Modulation of PP2A activity by Jacalin: is it through caveolae and ER chaperones?

Plant lectins have been reported to affect the proliferation of different human cancer cell line probably by binding to the specific carbohydrate moieties. In the present study, Badan labeled single cysteine mutant (present in the caveolin-1 binding motif) of jacalin (rJacalin) was found to penetrate the target membrane, indicating a protein-protein or protein-membrane interaction apart from its primary mode of binding i.e. protein-carbohydrate interaction. Further, Jacalin treatment has resulted in the movement of the GFP-Caveolin-1 predominantly at the cell-cell contact region with much restricted dynamics. Jacalin treatment has resulted in the perinuclear accumulation of PP2A and dissociation of the PHAP1/PP2A complex. PP2A was found to act as a negative regulator of ERK signaling and a significant decrease in the phosphorylation level of MEK and AKT (T308) in A431. In addition, we have also identified several ER resident proteins including molecular chaperones like ORP150, Hsp70, Grp78, BiP of A431 cells, which were bound to the Jacalin-sepharose column. Among various ER chaperones that were identified, ORP150 was found to present on the cell surface of A431 cells.     

Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin

Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal相似文献   

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.     

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin,jacalin

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1β, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.     

Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis

Although previous studies have shown that autoantigens such as Hsps have been implicated by induction of an autoimmune process in the development of atherosclerosis, the exact role of anti-Hsp70 antibody in atherosclerosis is unknown. In the present study, the levels of anti-Hsp70 autoantibodies and oxidized low density lipoprotein (OxLDL) were all significantly increased, and they were strongly correlated in an atherosclerosis model. After the endothelial cells were incubated with 20 μg/mL OxLDL for 12 h at 37 °C and followed by 90 min recovery, Hsp70 positive staining of OxLDL-treated endothelial cells was observed on the cell surface in immunostaining and flow cytometric analysis. This membrane Hsp70 was not from culture supernatant Hsp70 and binding of extracellular Hsp70 but was defined as endothelial surface membrane Hsp70. Furthermore, only in the OxLDL-treated group, but not in the untreated group, ⁵¹Cr-labeled endothelial cells were lysed by anti-Hsp70 antibody (BD091, Ig^AS) in the presence of either complement or peripheral blood mononuclear cells. Control antibodies, including Ig^Nor, mAb to Hsp70 (SPA-810), and mAbs to Factor VIII, α-actin, and CD3 showed no cytotoxic effects. In conclusion, anti-Hsp70 antibodies could be reacting with the endothelial surface membrane Hsp70 induced by OxLDL and were able to mediate endothelial cytotoxicity. There is a possibility that a humoral immune reaction to endothelial surface membrane Hsp70 may play an important role in the pathogenesis of atherosclerosis.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

Binding of Porphyrins by the Tumor-Specific Lectin, Jacalin [Jack Fruit (Artocarpus integrifolia) Agglutinin]

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes thetumor-associated T-antigenic disaccharide structure,Gal13GalNAc. Porphyrins and their derivatives are currently used asphotosensitizers in photodynamic therapy to treat malignant tumors. In thisstudy, the interaction of several free base porphyrins and their metalderivatives with jacalin is investigated by absorption and fluorescencespectroscopy. Each lectin subunit was found to bind one porphyrin moleculeand the association constants were estimated to be in the range of2.4×10³M^–1 to 1.3×10⁵M^–1 at room temperaturefor the interaction of different porphyrins with jacalin. These values arein the same range as those obtained for the interaction of monosaccharidesto jacalin. Both free lectin and lectin saturated with the specificsaccharide were found to bind different porphyrins with comparable bindingstrength indicating that porphyrin binding takes place at a site differentfrom the sugar binding site. Further, both anionic and cationic porphyrinswere found to interact with the lectin with comparable affinity, clearlyindicating that the charge on the porphyrin does not play any role in thebinding process and that most likely the interaction is mediated byhydrophobic forces. These results suggest that jacalin and other lectins maypotentially be useful for targeted delivery of porphyrins to tumor tissuesin photodynamic therapy.     

Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK(1) cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Instead of heat shock stress, TGF-beta1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-beta1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-beta1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.     

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.     

Jacalin: an IgA-binding lectin

We previously reported that seeds of Artocarpus integrifolia (jackfruit) contain a lectin, which we call jacalin, that is both a potent T cell mitogen and an apparently T cell-independent activator of human B cells for the secretion of immunoglobulins. During the above experiments we noted a massive precipitation in cell cultures stimulated with greater than or equal to 100 micrograms of lectin. In this paper, we show that the precipitate is formed after the interaction of jacalin and the serum protein added to the culture medium. More importantly, we demonstrate that IgA is probably the major serum constituent precipitated by the lectin and that no IgG or IgM can be detected in the precipitates. In secretions such as colostrum, IgA is the only protein precipitated by jacalin. On the basis of this specificity we describe a simple and reliable affinity chromatography procedure for the purification of both human serum and colostrum IgA. Jacalin is a D-Gal binding lectin and should be a useful tool for studying of serum and secretory IgA.     

Birth defects and anti-heat shock protein 70 antibodies in early pregnancy

Abstract It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223-406) microg/mL compared to 150 (88-207) microg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.     

