Different Glycosubstances and Galactans in the Albumin Gland and Eggs of Achatina fulica

z. Immun.-Forsch. vol.

152, pp. 220-230 (1976)

Department of Immunobiology, Medical University Clinic, Cologne (Federal Republic of Germany), and Department of Biochemistry, University of Lagos (Nigeria)

Different Glycosubstances and Galactans in the Albumin Gland and Eggs of Achatina fulica G.

UHLENBRUCK,

G.

STEINHAUSEN,

and H. A.

KAREEM

With 8 Figures Received May 24,1976· Accepted July 17,1976

Abstract In saline extracts from the eggs and the albumin gland of the snail Achatina fulica 3 different forms of glycosubstances have been found by using heterophile precipitins from different sources: 1. An alkali·stable galactan reacting with the anti.galactans from Axinella poly.

poides sponge and from the clam Tridacna maxima (Tridacnin) and with Concanavalin A. 2. Another glycosubstance giving cross-reactions with a second precipitin from Axinella polypoides, with the lectin from Ricinus communis, with murine myeloma anti.galactan, with pneumococcus Type XIV antiserum and with Tridacnin. 3. The second precipitin from Axinella polypoides detects a third glycosubstance, which reacts with the lectins from Abrus precatorius and wheat germ (Triti­ cum vulgaris). The importance of different snail galactans for the detection of myeloma anti-galactan specificity is discussed.

Introduction

Galactans are galactose-containing polysaccharides, which have first been discovered in the albumin gland and eggs of snails (1). They are poorly or not at all antigenic in most animals so far tested. Differ­ ent forms of galactans occur also in plants, microorganisms and in vertebrates too (2). Anti-galactans represent a group of heterophile precipitins from mainly invertebrate, but also from plant and vertebrate origin (2). They react strongly with certain non-reducing ~-galactosyl endgroups in these galactans (2). The immunochemical importance of the galactan - anti-galactan reaction can be deduced from essential facts:

Glycosubstances and galactans in Achatina fulica . 221

a) very potent anti-galactans are represented by certain mouse mye­ loma IgA proteins (3), b) different anti-galactans from different origin show, when compared in their competitive precipitin reaction with different galactans, closely related or nearly identical combining specificities (4). c) anti-galactans give quite a number of cross-reactions with carbo­ hydrate structures in bacterial (lipo-)polysaccharides or in certain glycoproteins, for instance human serum glycoproteins after remo­ val of neuraminic acid (5). A most useful and strong anti-galactan, the specificity of which is predominantly directed against a ~-(1-6)-galactobiose structure, is Tridacnin from the haemolymph of the elongate bivalve clam Tridacna maxima [RODING (6)]. It has previously already been mentioned (7), that Tridacnin detects a different form of galactan in the eggs of Achatina fulica. A second anti-galactosido precipitin fraction is avail­ able through the work of GOLD et al. (8) and BRETTING and REN­ WRANTZ (9) and was included in our investigations. In this communication, we want to report that, with 3 different anti-galactans, 3 different galactans or glycosubstances can be found in extracts of the albumin gland and eggs of the snail Achatina fulica. The heterophile reagents, listed in this paper, can also be used as serological tools when investigating snail eggs from different origin.

Material and Methods Precipitins from invertebrates a) Tridacnin: This precipitin represents more than 50% of the haemolymph proteins from Tridacna maxima. It was enriched and concentrated as de­ scribed before (7). b) Axinella precipitins: The raw extract from Axinella polypoides was used as described by BRETTING (10). c) Limulus polyphemus haemolymph was kindly supplied by Dr. E. COHEN, Roswell Park Memorial Institute, Buffalo, N.Y., USA.

Precipitins from plants a) b) c) d)

Abrus precatorius } Saline extracts of the seeds were used. Ricinus communis Wheat germ agglutinin from Calbiochem (Triticum vulgaris) Con A from Calbiochem (Concanavalin A).

Precipitins from vertebrates a) Anti-pneumococcus Type XIV antiserum from horse was kindly supplied by Dr. J. L. HENDRY, New York, State Department of Health Laboratories. b) Ascites from mice bearing IgA myeloma J 539 was kindly given to us by Dr. MICHAEL POTTER, NIH, USA. The precipitin was purified according to EICHMANN et al. (4).

222 .

G. UHLENBRUCK, G. STEINHAUSEN,

and H. A.

