Con A-binding Proteins Research Articles

Con A‐binding protein Zn‐α2‐glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility

Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation.

Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.

Evaluation of Caenorhabditis elegans glycoproteins as protective immunogens against Haemonchus contortus challenge in sheep

High levels of protection can be attained against Haemonchus contortus challenge infection in sheep using native antigens isolated from the gut of the adult parasite. However, vaccination with recombinant forms of these antigens, or components thereof, has disappointingly failed to generate similar levels of protection, suggesting that appropriate nematode glycosylation may be a prerequisite for protection. The free-living nematode, Caenorhabditis elegans is closely related to H. contortus and has been shown to share similar glycan moieties. In order to investigate the potentially protective role of these glycan moieties, a complex set of glycoproteins was isolated from C. elegans using ConA-lectin chromatography and their efficacy as immunogens against H. contortus challenge infection evaluated in sheep. Despite the generation of a high titre systemic IgG antibody response to the C. elegans glycoproteins and the ability of these antibodies to bind to the microvillar surface of the gut of H. contortus, no protection against challenge infection was observed. Serum antibodies to the C. elegans glycoproteins cross-reacted with the H. contortus host-protective antigen, H-gal-GP, by ELISA, although the level of cross-reactivity was not of a magnitude considered protective. Qualitative differences were also determined between the glycan epitopes of the C. elegans ConA-binding proteins and those of H-gal-GP, suggesting the presence of H. contortus-specific patterns of glycosylation.

Involvement of a 40-kDa Glycoprotein in Food Recognition, Prey Capture, and Induction of Phagocytosis in the Protozoon Actinophrys sol

A 40-kDa glycoprotein (gp40) was identified as a Con A-binding adhesive substance of the heliozoon Actinophrys sol for immobilizing and ingesting prey flagellates. Isolation and partial characterization of gp40 showed that: 1) gp40 is a major Con A-binding protein of Actinophrys with a molecular weight of 40 kDa, and is stored in secretory granules called extrusomes; 2) gp40 was purified by Con A-affinity chromatography, and the N-terminal amino acid sequence was determined as H2N-KVLK-FEDDFDTFDLQ; 3) prey flagellates became adhered to gp40-immobilized agarose beads; 4) phagocytosis of Actinophrys was induced against gp40-immobilized agarose beads; and 5) solubilized gp40 induced exocytosis of extrusomes and cell fusion of heliozoons. These results indicate that gp40 is a multi-functional secretory protein of Actinophrys, which is required in correct targeting of the heliozoon to food organisms as well as in self-recognition.

Evaluation of immunization with gut membrane glycoproteins of Ostertagia ostertagi against homologous challenge in calves and against Haemonchus contortus in sheep

Peanut and ConA lectins were used as ligands to isolate glycoproteins from detergent extracts of adult Ostertagia ostertagi membranes. As judged by their profiles following SDS-PAGE, these fractions closely resembled the equivalents from Haemonchus contortus which are derived from the nematode intestinal cell microvillar membranes and which are highly protective when used as antigens. Groups of calves were immunized with the peanut and ConA binding fractions of Ostertagia, either as separate or pooled antigens mixed with QuilA as adjuvant. All calves, including controls immunized with adjuvant only, were challenged with a single dose of infective Ostertagia larvae and faecal egg counts were monitored for 5 weeks. In two experiments where the antigen fractions were pooled, moderate (30-50%), but statistically significant reductions in egg output were observed, but the number of worms was not diminished. No significant protection was observed in a third trial where groups of calves were immunized with peanut or ConA binding proteins given separately. Two further trials were conducted in sheep immunized with the same Ostertagia fractions but challenged with Haemonchus. Irrespective of whether they were administered separately or together, the Ostertagia antigens cross protected efficiently against Haemonchus reducing egg counts by between 81% and 97% and worm numbers by between 57% and 84%.

Affinity-purification of concanavalin A-binding ciliary glycoconjugates of starved and feeding Tetrahymena thermophila.

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.

Concanavalin A Directly Stimulates Bone-Resorbing Activity of Osteoclasts and Their Gene Expression of Cathepsin K/OC-2

Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by α-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.

