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Aggregation of sponge cells: 23. Interaction of Geodia lectin with Tethya cell-surface glycoprotein
Aggregation of sponge cells: 23. Interaction of Geodia lectin with Tethya cell-surface glycoprotein W. E. G. Miiller*t, R. K. Zahn*t, A. Bernd*, B. Kurelect, I. Miiller*t, P. Vaith, and G. Uhlenbruek, *Institut J~r Physiolo,qische Chemie, Universitiit, Duesber,qwe,q, 6500 Mainz, W. German), +lahoratory.lbr Marine Molecular Biolo,qy, Institute Ruder Boskovi(, Centre )br Marine Research, 52210 Rovinj, Yu,qoslavia and +Medizinische Universitiitsklinik, Kerpener Strass 15, 5000 K61n, W.. Germany
(Received 13 November 1979) A macromolecule has been isolatedJrom the cell membranes of the sponge Tethya lyncurium. This macromolecule was purified and Jbund to bind to a D-,qalaetose specific lectin from the sponge Geodia cydonium. The lecrin receptor was charaeterized as a ,qlycoprotein with a molecular weight in the re,qion of155 000. Evidence is presented indicating that the hindin,q qllectin with the lectin receptor is caused by hydrophohic interactions.
Introduction In the preceding paper 1 we described the elucidation of the function of the D-galactose specific lectin, isolated from the siliceous sponge Geodia cydonium 2 in a heterologous biological system. It was shown that the Geodia lectin agglutinates cells from the siliceous sponge Tethya lyncurium. This agglutination process leads to a reduction of their viability as well as an inhibition of programmed syntheses in these cells 1. In the present study we describe the isolation procedure of a cell-surface glycoprotein from Tethya which functions as the receptor for the Geodia lectin.
Experimental
mM lithium diiodosalicylate, 20 mM Tris HCI, pH 9.0, 50 mM NaCI, 5 mM KCI and 2')~ (v/v) Triton X-100.
Determination q] the activity o1"the Tethya lectin receptor In a standard assay 3 150 stg (referred to protein) of
Geodia lectin was added to 0.5 ml C M F solution containing different concentrations of Tethya receptor and preincubated for 10 min at 20'JC. Then, 75+_ 15 × 106 Tethya cells were added to final reaction volume of 3 ml. In the absence of the Tethya receptor, cell agglutinates with a diameter of 720_+ 120 #m were formed after 60 min incubation. The diameter of the agglutinates was determined as previously described 4. One Tethya receptor unit was defined as the reciprocal value of that dilution which caused a 50% reduction in the diameter of the agglutinates in the standard assay.
Compounds The following materials were used: [8-3H] G M P (spec. act. 4.6 Ci/mmol) from The Radiochemical Centre, Amersham, England; lithium 3,5-diiodosalicylate from Eastman Kodak, Rochester, NY, USA; lactose from E. Merck, Darmstadt, W. Germany; Sepharose 6 B and dextran blue from Deutsche Pharmacia, Freiburg, W. Germany; fluoram (fluorescamine) from Hoffmann-La Roche, Grenzach, W. Germany; thyroglobulin, bovine serum albumin, aldolase and ~-chymotrypsinogen from Boehringer, Mannheim, W. Germany; D-glucuronic acid from Sigma Chemical Co., St Louis, Mo., USA. A description of the animals (siliceous sponges Geodia cydonium Jam. and Tethya lyncurium L.) 1, dissociation procedure of sponge tissue to single ceils 3, the composition of calcium- and magnesium-free artifical sea water (CMF) 4 and the Geodia lectin with a protein concentration of 2 mg/ml 2 have all been described.
Solutions Tris/NaCl solution contained 100 mM Tris-HCl, pH 8.2, and 500 mM NaC1. The salicylate solution contained 2 0141 8130/80/050302~)4502.00 © 1980 |PC Business Press
302
Int. J. Biol. Macromol., 1980, Vol 2, October
Solubilization of the Tethya cell-surface receptor Cell membranes were isolated from a single cell suspensionS: the membrane preparation contained 41.2~ protein, 36.8~o lipid and 1aJ.-/oT°/neutral carbohydrates. The absolute protein content in the membrane fraction of a preparation derived from 20 ml packed cells (corresponding to 300 mg of protein) was determined to be 4.1 mg. The activity of 5'-nucleotidase in the membrane fraction was equivalent to 9.3 ~tmol [ 3 H ] G M P hydrolysed (mg protein)- 1 h - 1. The cell membranes (300 mg protein) were solubilized with salicylate solution 5 and the resulting suspension was centrifuged (40000g; 30 min; 20"C). Dialysis of the supernatant against Tris/NaCl solution gave Fraction I (crude fraction).
