An isolated RBP with at least one of the following characteristics: a) a molecular weight of approximately 65-70 kDa and more preferably 66-69kDa; b) a pI of greater than 10 or less than 3; c) a dissociation constant of approximately 15 x 10-6 when bound to the rhamnose moiety of solamargine; d) adapted to bind to a rhamnose affinity column prepared according to example 1 and under the conditions set out therein ; e) adapted to be eluted from the column in example 1 with a 100mM rhamnose solution; f) insoluble in aqueous solution; and g) soluble in highly denaturing buffers containing greater that approximately 2% surfactant

Rhamnose binding protein

We have examined the existence of lectin activity in saline-extracted fractions of fish roe. Cell agglutination was observed in 13 of 19 fish species (68%), especially in Cypriniformes and Salmoniformes. A rhamnose-binding lectin was isolated from Osmerus eperlanus mordax (Olive rainbow smelt, Osmeridae, Salmoniformes) roe by affinity chromatography and ion-exchange chromatography. This lectin, termed OML, agglutinated rabbit and human ABO blood group erythrocytes and sarcoma 180 murine tumor cells. OML-induced hemagglutination was inhibited by L-rhamnose and α-galactosyl oligosaccharides. The apparent molecular weight of OML was estimated to be 26kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. The N-terminal 29 amino acid sequence of OML was determined as follows; VTTDIXEGQQATLNXGSSVINVVSANYGR (X: not determined).

Studies on fish roe lectins: Distribution, purification and characterization

L'invention concerne une proteine fixant le rhamnose RBP presentant au moins une des caracteristiques suivantes: a) une masse moleculaire d'approximativement 65-70 kDa et idealement de 66-69kDa; b) un pl superieur a 10 ou inferieur a 3; c) une constante de dissociation d'approximativement 1,5 x 10-6 lors d'une fixation a la fraction rhamnose de solamargine; d) une aptitude a se fixer a une colonne d'affinite de rhamnose preparee selon l'exemple 1 et dans les conditions y figurant; e) une aptitude a etre eluee a partir de la colonne dans l'exemple 1 avec une solution a 100 mM de rhamnose; f) une insolubilite en solution aqueuse; et g) une solubilite dans des tampons hautement denaturant contenant plus d'approximativement 2 % de tensioactif

Proteine fixant le rhamnose

A novel 36kDa rhamnose-binding lectin isolated from Plecoglossus altivelis roe (PA 36), a probe for L-rhamnose and α-D-galactosyl nonreducing termini, bound to and agglutinated murine tumor cells rather than human type B erythrocytes. PA 36 was purified by DEAE-cellulose ion exchange and rhamnose-Sepharose affinity chromatography. The most effective saccharide in the agglutination inhibition assay was L-rhamnose. The α-D-galactosyl containing saccharides, such as melibiose, raffinose and stachyose, were more effective than the β-D-galactosyl containing saccharide, lactose. Lectin binding glycoproteins were determined by SDS-PAGE and blotting, followed by reaction with horseradish peroxidase-labeled PA 36. The glycoproteins ranging in molecular weight from 66, 000 to 72, 000 and deriving from murine tumor cells and bands 4.1 and 4.2 deriving from human type B erythrocytes were PA 36-binding glycoproteins. These results raise the possibility that nonreducing terminal α-D-galactosyl-containing glycoproteins composed of heterogeneous carbohydrate chains may be present on the surface of a number of different murine tumor cells as well as on human type B erythrocytes.

Kouya Kusakari

Papers

Comparative studies of the agglutination of tumor cells and erythrocytes by Plecoglossus altivelis (Ayu fish) roe lectin