Binding of lectins to novel migration promoters on cardiac mesenchymal cells in the chick.

Chicken serum promotes migration of cardiac mesenchymal cells of chick embryos in vitro. In the present study, migration promotion of unknown migration promoters in chicken serum was examined by using lectins. Cardiac mesenchymal cells of the conotruncal and atrioventricular cushions were cultured on collagen type-I gel with medium including chicken serum. A concentration (100 micrograms/ml) of Concanavalin A (Con A), peanut agglutinin (PNA), pisum sativum agglutinin (PSA), soybean agglutinin (SBA) or wheat germ agglutinin (WGA) was added to the medium. Con A, PSA, and WGA inhibited migration, while PNA and SBA did not affect migration of cardiac mesenchymal cells. WGA inhibited migration in a concentration dependent manner. Preincubation of WGA with specific binding monosaccharides (alpha-D-N-acetylglucosamine and alpha-D-N-acetylneuraminic acid) clearly reduced the inhibition ability of WGA, while preincubation of Con A and PSA with alpha-D-mannose and alpha-D-glucose did not. On the other hand, Con A-binding proteins, eluted from a Con A affinity column with the buffer including alpha-D-mannose and alpha-D-glucose, promoted migration of cardiac mesenchymal cells, as did WGA-binding proteins. These proteins promoted migration in a concentration dependent manner. Western blotting showed that PSA bound the subunits of collagen type-I, but ConA and WGA did not. In migration inhibition assays by monosaccharides, only N-acetylneuraminic acid inhibited migration of cardiac mesenchymal cells. These results suggested that chicken serum contains novel migration promoters for chick cardiac mesenchymal cells. The promoters are proposed to have the terminal N-acetylglucosamine, N-acetylneuraminic acid, and glucose and/or mannose residues.

Secretion of type-1-fimbriae binding proteins from human neutrophil granulocytes.

Granule matrix proteins secreted from human neutrophils after ionomycin stimulation were separated by SDS-PAGE, blotted onto a polyvinylidene diflouride (PVDF) membrane and overlaid with the mannose-binding lectin concanavalin A (Con A) or Escherichia coli bacteria exposing type-I-fimbriae. Four proteins of approximately 30, 40, 70 and 80 kD, respectively, derived from both the azurophil and the specific granules were shown to expose mannose-containing structures by binding of Con A. Such reactivity was also shown for a 90-kD protein from the light membrane fraction enriched in plasma membrane and secretory vesicles. When blots of granule matrix proteins were exposed to type-I-fimbriated bacteria, a total of seven proteins was recognized; four of the five Con A-binding proteins (40, 70, 80 and 90 kD) was detected also by the bacteria in addition to three proteins not detected by Con A (50, 60 and 100 kD). The role of the secreted type-1-fimbriae binding proteins as anti-adhesin candidates is discussed.

Cholesterol microcrystals associated with concanavalin A-binding glycoproteins contribute artifactually to nucleating activity assays.

Concanavalin A (Con A) affinity chromatography is the standard procedure to separate cholesterol nucleating biliary proteins from lipids and pigment. We observed that even after extensive washing following application of bile, lipid contaminants remain. We have determined the contribution of lipid contamination to cholesterol nucleation and assessed a modified procedure to remove lipids from the column. Human gallbladder bile was spiked with [3H]cholesterol and [14C]phospholipid and applied to two sets of Con A-Sepharose columns. One column was washed in the usual manner with Tris-HCl buffer and the other with buffer containing 10 mM taurocholate prior to eluting bound glycoproteins with alpha-D-methylmannopyranoside. Eluted proteins were added to heated abnormal bile at a final concentration of 250 micrograms/ml to study the effect on cholesterol nucleation. A separate aliquot (20 microliter) of the protein solutions was counted for radioactivity. Cholesterol nucleating activity was less in samples from columns washed with 10 mM taurocholate than in samples from columns not washed with the bile salt. Lipid radioactivity was associated with Con A-binding proteins prepared without taurocholate, but not in those prepared with taurocholate wash. Light microscopy revealed the presence of cholesterol microcrystals and vesicles in some Con A-binding protein preparations prepared without a taurocholate wash. However, pellets from ultracentrifuged Con A preparations prepared without a bile salt wash revealed cholesterol crystals in all samples (n = 6). Washing with taurocholate did not affect recovery of protein mass and appearance of bands on SDS-PAGE gel showed an identical pattern in the two groups. This modified procedure to prepare potential nucleating proteins from gallbladder bile should eliminate erroneous results that may arise due to lipid contamination.