Purification of the Yethya lectin receptor Fraction I (19 ml) was further fractionated by gel chromatography on a Sepharose 6B column (2.5 × 50 cm) equilibrated with Tris/NaCl solution and eluted with the same buffer. Fractions of 4 ml were collected. The fraction
Interaction qf a sponge lectin with a cell-surface glycoprotein: W. E. G. Mi~ller et al. Table 1 Purification of the Tethya lectin receptor. The units of lectin receptor activity are defined in Experimental
Fraction
Description
I |I III
Crude fraction Gel chromatography Lectin chromatography
J, 3
2 o
P 7~
:=:L 0
L
b
P 2,
f'.~"l
12C
aooo
!1 "l
10C
~ 8c
v
u) c
"1 60
2
c
~fl)
4o
t3
i!/
n
!
b
2O
10
5 I
~
15 20 Froction n u m b e r
i 2
i 3
Neutral carbohydrate (/ag/ml)
Specific activity (units/~g carbohydrate)
Yield (,';,)
19 2 0.7
35.8 52.6 4.6
14.6 82.1 2134.9
100 87 61
was purified by gel chromatography, taking advantage of its partial binding capacity to the lectin. Lectin preparation (0.1 ml 200 #g protein) was added to 2 ml of Fraction I! and incubated in roller tubes (35 rev/min) for I h at 2ffC. The sample was then applied to a Sepharose 6B column and chromatographed (Figure la). Fractions 7 9, eluting near the void volume, were dialysed against salicylate solution for 24 h at 20°C and subsequently analysed by gel filtration using salicylate solution as the eluting buffer (Figure I b). The lectin/lectin receptor was destroyed by this procedure; the lectin eluted at Ve/Vo values in the range 2.92--3.03 (fractions 19-20) and the lectin receptor at Ve/Vo in the range 2.21 2.50 (fractions 14-16). The fractions containing the lectin receptor were pooled and concentrated by dialysis against poly(ethylene glycol) in Tris/NaCl solution to a final volume of 0.7 ml. This fraction was used for the experiments. The data for typical preparation are summarized in Table 1.
a
140
Total volume (ml)
25
i
v~/Vo Figure 1 Purification of lectin receptor. (a) The lectin receptor preparation (Fraction II) was coincubated with the lectin and chromatographed on a Sepharose 6B column (1 × 18 cm) previously equilibrated with Tris/NaC1. The column was loaded with 2.5 ml of the lectin/lectin receptor mixture and eluted with Tris/NaCl. Fractions of 0.8 ml eluting near the void volume (nos 7 9) were collected. (b) The combined sample was treated with salicylate solution and rechromatographed (sample volume 2 ml) on Sepharose 6B using salicylate solution as eluent (other conditions as described above). Fractions were assayed for carbohydrate content (e), for protein (x) and for receptor activity (o).J,, position of Vo (dextran blue marker). Scale gives V,,/Vo values °. The bar marked L gives the position of the purified lectin eluting with Ve/Vo values 6 between 2.36 and 2.70 containing the lectin receptor were collected and concentrated in a dialysis tubing with poly(ethylene glycol) (Carbowax 6000) to 2 ml. The total receptor activity recovered after these procedures was determined to be 8640 units (Table 1) (Fraction II). Fraction II, containing 105.2/~g neutral carbohydrates,
Other methods Protein was determined by the Fluoram method mS, neutral carbohydrates (D-glucose standard) according to the method of Dubois et al. 9, hexuronic acid (Oglucuronic acid standard) according to Avigad 1°, lipids according to Hinsberg et al. 11, the 5'-nucleotidase activity according to Miiller et al. ]2. Molecular weight was determined by dodecylsulphate/polyacrylamide (10%) gel electrophoresis, with bovine serum albumin, aldolase, c¢-chymotrypsinogen and thyroglobulin as reference standards. The gels were stained with periodic acid/Schiff stain 13. Determination of the molecular weight by gel chromatography was carried out on Sepharose 6B which was calibrated with the same protein markers mentioned above.
