Combination of fetuin and trehalose in presence of low glycerol has beneficial effects on freeze-thawed ram spermatozoa.

BACKGROUND: Freeze-thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze-thawing is suggested to improve sperm parameters. OBJECTIVES: The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant-related gene expressions and DNA integrity during the freeze-thawing process, in low glycerol concentration. METHODS: Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris-based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15). RESULTS: G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05). CONCLUSIONS: Co-supplementation of tris-based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze-thawing process is recommended.

Is hydroxypyridonate 3,4,3-LI(1,2-HOPO) a good competitor of fetuin for uranyl metabolism?

Uranium is widespread in the environment, resulting both from natural occurrences and anthropogenic activities. Its toxicity is mainly chemical rather than radiological. In the blood it is transported as uranyl UO22+ cation and forms complexes with small ligands like carbonates and with some proteins. From there it reaches the skeleton, its main target organ for accumulation. Fetuin is a serum protein involved in biomineralization processes, and it was demonstrated to be the main UO22+-binder in vitro. Fetuin's life cycle ends in bone. It is thus suspected to be a key protagonist of U accumulation in this organ. Up to now, there has been no effective treatment for the removal of U from the body and studies devoted to the interactions involving chelating agents with both UO22+ and its protein targets are lacking. The present work aims at studying the potential role of 3,4,3-LI(1,2-HOPO) as a promising chelating agent in competition with fetuin. The apparent affinity constant of 3,4,3-LI(1,2-HOPO) was first determined, giving evidence for its very high affinity similar to that of fetuin. Chromatography experiments, aimed at identifying the complexes formed and quantifying their UO22+ content, and spectroscopic structural investigations (XAS) were carried out, demonstrating that 3,4,3-LI(1,2-HOPO) inhibits/limits the formation of fetuin-uranyl complexes under stoichiometric conditions. But surprisingly, possible ternary complexes stable enough to remain present after the chromatographic process were identified under sub-stoichiometric conditions of HOPO versus fetuin. These results contribute to the understanding of the mechanisms accounting for U residual accumulation despite chelation therapy after internal contamination.

Proteomic analysis of plasma proteins after low-level laser therapy in rats.

The laser radiation absorbed by cells induces production of reactive oxygen species (ROS), followed by the development of oxidative stress. Proteins are major targets for ROS due to their abundance in biological systems. The aim of the present pilot study was to examine the effects of transcutaneous laser blood irradiation (TLBI), i.e., low-level laser therapy (LLLT) at 830 nm on plasma proteome in Wistar rats. Rats were irradiated in the heart area (i.e. coronary arteries) daily (i.e., for 9-day period), by commercially available GaAsAl diode laser (Maestro/CCM, Medicom Prague, Czech Republic, lambda=830 nm, power density 450mW/cm(2), daily dose 60,3 J/ cm(2), irradiation time 134 sec). The comparison of blood plasma proteome from irradiated and non-irradiated rats was performed utilizing 2D electrophoresis followed by MALDI TOF/TOF mass spectrometry. LLLT led to a quantitative change in the acute phase proteins with antioxidant protection i.e., haptoglobin (log(2) fold change (FC)=3.5), hemopexin (log(2) FC=0.5), fibrinogen gamma (log2 FC=1.4), alpha-1-antitrypsin (log(2) FC=-2.2), fetuin A (log2 FC=-0.6) and fetuin B (log2 FC=-2.3). In comparison to conventional biochemical methods, the changes in protein levels in blood plasma induced by LLLT offer a deeper insight into the oxidative stress response.

Herbal and polymeric approaches for liver-targeting drug delivery: novel strategies and their significance.