A study of protein and glycoprotein syntheses in pre-implantation mouse embryos using mini-2D-electrophoresis, video densitometer scanning and computer-image analysis.

In the present study, proteins and glycoproteins of mouse embryos at 2-cell, morula and blastocyst stages were analyzed. The techniques of 35S-Met incorporation, ConA antiserum-precipitating ConA-binding proteins, mini-2D-electrophoresis, fluorography, video densitometer scanning and the computer-image system were used for analyses. Results of the investigations indicated that proteins and glycoproteins were synthesized by the embryos in a gradual increase manner from 2-cell to blastocyst. A relatively large amount of glycoproteins was synthesized during the morula and blastocyst stages. Two specific small glycoproteins respectively with molecular weights about 6500 and 9000 and PIs at 5.0 and 6.5 were apparently synthesized in the blastocyst but not in the 2-cell or the morula.

Analysis of Con A-binding glycoproteins in synaptosomal membranes.

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51 kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.

Nuclear glycoproteins of hamster liver and kirkman-robbins hepatoma recognized by concanavalin A

1. 1. Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma. 2. 2. The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively. 3. 3. A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed.

Organisation of glycoproteins into plasma membrane domains on Fucus serratas eggs

ABSTRACT Fertilisation in the brown alga Fucus involves the interaction of motile, biflagellate sperm with spherical eggs. The eggs differ from those of animals in not having the equivalent of a vitelline layer, jelly coat or zona pellucida outside the plasma membrane, and in addition they are not surrounded by a cell wall. Previous studies on Fucus eggs have shown that the lectin Concanavalin A (ConA) binds in patches on the egg surface, suggesting that there is a non-uniform distribution of plasma membrane glycoproteins. In this paper we have further investigated the occurrence of domains on the Fucus egg plasma membrane using monoclonal antibodies (mAbs) and the lectins ConA and Fucose Binding Protein (FBP). Confocal laser scanning microscopy (CLSM) has been used to observe the binding of probes to the Fucus egg cell surface. Four mAbs (FS2, FS4, FS5 and FS9) raised to Fucus serratas sperm have previously been shown to cross-react with crude egg membrane vesicles. Three of these mAbs (FS2, FS4 and FS5) have now been shown to bind to the egg cell surface and they recognise glycoproteins which are organised into domains. mAb FS4 labels large areas of the egg surface, whereas mAbs FS2 and FS5 bind to smaller patches. The lectins ConA and FBP also bind to smaller, discrete domains. Western blotting results and competition binding assays have shown that mAbs FS2 and FS5 compete for binding to the same set of glycoproteins, and FS5 is used in subsequent experiments; FS4 also binds to several glycoproteins but produces a different pattern of labelling on Western blots compared to FS5, though there may be some common components. ConA labels a subset of the glycoproteins recognised by mAb FS5, and FBP recognises one major glycoprotein which is also recognised by ConA and FS5. Double labelling experiments using the CLSM, with FITC- and Au-labelled probes, have shown that the regions on the egg surface labelled by FS4 and FS5 are mainly exclusive, with small areas of overlap. There are also areas which are not labelled by either of these antibodies. The domains recognised by mAb FS5 contain smaller areas which are labelled by ConA. Overall the results show that the Fucus egg surface is heterogeneous with different sets of glycoproteins being organised into domains. With the probes used it is possible to distinguish between FS4+ FS5−, FS4− FS5+, FS4+ FS5+ and FS4− FS5” regions. Within the FS5+ domains smaller sets of glycoproteins are recognised by ConA and within these latter regions there are glycoproteins recognised by FBP.