Results The lectin receptor from Tethya lyncurium has been isolated and purified by gel chromatography and lectin chromatography. The final specific activity was found to be 2135 receptor units//~g carbohydrate. Physical characterization The purity of the lectin receptor preparation was checked on polyacrylamide gel electrophoresis containing sodium dodecylsulphate. Figure 2 shows that with the periodic acid/Schiff reaction the receptor contains only one band. From this finding we conclude that the lectin receptor was purified to homogeneity and consisted of glycosylated protein. The molecular weight determined by dodecylsulphate gel electrophoresis was found to be
Int. J. Biol. Macromol., 1980. Vol 2, October
303
Interaction o./'a sponge lectin with a cell-surface glycoprotein: W. E. G. Mfiller et al.
o n
n
[
l
I
0
[
[
I
I
i
05 R e l o t i v e mobil It3/
10
Figure 2 Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate, showing the lectin receptor. Gels were loaded with 50 /~1 of Fraction 1II and subjected to periodic acid/Schiff staining. The absorbance scans are shown. The peak represents an apparent molecular weight of 170 000, calculated relative to the migration of series of reference proteins (see Experimental)
004
003
~
÷0 0 0 5
8c OO2
S
t_
~0
<
c
g
o ol
c
C~ O 300
J
t
I
I 280
Wavelength
J
t
I
I 260
I
-0005
(nm)
Figure 3 Spectral analysis of lectin receptor, lectin and lectin lectin receptor complex: pure lectin preparation (25 /~g protein/ml) (•), Fraction III of the lectin receptor (1/zg protein/ml) (©) and lectin-lectin receptor complex ( x ) formed after incubation (20 min; 20°C) of the two components at the concentrations indicated above. Samples were dissolved in CMF. The difference spectrum (A) (change in absorbance) was calculated by subtraction of the absorbance of the complex from the sum of the absorbances of lectin and lectin receptor 170 000 (Figure 2). F r o m the elution pattern on Sepharose 6B (Ve/Vo=2.35) the apparent size of the receptor was 155000. Lectin receptor activity is 90% destroyed after heat treatment (3 min, 100°C).
values for the lectin and the lectin receptor has a maximum at 280 nm if the u.v. values from the lectinlectin receptor mixture are subtracted. This finding indicates that the lectin and the lectin receptor form a complex under quenching of the u.v. absorption of these chemical groups of the lectin and/or lectin receptor with a maximum around 280 nm. The kinetics of complex formation between lectin a~d lectin receptor are shown in Figure 4. The change in absorbance, which is a measure of complex formation, occurs immediately after mixing the two components and is terminated after 12 min incubation.
Discussion The D-galactose-specific lectin from the sponge Geodia cydonium z seems to have distinct functions both in the homologous biological model (control of the cell aggregation process; Ref 13) as well as in heterologous systems (to support the self-non-self mechanism: Ref. 1). In the present work using the heterologous system Geodia cydonium and Tethya lyncurium, the relation between the Geodia lectin and the Tethya cell membrane could be elucidated at a molecular level. Geodia lectin was found to react with a distinct receptor macromolecule from Tethya cell membrane. From its chemical composition the lectin receptor is a glycoprotein containing neutral carbohydrates as major components. The molecule has a molecular weight of 155 000 as determined by gel chromatography on Sepharose 6B; the higher value for the apparent molecular weight (170000) determined by dodecylsulphate electrophoresis is most likely due to the" high presence of carbohydrates in the receptor molecule, resulting in an anomalous migration 14. It is interesting that during complex formation between the lectin and the lectin receptor the u.v. absorption is quenched at a wavelength of 280 nm. At present we explain this result by assuming that aromatic amino acids (phenylalanine, tyrosine, tryptophan) which are characterized by a pronounced peak between 240 and 300 nm 15 are involved in the process of complex formation. Because complex formation is also independent of divalent ions (Ref 1, and this work) we have some evidence that the physico-chemical basis for the binding of the lectin to the
I
,7.
I
I
÷0.003
Chemical characterization Chemical analysis showed that the tectin receptor consists mainly of neutral carbohydrates (53.4~o dry weight); protein (35.4~o), lipid (6.5~o) and hexuronic acid (1.8~o) are present in lower concentrations.
Lectin-lectin receptor complex The spectrum of the lectin shows a sharp maximum at 274 nm while the spectrum of the lectin receptor has no pronounced peak within the range 300-250 nm (Figure 3). A mixture of the two components has an absorption spectrum with a maximum at 272 nm. The difference spectrum, calculated from the three spectra shows a characteristic pattern (Figure 3); the sum of the absorption
304
Int. J. Biol. Macromol., 1980, Vol 2, October
O,
I
0
I
I
m
1
I
I
10 Incubation t i m e (rain)
Figure 4 Kinetics of formation of the lectin-lectin receptor complex. The change in the difference spectrum with time between lectin and lectin receptor in separate cuvettes and an assay containing these components together was determined at 280 nm. Concentrations of the components were the same as described in Fi.qure 3. The incubation was performed at 20°C
20
Interaction of a sponge lectin with a cell-surface glycoprotein: W E. G. Miiller et al.