KAREEM

Achatina fulica extracts Crude saline extracts were used from the eggs and the albumin glands from Achatina fulica. The material was shipped by air in a dried state and obtained from districts of Nigeria and Madagascar, Africa. Helix pomatia galactan was used like in previous papers of this series (2, 4, 5, 6, 11). Agar gel diffusion and electrophoresis was performed according to GAUWERKY et al. (11).

Results

1. Electrophoretic heterogeneity of Achatina fulica galactan A sample of the saline extract was submitted to agar gel electro­ phoresis in order to locate the galactan component. Here it first turned out, that the extract contained 2 galactans, which are preci­ pitated by Tridacnin (Fig. 1).

Fig. 1. Agar gel electrophoresis of the crude Achatina fulica extract. Always the same picture was obtained whether the extract was from the eggs or from the albumin gland of the snail. Upper and lower well: crude extract Middle well: extract after alkali-treatment In both troughs: Tridacnin Anode: left side

Alkali-treatment destroys the genuine galactan and leaves 1 arc. Boiling with 20% KOH for 2 hours with subsequent dialysis has been used since MAY (1) for the purification of galactans. The picture in Figure 1 shows the conversion of the genuine galactan into an artifi­ cial product: the question arises, whether by this treatment glyco­ protein or protein parts, which are associated with the galactan in form of (glyco-)proteo-galactans may be completely degraded. This problem can only be solved by isolating both components of the galactan fraction detected by Tridacnin by affinity chromato­ graphy. In this connection, however, it was of interest, whether both arcs had different serological specificities as revealed by other hetero­ phile anti-galactan reagents.

Glycosubstances and galactans in Achatina fulica . 223 Fig. 2. Heterophile receptors in the Achatina fulica extract. Center: Achatina fulica extract Outer wells (always from 12 o'clock on clockwise numerated 1-6, also in the following pictures): 1,4 = Tridacnin 2,5 = Abrus precatorius 3,6 = Anti.pneumococcus Type XIV serum

2. Serological heterogeneity of the Achatina fulica extract The purpose of this experiment was, to demonstrate, whether other known serological specificities of galactans (2) can be found with other heterophile anti-galactan precipitins in this extract. The result is given in Figure 2. As can be seen from that picture, all 3 reagents do not give an identity reaction at all and do not fuse. In this respect however, it is remarkable, that Abrus and the pneumococcus antiserum seem to have no realationship to a common receptor. This was expected according to the work of BIRD (12), who provided experimental evi­ dence, that the receptors detected by Abrus and by Type XIV anti­ serum were closely related or nearly identical. This indicates, on the other side, that in the Achatina fuIica extract, both precipitins do react with receptors on different molecules. 3. Agar gel electrophoresis, using different anti-galactan reagents

from different origin

In the next experiment it should be determined, which of the hetero­ phile anti-galactans reacted with which part of the double-arc as has been detected by Tridacnin. The result is visualized in Figure 3.

•

----­

...

----~

Fig. 3. Agar gel electrophoresis of the Achatina fulica extract (eggs from the Madagascar sample). Well: Achatina fulica extract Anode: right side Upper trough: Anti pneumococcus Type XIV antiserum Lower through: Tridacnin

224 . G.

UHLENBRUCK,

G.

STEINHAUSEN,

and H. A.

KAREEM

It is evident from this picture that only the small arc did give a precipitin reaction with the heterophile pneumococcus antiserum. In exactly the same way did react: Abrus precatorius, Ricinus communis and the myeloma IgA anti-galactan, whereas on the other side Limu­ Ius polyphemus anti-galactan or eelserum, which react strongly with the Helix pomatia galactan (2), gave no precipitate at all. It is re­ markable that even the mouse myeloma anti-galactan did give a fine precipitin reaction; although it does not so with all other galactans [arabinogalactan from plants, bovine pneumogalactan, purified galac­ tan from Helix pomatia etc. (2)] when tested in agar gel diffusion. This is astonishing, as the specificity, anti-~-(1-6)-digalactobiose, which is the main terminal receptor in the above mentioned galactans, is the same as the specificity of other anti-galactans like those from Axinella polypoides and Tridacna maxima (4). Therefore, it was also unexpected, that the myeloma IgA reacted only with 1 arc of the Achatina fulica galactan-fraction, and not with the other like Tri­ dacnin. Obviously, ~-galactosyl groups of the agar, may interfere with the galactan - anti-galactan precipitin reaction (9). Another anti-galactan, namely the one ofAxinella polypoides, is also known not to give a precipitin reaction in agar with galactans (9). This prompted us, to include this anti-galactan too in our investiga­ tions, the more, as another anti-galactan, the one from Cerianthus sp. (11), proved to be instable and had lost its precipitating activity during storage. 4. The reaction oj Axinella polypoides precipitins with the Achatina julica extract

