Hagen Bretting

Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt)

SummaryThe localization of two carbohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the “spherulous cells”. After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water.Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the mesh work of the spongin fibres of the central axis. They are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.

Chemical and immunochemical studies on the structure of four snail galactans

Abstract The four snail galactans studied are polysaccharides of high molecular weight that are composed entirely of d - and l -galactosyl residues. AaG and CnG, which had not previously been studied, are highly branched galactans composed mainly of (1→3)- and (1→6)-linked galactosyl residues, as shown by the results of periodate oxidation and permethylation studies. On methylation, HpG, CnG, and AaG yielded ∼40% of 2,3,4,6-tetra-, 40% of 2,4-di-, 7–14% of 3,4,6-tri-, and 8–12% of 2,4,6-tri-O-methylGal derivatives. BgG gave equal amounts of tetra- and di-O-methyl derivatives, and 8.5% of 2,4,6-tri-O-methylGal, and 10% was unmethylated Gal, indicating 1, 2, 3, 4, and 6 substitution not previously reported in nature. Antisera to the four galactans showed various degrees of cross-reactivity, indicating structural differences ascribable partially to determinants involving a galactose phosphate and, probably, to the linkage and the position of l -Gal in the molecule. The galactans differed in susceptibility to d -galactose oxidase, and some of the immunochemical observations are most probably attributable to species-specific differences in distribution of linear stretches and branches. The first stages of Smith degradation of HpG and AaG showed a substantial increase in unsubstituted (1→3)- and (1→6)-linked residues. These results, and the appearance of linear stretches within the native galactans preclude the strictly dichotomously-branched structure proposed earlier.

Immunohistochemical studies on the distribution and the function of the d-galactose-specific lectins in the sponge Axinella polypoides (Schmidt)

SummaryThe distribution of the two d-galactose-specific lectins within the sponge tissue of Axinella polypoides was studied by autoradiography and by an immunohistochemical method on paraplast- and cryosections. Both techniques revealed that the lectins are stored inside the vesicles of the spherulous cells. All spherulous cells, regardless of their appearance in the different types of tissue contained the lectins. Antibodies were purified from an antiserum that reacted with both lectin I and lectin II and from the same antiserum rendered monospecific for lectin I. The purified antibodies were used to demonstrate that lectin II is predominantly present in spherulous cells with small vesicles, and lectin I in those with large vesicles.Electron-microscopic studies revealed that the spherulous cells with small vesicles are derived from archaeocytes and transformed into spherulous cells with large vesicles, a process accompanied by the conversion of lectin II to lectin I.Histological investigations showed that the tips of the bush-like, branched sponge lack the central axis, a spongin fiber network that provides support and stability to the sponge tissue. However, the missing spongin network is already preformed by cell bundles that ultimately produce the numerous fiber strands of the central axis. These bundles are composed exclusively of spindle-shaped cells and the spherulous cells.Other areas where production of spongin fibers is expected are also enriched with spherulous cells. These findings and the reaction of lectin-specific antibodies with the spongin fibers indicate that spherulous cells, and thus the lectins, are involved in synthesis of spongin fiber.Sponges lacking spongin fibers, e.g. Aaptos aaptos and Geodia cydonium, produce lectins with different carbohydrate specificity and possess large numbers of spherulous cells.

The occurrence of l-galactose in snail galactans

Abstract The occurrence of l -galactose ( l -Gal) was investigated in twelve preparations of snail galactans from nine different species. l -Gal (11–15%) was found not only in members of the family Helicidae , a highly developed group among the Pulmonata , but was detected also in a galactan of the oldest phylogenetic subclass of snails in the Prosobranchiata . To determine the position of the l -Gal residues, the galactans of Arianta arbustorum , Cepaea nemoralis , and Helix pomatia were methylated and hydrolysed, and the partially methylated Gal derivatives were isolated. Each derivative was O -demethylated and the ratio of d - and l -galactose was determined by g.l.c. of the trifluoroacetylated methyl glycosides using a chiral stationary phase. l -Gal (22–34%) was detected only in the tetra- O -methyl fractions, and hence l -Gal occupies exclusively terminal non-reducing positions.

Investigations on the occurrence of lectins in marine sponges with special regard to some species of the family axinellidae

1. 1. Thirty-two species of eighteen families of sponges were screnned for their content of lectins. Twenty-one produced agglutinins reacting with human and animal red blood cells. Hemagglutiation inhibition tests were carried out with six species of different families to look for possible purification procedures. 2. 2. All species available of the family Axinellidae were compared by Ouchterlony tests, as well as by immuno- and disc electrophoresis. Axinella polypoides and Axinella dissimilis showed close structural relationships, whereas Axinella flustra had only restricted immunological similarities. The other three species displayed no actions with anti-Axinella polypoides antisera. 3. 3. Within the family Axinellidae lectins were detected only in Axinella polypoides and Axinella dissimilis. They were purified and their carbohydrate specificities studied, which are directed against dGal moieties in terminal non-reducing position. dGal β linked is of higher inhibitory potency than g linked dGal residues. A major difference between the lectins of both species is seen in inhibition studies with dGalNAc, which is 250 times more active against Axinella dissimilis than Axinella polypoides.