lectin receptor might be hydrophobic interactions. The involvement of this type of force in the adhesive behaviour of some biological membranes was first demonstrated experimentally in the system of cuvierian tubules of
5 6
Holothuria forskali“j 8
Acknowledgement This work was supported by a grant of the Stiftung Volkswagenwerk (35850; W.E.G.M.). References 1 2 3 4
Miiller, W. E. G., Zahn, R. K., Kurelec, B., Miiller, I., Vaith, P. and Uhlenbruck, G. Int. J. Biol. Macromol. 1980, 2, 297 Vaith, P., Uhlenbruck, G.. Miiller, W. E. G. and Holz, G. Deu. Comp. Immunol. 1979, 3, 399 Miiller, W. E. G. and Zahn, R. K. Exp. Cell. Res. 1973, 80, 95 Miiller, W. E. G.,Zahn, R. K., Kurelec, B., Miiller, I., Uhlenbruck, G. and Vaith, P. J. Biol. Chem. 1979, 254, 1280
9 10 11 I2 13 14 15 16
Miiller, W. E. G., Zahn, R. K., Kurelec, B., Miller, I., Vaith, P. and Uhlenbruck, G. Eur. J. Biochem. 1979, 97, 585 Determann, H. ‘Gelchromatographie’, Springer-Verlag, Berlin, New York, 1967 Udenfried, S., Stein, S., Biihlen, P., Dairman, W., Leimgruber, W. and Weigele, M. Science 1972, 178, 871 Weigele. M., DeBernardo, S. L. and Leimgruber, W. Biochem. Biophys. Rex Cornmutt. 1973, 50. 352 Dubois. M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith. F. A&. Chem. 1956. 28, 350 Avigad, G. Methods Enzymol. 1975. 4lB, 29 Hinsberg, K. and Lang, K. in ‘Medizinische Chemie’, Urban and Schwarzenberg, Miinchen, Munich. 1951, pp. 245- 307 Miiller, W. E. G., Schriider, H. C., Stetlen, R., Zahn, R. K. and Dose, K. Mol. Biol. Rep. 1977, 3, 331 Miiller. W. E. G.. Kurelec. B.. Zahn. R. K.. Miiller. 1.. Vaith. P. and Uhlenbruck, G. J. Biol. Chem. 1979, 254, 7479 Segrest, J. P. and Jackson, R. L. Methods Enzymol. 1972, 28B, 54 Sober, H. A. ‘Handbook of Biochemistry’, The Chemical Rubber Co., Cleveland, 1968, p. B 18 Miiller, W. E. G., Zahn. R. K. and Schmid, K. Cytohiol. 1972.5, 335
Int. J. Biol. Macromol., 1980, Vol 2, October
305
(Received 13 November 1979) A macromolecule has been isolatedJrom the cell membranes of the sponge Tethya lyncurium. This macromolecule was purified and Jbund to bind to a D-,qalaetose specific lectin from the sponge Geodia cydonium. The lecrin receptor was charaeterized as a ,qlycoprotein with a molecular weight in the re,qion of155 000. Evidence is presented indicating that the hindin,q qllectin with the lectin receptor is caused by hydrophohic interactions.
Introduction In the preceding paper 1 we described the elucidation of the function of the D-galactose specific lectin, isolated from the siliceous sponge Geodia cydonium 2 in a heterologous biological system. It was shown that the Geodia lectin agglutinates cells from the siliceous sponge Tethya lyncurium. This agglutination process leads to a reduction of their viability as well as an inhibition of programmed syntheses in these cells 1. In the present study we describe the isolation procedure of a cell-surface glycoprotein from Tethya which functions as the receptor for the Geodia lectin.
Experimental
mM lithium diiodosalicylate, 20 mM Tris HCI, pH 9.0, 50 mM NaCI, 5 mM KCI and 2')~ (v/v) Triton X-100.
Determination q] the activity o1"the Tethya lectin receptor In a standard assay 3 150 stg (referred to protein) of
Geodia lectin was added to 0.5 ml C M F solution containing different concentrations of Tethya receptor and preincubated for 10 min at 20'JC. Then, 75+_ 15 × 106 Tethya cells were added to final reaction volume of 3 ml. In the absence of the Tethya receptor, cell agglutinates with a diameter of 720_+ 120 #m were formed after 60 min incubation. The diameter of the agglutinates was determined as previously described 4. One Tethya receptor unit was defined as the reciprocal value of that dilution which caused a 50% reduction in the diameter of the agglutinates in the standard assay.