The liver is a vital organ present in vertebrates, which performs many functions including detoxification, protein synthesis and production of various bio-chemicals which are very important for digestion. A large number of serious liver disorders affect millions of people worldwide which are very difficult to treat properly despite many efforts. There are several factors which are responsible for liver injuries, include plants (Crotalaria Senecio Heliotropium Symphytum officinale), drugs (analgesic and antibiotics), industrial toxins (mercury and lead), water, alcohol and so on. Herbal medicinal preparations can be used for the treatment of a large number of human liver disorders like cirrhosis, hepatitis, carcinomas, etc. Indian Medicinal Practitioner's Co-operative pharmacy and Stores (IMPCPS) approved herbal-based systems (Unani, Siddha and Ayurveda) for the treatment of various chronic liver disorders. Different types of the receptors are found on the surface of hepatocytes, Kupffer cell, hepatic stellate cell and sinusoidal endothelial cells, etc., which can be used for achieving liver targeting. These receptors bind to different types of ligands (galactosylated, lactobionic acid, asialofetuin, etc.) which can be used in the formulation to achieve targeted delivery of the drug. Various novel particulate approaches (liposomes, niosomes, nanoparticles, micelles, nanosuspensions, etc.) can be used to enhance the targeting efficiency of systems to receptors found on the surface of different cells present in the liver. In this review, we focused on the status of liver targeting via herbal and nanotechnology inspired formulation approaches.

Antioxidative effects of cysteamine, hyaluronan and fetuin on post-thaw semen quality, DNA integrity and oxidative stress parameters in the Brown Swiss bull.

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm), hyaluronan (0.25, 1 mg ml(-1) ) and fetuin (5, 10 mg ml(-1) ) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo-osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml(-1) of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml(-1) of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze-thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml(-1) of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml(-1) of hyaluronan and 7.5 mm of cysteamine after the freeze-thawing process (P < 0.001).

Improvement of an enzyme linked lectin assay to determine recombinant mistletoe lectin I.

Extracts from Viscum album leaves, with mistletoe lectin I (ML I) as the main therapeutic agent, are commonly used as an immunomodulating adjuvat in tumour therapy. Because of its popularity against cancer and the possibility for a better standardisation a recombinant ML I (rML I) was developed by Eck et al. To improve the sensitivity of an already established enzyme linked lectin assay (ELLA) for rML I human haptoglobins with different phenotypes (1.1, 2.1 and 2.2) are used to replace asialofetuin as matrix. To determine the carbohydrate binding specificity of the tested glycoproteins the ELLA was realised in the presence of the competitive carbohydrate beta-d-lactose. It could be shown that using haptoglobin phenotype 1.1 instead of asialofetuin improved the test results markedly. Both, the limit of detection and the limit of quantitation were decreased by an order of magnitude. However, this positive result was obviously accompanied by a loss in specificity of the test. The specificity of asialofetuin for rML I is almost six-fold higher than for the tested haptoglobins. Thus, in cases where high specificity and less sensitivity values for rML I is required the ELLA should still be run with asialofetuin as binding partner.

AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium.

Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).

Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus).

A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [Osmerus (Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannose-specific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca(2+) ion, and this activity was inhibited by l-rhamnose and d-galactose derivatives, but not by d-mannose and d-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins.

Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica).

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel beta-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of approximately 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca(2+) ions. SDS/PAGE showed that native eCL-1 and eCL-2 have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.

The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.

Characterization of recombinant and plant-derived mistletoe lectin and their B-chains.

Mistletoe lectin I (pML) and its isoforms ML II and III constitute the active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy. The heterodimeric disulfide-linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP). Recently, the sequence coding for the mistletoe lectin prepro-protein was identified and the existence of a single intron-free gene was shown [Eck, J., Langer, M., Möckel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, H. (1999) Eur. J. Biochem. 264, 775-784]. The aim of this study was to prepare pure and homogeneous rMLB-chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asialofetuin was able to compete with binding of both chains (50% at 0.8 microM). The specificity of the B-chains to lactose was more distinct with halfmaximal competition at 4.9 mM (pMLB) and > 90 mM (rMLB), respectively. Furthermore, in a coassociation process rMLA- and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML-heterodimer. The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared. Cytotoxicity was determined using MOLT-4 cells and enzymatic rRNA N-glycosidase activity was measured in a coupled transcription/translation assay. The IC50 values of the two heterodimers were similar in both assays; rMLB-chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved at 1.6 mM (rML) and 1.8 mM (pML). Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML. Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activity and the rRNA N-glycosidase activity, are prerequisites for the cytotoxic effects on target cells.

