulating activity on lymphocyte proliferation

Pectin is a group of carbohydrate polymers constructing the primary cell walls and the middle lamella of terrestrial plants. Herein, we demonstrated the structure and immunomodulatory activity of the major pectic polysaccharide DL‐3B₂ isolated from the leaves of Diospyros kaki. Based on composition analysis, methylation analysis, two‐step acid hydrolysis, lithium‐mediated selective degradation, ¹³C NMR spectroscopy, and electrospray ionization mass spectrometry, DL‐3B₂ was found to contain an α‐1, 4‐linked galacturonic acid (GalA) backbone with some insertions of α‐1, 2‐linked rhamnose residues. The arabinan‐ and arabinogalactan‐side chains were attached to O‐4 of the rhamnose residues, whereas the linear arabinoxylan was probably linked to O‐3 of the GalA residues. Immunological tests in vitro showed that DL‐3B₂ could help stimulate lipopolysaccharide‐induced B lymphocyte proliferation, but not ConA‐induced T lymphocyte proliferation, and that the arabinose residues play a role in maintaining this immunological activity. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 649–656, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Complete structure determination of N‐acetyl‐D‐galactosamine‐binding mistletoe lectin‐3 from Viscum album L. album

The primary structure of the B chain of the N‐acetyl‐D ‐galactosamine‐recognizing mistletoe lectin‐3 (ML‐3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC‐separation and Edman degradation and confirmation of the peptide sequences by MALDI‐MS. ML‐3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N‐glycosylation sites at positions Asn⁹⁵ and Asn¹³⁵ of the lectin, were determined by a combination of glycosidase treatment and MALDI‐MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type‐II RIPs, although there are remarkable differences in the D ‐galactose‐specific mistletoe lectin‐1B chain. The recently published primary structure of the mistletoe lectin‐3A chain 1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML‐3. The model demonstrates, unequivocally, that ML‐3 is a member of the type‐II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML‐1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin‐3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

The Sperm‐surface glycoprotein,SGP, is necessary for fertilization in the frog,Xenopus laevis

To identify a molecule involved in sperm‐egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm‐surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti‐SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti‐SGP antibody recognized large molecules, with molecular masses of 65–150 kDa and minor smaller molecules with masses of 20–28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle‐binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm‐egg membrane binding and is responsible for the establishment of fertilization in Xenopus.