Siva Kumar Nadimpalli

Purification in high yield and characterisation of the galactose‐specific lectin from the seeds of snake gourd (Trichosanthes anguina)

The galactose‐specific lectin present in the seeds of snake gourd (Trichosanthes anguina) was purified in high yield by affinity chromatography on cross‐linked guar gum. The purified snake gourd seed lectin (SGSL) yielded a single symmetrical peak on gel filtration with an Mr of 62 kDa and gave a single band in PAGE under non‐denaturing conditions. In SDS‐PAGE, SGSL gave a single band of Mr 53 kDa in the absence of β‐mercaptoethanol, and two bands of Mr 32 and 23 kDa in its presence, indicating that the lectin is a heterodimer in which the subunits are linked by a disulphide bridge. The lectin gave a single precipitin line in immunodiffusion experiments with antiserum raised against the purified SGSL. No cross‐reactivity was found between SGSL and antiserum raised against the Momordica charantia lectin and vice versa, suggesting that the two lectins are antigenically dissimilar. Haemagglutination‐inhibition data show that MeβD‐Gal is the best monosaccharide inhibitor of SGSL and indicate that an equatorial hydroxyl at C‐2 and axial hydroxyl at C‐4 in the pyranose form are important binding loci for the lectin.

Purification in high yield and characterisation of a new galactose-specific lectin from the seeds of Trichosanthes cucumerina

Abstract Ten Cucurbitaceae species have been investigated for the presence of seed lectins of which only two species, Trichosanthes cucumerina and T. palmata, have displayed agglutination activity which was inhibited by galactose. The lectin from T. cucumerina seeds has been purified in high yield (∼350 mg lectin/100 g deshelled seeds) by affinity chromatography on cross-linked guar gum. The purified T. cucumerina seed lectin (TCSL) moved as a single symmetrical peak on gel filtration on Sephadex G-150 with an apparent molecular weight of 62 (±5) kDa and gave a single band on PAGE under non-denaturing conditions. In SDS-PAGE, TCSL gave a single band at 69 kDa in the absence of 2-mercaptoethanol, whereas in the presence of 2-mercaptoethanol two bands corresponding to 41 and 22 kDa were observed, suggesting that the lectin is made up of two non-identical subunits that are linked by one or more disulphide bridges. TCSL is a glycoprotein with about 3.0% covalently bound neutral sugar. The lectin cross-reacted with rabbit antiserum raised against the Trichosanthes anguina (snake gourd) seed lectin (SGSL), yielding a single precipitin line and SGSL cross-reacted with the anti-TCSL antiserum raised in rabbits, indicating that the two lectins are antigenically very similar. This was further confirmed by Western blot analysis where the two subunits of TCSL were found to react with both anti-TCSL and anti-SGSL antisera and vice versa. On the other hand, TCSL did not cross-react with the antiserum raised against Momordica charantia lectin and vice versa, suggesting that these two cucurbit seed lectins are antigenically dissimilar. Haemagglutination-inhibition data show that TCSL is specific for the β-anomer of galactose with MeβGal and lactose being the best mono- and disaccharide inhibitors, respectively.

Mannose 6-phosphate receptors (MPR 300 and MPR 46) from a teleostean fish (trout).

Mannose 6-phosphate receptors (MPRs) are known to occur in mammals, birds, reptiles and amphibians. Here we provide evidence for the presence of two MPRs in fish, the earliest vertebrates. Using phosphomannan-Sepharose affinity chromatography, MPR 300 was purified from liver membrane extract of trout. The purified trout liver MPR 300 showed similar electrophoretic mobility as the goat liver receptor and a pH optimum of 7.0 for binding to phosphomannan. The presence of MPR 46 in fish was shown by metabolically labelling embryonic fish cells (Xiphophorus) and immunoprecipitation with an antibody against the cytoplasmic tail of human MPR 46 (anti-MSC1). This antibody had recently been shown to immunoprecipitate MPR 46 also from reptiles and amphibians.

Identification of the Putative Mannose 6-Phosphate Receptor (MPR 46) Protein in the Invertebrate Mollusc

Mannose 6-phosphate receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403–409). In the present study we demonstrate that the unio also contains the putative mannose 6-phosphate receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & Physiol. 123B:261–265). The authenticity of the receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [35S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with mannose 6-phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same receptor protein.

Identification of histidine residues in the sugar binding site of snake gourd (Trichosanthes anguina) seed lectin.

Chemical modification studies have been carried out on the galactose‐specific lectin (SGSL) purified from snake gourd (Trichosanthes anguina) seeds. Modification of the imidazole side chains of histidine residues with ethoxyformic anhydride resulted in a complete loss of activity of the lectin. A total of 9.5 (±0.7) histidine residues were modified per dimer of Mr 55,000 when the reaction was carried out for 2 hours. A partial protection was observed when the modification was done in the presence of 0.1M galactose, indicating that histidine residues are directly involved in the sugar‐binding activity of the lectin. Complete recovery of the lectin activity was observed when the modification was reversed by treatment with hydroxylamine. In immunodiffusion experiments, the histidine‐modified lectin reacted with rabbit antiserum raised against the native SGSL forming a precipitin line, indicating that the loss of activity upon modification was not due to changes in the overall conformation of the lectin. Modification of the side chains of lysine, cysteine and tyrosine residues did not result in any change in the activity of SGSL.

Mannose 6-Phosphate Receptor Proteins from Reptiles and Amphibians: Evidence for the Presence of MPR 300 and MPR 46

Abstract Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate Asterias rubens (Echinodermate): biochemical and functional characterization of MPR 46 protein

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr(−/−) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.

Purification of α‐mannosidase activity from Indian lablab beans

Seeds of Dolichos lablab var. typicus (Indian lablab beans contain a glucose/mannose specific lectin that was affinity purified on Sepharose mannose columns in our laboratory. The unbound fraction from this matrix showed α‐mannosidase activity. In the present study this has been purified to homogeneity by a combination of ion‐exchange, hydrophobic chromatography and gel filtration. Purified α‐mannosidase had an apparent molecular weightof 195,000±5,000 with 4.5% carbohydrate. On SDS‐PAGE under reducing conditions, the enzyme dissociated into two major bands corresponding to Mr 66,000 and Mr 44,000. An antibody to the well studied jack bean α‐mannosidase cross‐reacts with the enzyme from the lablab beans suggesting antigenic similarity between these two legume mannosidases.

The early vertebrate Danio rerio Mr 46000 mannose-6-phosphate receptor: biochemical and functional characterisation

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, γ-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish.

Molecular cloning of goat mannose 6-phosphate receptors, MPR 300 and 46

Mannose 6-phosphate receptor proteins (MPR 300 and 46) are type 1 transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. In a recent study we have purified both receptors from goat liver and raised antibodies. An ELISA method was developed that allowed quantification of both receptors in different tissues of goat and chicken and an immuno-affinity method was also developed to purify the receptors. In the present study to understand the structural similarities of the goat receptors to other known receptor proteins, we have prepared cDNA clones for both receptors by RT-PCR approach. A partial cDNA clone (1.368 kb) for the MPR 300 protein, and a full length cDNA clone (0.84 kb) for the MPR 46 protein were obtained. MPR 300 exhibits typical conserved cassette structure in the amino terminal domain similar to other known vertebrate MPR proteins with the conserved cysteine residues and the ligand binding arginine residue in the third domain. MPR 46 exhibits high degree of sequence homology to other known MPR proteins with the conserved cysteine residues, the transmembrane domain and the cytoplasmic tail. mRNA transcript size for both receptors were comparable with that of other vertebrates. Published in 2004.