Cell spreading on laminin substrate involves Con A-binding proteins

Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.

Detection of a Concanavalin A binding protein in the mollicute Spiroplasma citri and purification from the plasma membrane

A protein that binds Concanavalin A (Con A) was detected on Western blots of Spiroplasma citri proteins. Its apparent molecular weight was 84000. It was localized in the plasma membrane. Affinity chromatography on Con A-agarose was used to isolate this protein. The glycosylation inhibitor, tunicamycin, inhibits S. citri growth and seems to block the glycosylation of the Con A-binding protein.

Changes in Protein Patterns and Protein Synthesis during Anther Development in Brassica oleracea

Summary To study male gametophyte development in Brassica oleracea L., the various stages of microspores and pollen in fresh anthers were assessed with the use of fluorescent dyes and by cytological observations. Developmental variations in SDS-PAGE protein patterns of anthers and pollen grains were then analysed from the late vacuolate microspore stage to the end of the pollen-maturation period. Total protein staining and concanavalin A-binding glycoprotein detection showed that a specific set of developmental polypeptides appears during the tricellular pollen stages. Protein synthesis was studied by [ 31 S] methionine incorporation, SDS-PAGE, fluorography. Two periods of protein synthetic activity were detected: the first one corresponding to the microspore and bicellular pollen stages; the second one corresponding to the mid, late, mature tricellular pollen stages. During this second period, new polypeptides were synthesized and most of them could be correlated with the developmental polypeptides that appeared in total and Con A-binding protein patterns. These results suggest the occurrence of a metabolic reorientation after the second pollen mitosis, at the time of sperm cell maturation. The biochemical data are discussed in terms of diploid and haploid genome expression.

Detection of tumor-associated membrane proteins in prostate and bladder carcinomas by means of protein blotting.

Analysis of membrane proteins by Western blotting has revealed both overexpression of proteins of molecular weight 10-200 kD (in particular, of proteins of MW less than 43 kD) and increased glycosylation in a xenografted human small cell undifferentiated prostatic carcinoma, and in two xenografted human bladder tumor cell lines compared with preparations from normal human tissue. Of potential functional significance were: a) a 43 kD protein in the bladder line, UCRU-BL-13, which demonstrated increased synthesis and a marked increase in the degree of glycosylation, and b), a 28 kD ConA-binding protein in prostatic tissue which was absent in normal tissue, present in intermediate quantity in a benign hyperplasia and greatly overexpressed in small cell carcinoma. This study demonstrates the utility of the protein blotting/autoradiography technique for the investigation of tumor membrane proteins.

Glycoprotein glycans may not be necessary for the differentiation of skeletal myoblasts

Previous work using glycosylation inhibitors has suggested that high-mannose type but not complex type oligosaccharides on the surface of cells may play a role in the differentiation of skeletal myoblasts. Earlier, we had shown that a concanavalin A-resistant mutant derived from an L6 myoblast line fails to differentiate in a medium containing 10% horse serum. Here we show that one such concanavallin A-resistant mutant (D-1) which was reported to have oligosaccharides of the type Man (3–5)G1cNAc 2, shows significant fusion ability when grown in media containing 1% horse serum. Lowering the serum concentration did not alter the dolichol-phosphate mannosyltransferase activity in D-1 which remained at low levels compared to L6. The incorporation of [ 3H]mannose in D-1 was found to be 60% of L6 in 10% serum whereas in 1% serum the incorporation into D-1 was further reduced to 30% of L6. [ 3H]mannose-labeled ConA-binding proteins isolated from L6 were quantitatively and qualitatively similar in cells grown in either 10 or 1% serum. However, in D-1 cells a further decrease in the ConA-binding ability of these glycoproteins was observed. Biochemical differentiation also occurs in D-1 upon fusion in 1% serum as seen by the increase in mRNA levels of the muscle-specific markers myosin light chain and troponin T. These results suggest the high-mannose type of oligosaccharides may not be involved in myoblast differentiation.

Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X

Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?