A new high molecular weight agglutinin from garlic (Allium sativum).

Erythrocyte agglutination by lectins from Allium sativum was inhibited only by mannose of the sugars tested. However, asialofetuin was more effective inhibitor of agglutination as compared to mannose. This led to the use of an asialofetuin-silica affinity column to isolate agglutinins of 110 and 25 kDa (ASA110 and ASA25). While ASA25 is a dimeric protein comprising of subunits of 12.5 and 13.0 kDa, ASA110 is a glycoprotein of two identical subunits of 47 kDa. ASA110 revealed to have a high content of aspartic acid, glycine, leucine and serine but low content of cysteine and methionine. It contains 14 residues of neutral sugars in addition to 43 residues of hexosamines per mole of lectin and requires metal ions for its functional conformation. Serological cross-reactions with other species showed some common epitopes of ASA110 and ASA25 present in A. porrum, A. ascalonicum, Narcissus alba, PHA and Con A but not in A. cepa. ASA110 with CHO cells indicated it to be weakly cytotoxic with LD50 of 160 microg/ml.

Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin.

Plant and animal lectins bind and cross-link certain multiantennary oligosaccharides, glycopeptides, and glycoproteins. This can lead to the formation of homogeneous cross-linked complexes, which may differ in their stoichiometry depending on the nature of the sugar receptor involved. As a precisely defined ligand, we have employed bovine asialofetuin (ASF), a glycoprotein that possesses three asparagine-linked triantennary complex carbohydrate chains with terminal LacNAc residues. In the present study, we have compared the carbohydrate cross-linking properties of two Lac-specific plant lectins, an animal lectin and a naturally occurring Lac-binding polyclonal immunoglobulin G subfraction from human serum with the ligand. Quantitative precipitation studies of the Lac-specific plant lectins, Viscum album agglutinin and Ricinus communis agglutinin, and the Lac-specific 16 kDa dimeric galectin from chicken liver demonstrate that these lectins form specific, stoichiometric cross-linked complexes with ASF. At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexes formed with both plant lectins and the chicken lectin. With increasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexes formed with the lectins irrespective of their source or size. The naturally occurring polyclonal antibodies, however, revealed a different cross-linking behavior. They show the formation of 1:3 ASF/antibody (per Fab moiety) cross-linked complexes at all concentrations of ASF. These studies demonstrate that Lac-specific plant and animal lectins as well as the Lac-binding immunoglobulin subfraction from specific stoichiometric cross-linked complexes with ASF. These results are discussed in terms of the structure-function properties of multivalent lectins and antibodies.

A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin.

Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens. We report here a novel Ca(2+)-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins. P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains. The latter contained two potential Ca(2+)-binding sites that may be involved in carbohydrate binding. The overall sequence of P35 was highly homologous to porcine ficolins alpha and beta. Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region. P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides. Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.

[Soluble beta-galactoside-binding lectins]. / Lectinas solubles beta-galactosídicas.

Animal lectins are classified on the basis of structural and functional studies in two types: the C-type, characterized by their dependence on calcium ions and the S-type which are not calcium-dependent, but thiol-dependent. In this late one, a group has been extensively studied as the S-Lac type. They are extracted with saline buffers added with lactose in presence of thiol agents, and constitute a family of structurally related protein which contain a series of conserved amino acids. They specifically bind to complementary glicoconjugates, and their biosynthesis and localization are developmentally regulated. Their role could be related to several biological activities in different organs.