The precipitin reaction between Achatina fulica extract and the Axinella polypoides is demonstrated in Figure 4. As can be observed in this picture, Axinella polypoides detects at least 3 galactans in the Achatina fulica extract. However, 2 of them are of importance in our context: one is crossreacting with pneumo­ coccus Type XIV antiserum and the other gives an identity line with Abrus precatorius. This again underlines the assumption, that we are here dealing with at least 2 different galactans.

Fig. 4. Center well: Achatina fulica extract Wells around: 1,3,5 = Axinella polypoides 2 = Abrus precatorius 4 = Limulus polyphemus 6 = Pneumococcus Type XIV antiserum

Glycosubstances and galactans in Achatina fulica . 225 Fig. 5. Agargeldiffusion of Achatina fulica extract (center). 1,3,5 = Tridacnin 2,4,6 = Axinclla extract

In order to elucidate the relationship of these galactans to the 2 arcs detected by Tridacnin, a second test was made. The precipitation picture obtained in this test (Fig. 5) may be interpreted in the follow­ ing way: in the simple agar gel diffusion Tridacnin gives mainly 1 arc with the Achatina fulica extract, but we must assume from Figure I of this paper, that there are 2 closely related molecules of galactan. Axinella polypoides does react weakly with one of these galactans, namely galactan I (see Fig. 6). Also Ricinus reacts weakly, a fact which can be shown in gel electrophoresis, and Con A. The second galactan II (see Fig. 5) does react with anti pneumococcus Type XIV serum, with Axinella, with Ricinus communis and (see Fig. 8) with myeloma IgA. This glycosubstance, and this is evident from Figure 5, has no connection to glycosubstance III, which is detected closely to the center well by Axinella too and by the Abrus lectin and the one from wheat germ. In the agar gel electrophoresis, however, II and III migrate together. Therefore, by both methods, gel diffusion and electrophoresis, only 2 galactans seem to be present, although there are 3. In order to establish further this assumption, it was necessary to compare the reaction of Tridacnin and Axinella with the Achatina fulica extract in the agar gel electrophoresis. 5. Comparison of Tridacna maxima and Axinella polypoides anti-galactan in their precipitin reaction with the Achatina fulica galactans

The Achatina fulica extract was again analysed by agar gel electro­ phoresis and the galactans made visible by the precipitin reaction with the anti-galactans from Tridacna maxima and Axinella polypoides, respectively. The result is shown in Figure 6a and b, where the preci­ pitation arcs formed in the agar gel electrophoresis are schematically drawn in order to clarify the situation for further discussion. It is evident that Axinella detects at least 2 glycosubstances, as already has been shown in Figure 5. The very faint precipitate (Fig. 5 and Fig. 6 near the Axinella well) is not related to either glycosubstances I, II and III and not considered in our discussion. It can be postulated, that Tridacnin precipitates a galactan I (great and long arc) (see also Fig. 1) and a small arc, containing galactan II,

226 . G. UHLENBRUCK, G. STEINHAUSEN, and H. A. KAREEM

Tridacna

--­ strong arcs I

+ Axinella

ll, II[

~ 1

weak

A. fulica

2

strong arc

a) Schematic picture: Upper trough: Tridacna maxima (Tridacnin) Lower trough: Axinella polypoides extract Center well: Achatina fulica extract I, II, III = different glycosubstances (galactans) in Achatina fulica extract 1 and 2 = the corresponding arcs, reacting with Axinella precipitins 1 and 2 (see Fig. 7a, b)

b) Upper trough: Axinella polypoides extract Lower trough: Tridacna maxima Center well: Achatina fulica extract The picture corresponds with respect to the lines to the above Fig. 6a. Anode: left side Fig. 6. Agar gel electrophoretic analysis of Achatina fulica extract with the anti· galactans from Tridacna maxima and Axinella polypoides.