Major stress protein Hsp70 interacts with NF-kB regulatory complex in human T-lymphoma cells

Polypeptides belonging to the Hsp70 major stress protein family and to the NF-kB/Rel multi-functional regulatory complex are known to be involved in cellular defense mechanisms. It was suggested that both systems may interact in cells that respond to injuring stimuli. To check this, Molt4 human lymphoma cells were heated at 43°C for 15 min and, after a 6 h post-shock recovery period, the cells were activated with phorbol ester or bacterial lipopolysaccharide. It as found that mild heat shock caused a substantial increase of the intracellular Hsp70 content with the concomitant suppression of NF-kB complexes, though the latter was properly activated in non-stressed cells. After a 24 h period of being inactive the complex fully recovered its activity and p65 and c-Rel subunits migrated to the nucleus. This new active period lasted even longer than that in non-heated control cells. As this suggested the existence of a Hsp70-related mechanism of NF-kB/Rel complex retention in cytoplasm, we carried out immunoprecipitation with the use of anti-Hsp70 and anti-Rel antibodies. All three Rel family members p65, c-Rel, p50, but not their precursors and IkBα inhibitory protein were shown to co-precipitate with the stress protein and anti-Hsp70 antibodies from both heated and non-heated cells. We conclude that the Hsp70 stress protein may confer a new mechanism of NF-kB regulation in cells affected by elevated temperature or other factors related to the cellular response to stress.     

Extracellular heat shock protein 70 (HSPA1A) and classical vascular risk factors in a general population

Atherosclerosis is a chronic inflammatory and autoimmune disease. Candidate molecules/autoantigens include heat shock proteins (HSPs); Hsp70 (HSPA1A) is one of the best studied HSPs. Various studies have shown a correlation between extracellular Hsp70 (eHsp70) and anti-Hsp70/anti-Hsp60 antibody concentration and development of atherosclerosis. A random sample of 456 people aged 40–60 (218 males, 234 females) was studied to investigate the prevalence of traditional vascular risk factors and eHsp70 and anti-Hsp70/anti-Hsp60 antibodies levels, according to the risk of vascular disease. Task Force Chart was applied for classification. Subjects were divided into three groups: G0 (with no vascular risk factor or a risk lower than 5%), n = 239; G1 (moderated 10–20% risk, who do not have established disease) n = 161; and G2 (established atherosclerosis disease) n = 52. eHsp70 and anti-Hsp70 were significantly lower in the atherosclerosis group (group 2) with respect to the other groups. Disease-free people showed the highest anti-Hsp60 concentration compared with the other two groups. A correlation has not been demonstrated between the concentrations of circulating Hsp70 (HSPA1A), anti-Hsp70, and anti-Hsp60 and classical vascular risk factors and C-reactive protein. Low levels of eHsp70 and anti-Hsp70 antibodies should be considered as candidate FRV. Simultaneous decrease of eHsp70 and anti-Hsp70 antibodies would be explained by circulating immune complex formation, and both could be proposed as biomarkers for the progression of atherosclerotic disease. Levels of circulating anti-Hsp60 antibodies may constitute a marker of inflammation in atherosclerosis.     

Plasma levels of Hsp70 and anti-Hsp70 antibody predict risk of acute coronary syndrome

Although immune reactions against heat shock proteins have been implicated in the pathogenesis of atherosclerosis, conflicting associations between Hsp70, anti-Hsp70 antibody and coronary heart disease (CHD) have been reported. This study assessed whether there is a significant association between extracellular human Hsp70, anti-Hsp70 antibody and acute coronary syndrome (ACS) and stable angina (SA), and examined dynamic changes in Hsp70 and anti-Hsp70 antibody levels induced by acute myocardial infarction (AMI). Plasma Hsp70 and anti-Hsp70 antibody levels in 291 patients with ACS (179 AMI, 112 unstable angina), 126 patients with SA and 417 age and sex-matched healthy subjects, and in 40 patients after admission for AMI, and on day 2, 3, and 7 after the onset of AMI were determined using enzyme-linked immunosorbent assays. Hsp70 levels were significantly higher in ACS and SA and anti-Hsp70 antibody levels were only markedly lower in ACS than controls. After adjustment for traditional CHD risk factors, increasing levels of Hsp70 were significantly associated with an increased risk and severity of ACS (P for trend < 0.001), whereas increasing levels of anti-Hsp70 antibody were associated with a decreased risk of ACS (P for trend = 0.0003). High levels of Hsp70 combined with low levels of anti-Hsp70 antibody had a joint effect on the risk of ACS (OR, 5.14, 95% CI, 3.00-8.79; P < 0.0001). In patients with AMI, Hsp70 levels decreased rapidly from days 1-7 after onset, whereas anti-Hsp70 antibody levels increased in patients with AMI. These findings suggest that higher Hsp70 levels or lower anti-Hsp70 antibody levels are independently associated with a higher risk of ACS. Higher Hsp70 levels and lower anti-Hsp70 antibody levels combine to further increase this risk.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

木菠萝凝集素结合部位糖的特异性

用定量免疫沉淀法和定量免疫沉淀抑制法研究了从木菠萝(Arthrocarpus integrifolia)种子提取的凝集素(jacalin)结合部位糖的特异性。Jacalin最强烈地沉淀含有DGalβ1→3DGalNAc结构的无活性抗冻糖蛋白,不同程度非特异地沉淀各种血型物质。研究发现凝集素结合部位对DGalβ→3DGalNAc有最高特异性。最强抑制剂是DGalβ1→3DGalNAcal→φNO_2,其抑制活性分别比DGalNAc和DGal高380倍和1000倍。对于各种甲基化或对硝基酚化的糖苷以及寡糖,除methylaDGalNAc_f外,仅α-构型表现出抑制活性,所有β-构型的糖苷均无抑制活性,jacalin结合部位对糖的结合是构型依赖性的。     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.