[Study of plant lectins from Viscum album using monoclonal antibodies]. / Issledovanie rastitel'nykh lektinov iz omely beloi s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

[Chimeric toxin of the A-subunit of viscumin and the B-subunit of ricin]. / Khimernyi toksin A-sub''edinitsy viskumina i B-sub''edinitsy ritsina.

A chimeric toxic protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. The chimeric protein was more toxic for Jurkat cells than native mistletoe lectin I, but not as effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain from degradation during delivery from the cell surface to the place where it is translocated into the cytosol.

Differences in the cross-linking activities of native and recombinant Erythrina corallodendron lectin with asialofetuin. Evidence for carbohydrate-carbohydrate interactions in lectin-glycoprotein complexes.

A previous study showed that several multivalent galactose-specific lectins including the 14-kDa lectin from calf spleen and the lectins from Erythrina indica, Erythrina cristagalli, and soybean agglutinin formed specific cross-linked complexes with the glycoprotein asialofetuin (ASF) [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472]. In the present study, we have used quantitative precipitation analysis to compare the cross-linking activities of the Gal/GalNAc-specific lectin from Erythrina corallodendron (ECorL) and the recombinant protein (rECorL) which lacks the covalently linked heptasaccharide chains of the native lectin, with ASF. At low concentrations of ASF relative to the lectin, native dimeric ECorL binds to each of the three terminal Gal residues of the three N-linked triantennary chains of ASF and precipitates as a cross-linked complex at a ratio of 1:9 ASF/lectin (monomer). With increasing concentrations of ASF, the 1:9 complex changes to a 1:3 ASF/lectin complex, and at higher ASF concentrations, a 1:1 cross-linked complex forms. However, rECorL, which possesses the same specificity and binding affinity as the native lectin, forms only the 1:9 and 1:3 ASF/lectin complexes. Other Erythrina lectins examined, all of which have covalently attached carbohydrate and are structurally similar to ECorL, show the same cross-linking behavior as native ECorL. On the other hand, the dimeric 14-kDa calf spleen lectin which lacks covalently attached carbohydrate forms only 1:9 and 1:3 cross-linked complexes with ASF [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472].(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the adenylylation of liver plasma membrane-bound proteins by plant and mammalian lectins.

Liver plasma membrane contains four major (130-kDa, 120-kDa, 110-kDa and 100-kDa) sialic acid-containing glycopolypeptides that are able to undergo adenylylation, as well as phosphorylation (San José et al. (1990) J. Biol. Chem. 265; 20653-20661). To gain insight into the regulation of these processes, lectins are employed to probe the extent of influence of their interaction with membrane fractions for these reactions. We demonstrate that the beta-galactoside-specific lectins from bovine heart and mistletoe at low concentrations inhibit the adenylylation of this set of plasma membrane glycopolypeptides. The extent of phosphorylation of these polypeptides is also reduced although to a lesser degree. Concanavalin A, too, inhibits the adenylylation of the plasma membrane glycopolypeptides, although higher concentrations of this lectin were required, whereas wheat germ lectin has only a very small inhibitory effect. The adenylylable polypeptides were isolated by concanavalin A-agarose chromatography upon elution with mannose. In agreement with this result, control experiments with a panel of neoglycoproteins indicate that mannose residues appear to be required for the concanavalin A-induced inhibition of the adenylylation. Neoglycoproteins containing mannose 6-phosphate, lactose, fucose, or sialic acid instead of mannose lack the ability to protect the adenylylation from the inhibitory action of concanavalin A. In contrast, none of the above-mentioned neoglycoproteins, nor asialofetuin, nor galactose-containing saccharides protect the adenylylation against the inhibitory effect of both the mistletoe and bovine heart lectins, emphasizing the importance of either high affinity carbohydrate ligands in the overall process, or other ligand sites for the lectins beside carbohydrates to affect the regulation of the adenylylation system.