which reacts with myeloma IgA, pneumococcus Type XIV antiserum and Ricinus communis, and finally a glycosubstance III, the Abrus reactive one, which reacts also with wheat germ agglutinin from Triticum vulgaris as we found. Axinella polypoides gives also 2 precipitation arcs: one very weak and corresponding to arc I of Tridacnin (such a faint reaction can also be seen with Ricinus communis, a much stronger reaction the arc I does also give with Con A), the other very strong precipitin corre­ sponds to the small arc of the Tridacnin-reactive Achatina fulica galactan extract, which contains the galactans II and III. The question now arises, whether the Axinella extract represents a single precipitin, like Tridacnin, or has more than I precipitin, as already has been found in Cerianthus spec. (II). 6. Heterogeneity otAxinella precipitins

In order to find out the occurrence of more than I Axinella precipi­ tin, another agar gel electrophoresis experiment in connection with

Glycosubstances and galactans in Achatina fulica . 227

Achatina fulica extract

strong 2

-

Axinella

...-­ 0

weak arc 1

+

Helix pomatia galactan a) Schematic drawing

,I'

---~:j

··1

b) Photograph of agar gel electrophoresis outlined in 7 a. Arc 1 can also be precipitated by Con A, that means, precipitin 1 is a glycoprotein Anode: right side Fig. 7. Agar gel electrophoresis of the Axinella polypoides extract (center well). Two precipitins can be made visible in agar by the Achatina fulica extract (upper trough). All other galactans fail to precipitate (in the lower trough in this case was purified Helix pomatia galactan).

agar gel diffusion was carried out. The result is given in Figure 7 a and 7 b and can be interpreted in the following way: In the above mentioned Figure, 2 different precipitins occuring in the Axinella poly­ poides extract can be demonstrated by the Achatina fulica extract but not with any other galactan, for instance the one from Helix pomatia. The strongly reacting component 2 seems to be neutral, the weakly reacting precipitin 1 migrates to the anode. These 2 precipitins correspond to arc 1 and 2 in Figure 6a and 6b. Precipitin 1 reacts with the galactan I from Achatina fulica; precipi­ tin 2, the strong precipitin, with glycosubstances II and III. Discussion

Our results have revealed, that different glycosubstances occur in the albumin gland of Achatina fulica, which can be detected by differ­ ent heterophile anti-carbohydrate precipitins: 1 galactan, and 2 other glycosubstances, whose nature still has to be identified and which may be protease inhibitors of the glycoprotein-type (13).

228 . G. UHLENBRUCK, G. STEINHAUSEN, and H. A. KAREEM

Tridacna maxima

o

Abrus precatorius

o

Typ XIV AS

Axinella polypoides

o Ricinus communis Center: Achatina fulica a) Schematic drawing of the agar gel diffusion of Achatina fulica extract.

b) Photographic picture of this precipitin reaction. For explanation see text Fig. 8. Different galactans or glycosubstances in the eggs (and albumin gland extract) of Achatina fulica as demonstrated by heterophile anti-galactans and lectins.

When analysing and interpreting the different precipitin reactions with different reagents in agar gel diffusion and in the agar gel electro­ phoresis, one can deduce the following conclusions (Fig. 8): Tridacna maxima precipitates with galactan I and II. With galactan I also reacts anti-galactan 1 from Axinella polypoides; this reaction does come out also in Figure 4. No identity between Tridacnin and the 2 Axinella precipitins can be stated in spite of their similar combining specificity (4). Anti-galactan 2 from Axinella detects galactan II from Achatina fulica, which also strongly reacts with myeloma IgA from mouse ascites, with pneumococcus Type XIV antiserum and with Ricinus communis. It precipitates, however, also glycosubstance III, which reacts with Abrus precatorius and Triticum vulgaris lectin. Interesting B,re the identity reactions between this second precipitin (Fig. 1 and 6) and myeloma IgA, Axinella, pneumococcus antiserum and Ricinus communis. These 3 glycosubstances of the Achatina fulica eggs and

Glycosubstances and galactans in Achatina fulica . 229 Tab. 1. Precipitin reactions of 3 glycosubstances from Achatina fulica eggs and albumin glands with anti·galactans and heterophile precipitins of different origin. Anti-galactans from

Lectins

Axinella polypoides 1 2

+

I

II III

+

=

( +)

=

-

=

+ +

+ +

+

+

+

(+)