Comparative investigations on the amino-acid sequences of different isolectins from the sponge Axinella polypoides (Schmidt)

The sponge Axinella polypoides contains four different D-galactose binding lectins and one, termed lectin IV, which is specific for hexuronic acids. Only the D-galactose binding lectins were investigated in this study. The complete amino-acid sequence of lectin I, the main component in the crude extract was determined. Lectin I is a homodimer and each subunit comprises 144 amino acids with a M(r) of 15,847 +/- 10, as calculated from the sequence data and determined by mass spectrometry. Each subunit contains one intrachain disulfide bridge between positions 4 and 46. Of lectin II, only the first 49 amino acids of the NH2-terminal end were analysed. This part has 29 amino acids in common with lectin I, including a cysteine residue at position 4, also suggesting an intrachain loop in a identical position as in lectin I. The molecular mass of its subunit is 16,235 +/- 10 Da. Only the first 15 NH2-terminal amino acids of lectins III and V could be sequenced. Lectin V was identical to lectin II in all positions, whereas lectin III showed only 5 residues identical to lectins I or II. Thus, lectins I, II and III are derived from three different genes, whereas lectin V may either be a proteolytic cleavage product, or result from different splicing events or may be derived also from a separate gene. Neither of the four lectins showed any similarity to known lectin sequences of animal or plant origin.

The reactivity of galactose oxidase with snail galactans, galactosides and D-galactose-composed oligosaccharides

The enzymic oxidation of snail galactans, of their first and second Smith degradation products and of some structurally related polysaccharides was studied. Lymnaea stagnalis galactan after one cycle of Smith degradation reacted best and native Helix pomatia galactan was almost inactive. Investigations on the structural requirements for oligosaccharides to bind to galactose oxidase showed that the branched tetrasaccharide, Gal-beta-1----6-[Gal-beta-1----3]-Gal-beta-1----1 L-Gro, in the terminal nonreducing position was the most complementary structure in the native galactan to associate with the enzyme. All nitrophenyl alpha-galactosides reacted better, and the ortho-form was 10-times more potent compared with this tetrasaccharide, indicative of the involvement of a hydrophobic region in binding. However, the beta-linked isomers were only equally or less reactive than galactose. The enzymic oxidation determined colorimetrically by transferring the peroxide formed to o-dianisidine ceased at a maximum typical for each substrate and independent of the reaction time. When the absolute turn-over rates for ortho- and para-nitrophenyl alpha-galactoside and for the beta-isomer were determined by HPLC, it could be demonstrated that the oxidation had not finished at the maximum of the colour reaction, but proceeded until the substrate was consumed. The initial speed of the colour reaction paralleled the absolute oxidation rate.

Isolation and characterization of a lectin from the snail Biomphalaria glabrata and a study of its combining site

The hemagglutinins from the spawn of the water snail Biomphalaria glabrata were isolated by affinity chromatography on hog gastric mucin coupled to Sepharose 4B. The N-acetyl-D-glucosamine eluate (0.1 M) was fractionated further on Bio-Gel P-300, yielding two fractions. Fraction 1 had an Mr of 350 000 and displayed one band in immunoelectrophoresis, but was heterogeneous in discontinuous electrophoresis. It agglutinated human red blood cells with A1 and B specificity at concentrations of 12 and 72 micrograms nitrogen/ml, respectively. Fraction 2 had an Mr on gel filtration of 67 000 and was homogeneous in immuno- and polyacrylamide electrophoresis, and in isoelectrofocusing. It is composed of three subunits with Mr of 17 000 and one smaller subunit of 15 000. This fraction (lectin I) is a glycoprotein containing 6% hexoses and 2.5% hexosamines. For minimal agglutination of human A1 and B red blood cells 2.4 and 72.0 micrograms nitrogen/ml, respectively, of lectin I were required. O red blood cells were not agglutinated. Lectin I precipitated well with a human blood group substance of A1 specificity, moderately with a B- and poorly with an H-substance. Precipitin-inhibition studies revealed that among other sugars N-acetylneuraminic acid was the most potent inhibitor. Immunofluorescence studies confirmed the good interaction of lectin I with receptors of A1 and B erythrocytes and the failure of lectin I to attach to O-erythrocytes. Since N-acetylneuraminic acid is present on the cell surface of all human erythrocytes, it cannot be the dominant part of the receptor for the B. glabrata lectin I, despite its effectiveness as an inhibitor.

Structural investigations of the galactan of the snail Lymnaea stagnalis

Abstract A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d -galactose and 0.9% of nitrogen, but neither l -galactose nor phosphate groups. The [α]D20 values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β- d -Gal-(1→1)- l -Gro, β- d -Gal-(1→3)-β- d -Gal-(1→1)- l -Gro, β- d -Gal-(1→6)-β- d -Gal-(1→1)- l -Gro, β- d -Gal-(1→6)- d -Gal-β- d -Gal-(1→3)-β- d -Gal-(1→1)- l -Gro, β- d -Gal-(1→3)-[β- d -Gal-(1→6)]-β- d -Gal-(1→1)- l -Gro, β- d -Gal-(1→3)-β- d -Gal-(1→6)-β- d -Gal-(1→1)- l -Gro, and β- d -Gal-(1→3)-β- d -Gal-(1→3)-β- d -Gal-(1→1)- l -Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.

Studies on the relationship between lectins from Axinella polypoides agglutinating bacteria and human erythrocytes

Abstract A new lectin, called Axinella IV, with bacteria agglutinating activity was detected in the crude extract of the sponge Axinella polypoides. Immunofluorescence studies and immunoelectrophoresis revealed no cross-reactivities with the already known lectins Axinella I. II, and III. d -Galactose and oligosaccharides with d -Galactose in terminal nonreducing position are inactive in agglutination inhibition tests demonstrating also different specificity of Axinella IV lectin when compared with the three aforementioned lectins.