+

+

(+)

strong precipitin reaction weaker precipitin arc no visible precipitate

albumin glands do not occur, as we examined, in the haemolymph of these snails. There, however, a strong and different migrating Con­ canavalin A reactive glycosubstance can be detected, the isolation of which is now in progress. In Table 1 all these results are summarized again. The 2 different Axinella precipitins can be clearly distinguished by these serological reactions and the 3 different galactans or glycosubstances, too. Future work, using affinity chromatography, is now necessary in order to isolate and purify these compounds separately. Noteworthy is the precipitin reaction of murine myeloma IgA in agar with the Achatina fulica extract, similar is the reaction of the Axinella precipitins, which also, like myeloma IgA, do not precipitate with any other galactans in agar gel diffusion. This fact provides in Achatina fulica galactans an useful tool for a screening test with human myeloma proteins in order to find out an anti-galactan specificity in human myeloma proteins too (12). Acknowledgement These investigations were supported by a grant from Deutsche Forschungs­ gemeinschaft.

References 1. MAY, F. 1934. Chemische und biologische Untersuchungen liber Galaktogen.

z. BioI. 95: 277. 2. UHLENBRUCK, G., G. STEINHAUSEN und B. A. BALDO. 1975. Galactane und Anti-Galactane. Forsch. u. Lehre Medizin 7, Verlag Josef Stippak, Aachen.

230 . G. UHLENBRUCK, G. STEINHAUSEN, and H. A. KAREEM 3. GLAUDEMANS, C. P. J. 1975. The interaction of homogeneous, murine mye­ loma immunoglobulins with polysaccharide antigens. Advances in carbo­ hydrate Chemistry and Biochemistry. R. S. TIPSON, and D. HORTON (Eds.) Academic Press 31: 313. 4. EICHMANN, K., G. UHLENBRUCK, and B. A. BALDO. 1976. Similar combin­ ing specificities of invertebrate precipitins and mouse myeloma protein J 539 for 8-(1-6)galactans. Immunochemistry 13: 1. 5. STEINHAUSEN, G., G. UHLENBRUCK und H. G. SCHWICK. 1976. ttber das Agglutinin und Prazipitin aus Tridacna. KongreJ3bd. Deutsch. Ges. Blut­ transfusion u. Immunhaematol. Frankfurt (im Druck). 6. UHLENBRUCK, G., B. A. BALDO, and G. STEINHAUSEN. 1975. Anticarbo­ hydrate precipitins and haemagglutinins in haemolymph from Tridacna maxima (Roding). Z. Immun.-Forsch. 150: 354. 7. UHLENBRUCK, G., G. STEINHAUSEN, and B. A. BALDO. 1976. Galactans and anti-galactans from invertebrates. Z. Naturforsch. 31 J: 205. 8. GOLD, E. R., C. F. PHELPS, S. KHALAP, and P. BALDING. 1974. Observations on Axinella sp. haemagglutinin. Ann. N.Y. Acad. Sci. (Wash.) 234: 122. 9. BRETTING, H., und L. RENWRANTZ. 1974. Weitere Untersuchungen zur Natur der Hamagglutinine aufgefunden in den Schwammen Aaptos papillata und Axinella polypoides. Z. Immun.-Forsch. 147: 250. 10. BRETTING, H. 1973. Serologische und immunelektrophoretische Unter­ suchungen tiber 2 Agglutinine, aufgefunden in den Schwammen Aaptos papillata (Keller) und Axinella polypoides (Schmidt). Z. Immun.-Forsch. 146: 239. 11. GAUWERKY, C., G. UHLENBRUCK und L. RENWRANTZ. 1974. Blutgruppen­ ahnliche Substanzen aus einigen marinen Invertebraten. IV. H- und A­ ahnliche Substanzen, Agglutinine und Prazipitine: Ihre Verteilung bei Cerianthus sp. Marine Biology 26: 369. 12. BIRD, G. W. G. 1961. Specific precipitins for Type XIV pneumococcus poly­ saccharide from Abrus precatorius seeds. Experientia 17: 71. 13. UHLENBRUCK, G., I. SPRENGER, and I. ISHIYAMA. 1971. A new polyvalent proteinase-inhibitor occuring in the albumin gland of Helix pomatia. Z. Klin. Chern. 9: 361. 14. FREEDMAN, M., R. MERRET, and W. PRUZANSKI. 1976. Human monoclonal immunoglobulins with antibody-like activity. Immunochemistry 13: 193. Prof. Dr. G. UHLENBRUCK, Abteilung Immunbiologie, KerpenerstraJ3e 15, D - 5000 Koln 41.