Compounds The following materials were used: [8-3H] G M P (spec. act. 4.6 Ci/mmol) from The Radiochemical Centre, Amersham, England; lithium 3,5-diiodosalicylate from Eastman Kodak, Rochester, NY, USA; lactose from E. Merck, Darmstadt, W. Germany; Sepharose 6 B and dextran blue from Deutsche Pharmacia, Freiburg, W. Germany; fluoram (fluorescamine) from Hoffmann-La Roche, Grenzach, W. Germany; thyroglobulin, bovine serum albumin, aldolase and ~-chymotrypsinogen from Boehringer, Mannheim, W. Germany; D-glucuronic acid from Sigma Chemical Co., St Louis, Mo., USA. A description of the animals (siliceous sponges Geodia cydonium Jam. and Tethya lyncurium L.) 1, dissociation procedure of sponge tissue to single ceils 3, the composition of calcium- and magnesium-free artifical sea water (CMF) 4 and the Geodia lectin with a protein concentration of 2 mg/ml 2 have all been described.
Solutions Tris/NaCl solution contained 100 mM Tris-HCl, pH 8.2, and 500 mM NaC1. The salicylate solution contained 2 0141 8130/80/050302~)4502.00 © 1980 |PC Business Press
302
Int. J. Biol. Macromol., 1980, Vol 2, October
Solubilization of the Tethya cell-surface receptor Cell membranes were isolated from a single cell suspensionS: the membrane preparation contained 41.2~ protein, 36.8~o lipid and 1aJ.-/oT°/neutral carbohydrates. The absolute protein content in the membrane fraction of a preparation derived from 20 ml packed cells (corresponding to 300 mg of protein) was determined to be 4.1 mg. The activity of 5'-nucleotidase in the membrane fraction was equivalent to 9.3 ~tmol [ 3 H ] G M P hydrolysed (mg protein)- 1 h - 1. The cell membranes (300 mg protein) were solubilized with salicylate solution 5 and the resulting suspension was centrifuged (40000g; 30 min; 20"C). Dialysis of the supernatant against Tris/NaCl solution gave Fraction I (crude fraction).
Purification of the Yethya lectin receptor Fraction I (19 ml) was further fractionated by gel chromatography on a Sepharose 6B column (2.5 × 50 cm) equilibrated with Tris/NaCl solution and eluted with the same buffer. Fractions of 4 ml were collected. The fraction
Interaction qf a sponge lectin with a cell-surface glycoprotein: W. E. G. Mi~ller et al. Table 1 Purification of the Tethya lectin receptor. The units of lectin receptor activity are defined in Experimental
Fraction
Description
I |I III
Crude fraction Gel chromatography Lectin chromatography
J, 3
2 o
P 7~
:=:L 0
L
b
P 2,
f'.~"l
12C
aooo
!1 "l
10C
~ 8c
v
u) c
"1 60
2
c
~fl)
4o
t3
i!/
n
!
b
2O
10
5 I
~
15 20 Froction n u m b e r
i 2
i 3
Neutral carbohydrate (/ag/ml)
Specific activity (units/~g carbohydrate)
Yield (,';,)
19 2 0.7
35.8 52.6 4.6
14.6 82.1 2134.9
100 87 61
was purified by gel chromatography, taking advantage of its partial binding capacity to the lectin. Lectin preparation (0.1 ml 200 #g protein) was added to 2 ml of Fraction I! and incubated in roller tubes (35 rev/min) for I h at 2ffC. The sample was then applied to a Sepharose 6B column and chromatographed (Figure la). Fractions 7 9, eluting near the void volume, were dialysed against salicylate solution for 24 h at 20°C and subsequently analysed by gel filtration using salicylate solution as the eluting buffer (Figure I b). The lectin/lectin receptor was destroyed by this procedure; the lectin eluted at Ve/Vo values in the range 2.92--3.03 (fractions 19-20) and the lectin receptor at Ve/Vo in the range 2.21 2.50 (fractions 14-16). The fractions containing the lectin receptor were pooled and concentrated by dialysis against poly(ethylene glycol) in Tris/NaCl solution to a final volume of 0.7 ml. This fraction was used for the experiments. The data for typical preparation are summarized in Table 1.
a
140
Total volume (ml)
25
i
v~/Vo Figure 1 Purification of lectin receptor. (a) The lectin receptor preparation (Fraction II) was coincubated with the lectin and chromatographed on a Sepharose 6B column (1 × 18 cm) previously equilibrated with Tris/NaC1. The column was loaded with 2.5 ml of the lectin/lectin receptor mixture and eluted with Tris/NaCl. Fractions of 0.8 ml eluting near the void volume (nos 7 9) were collected. (b) The combined sample was treated with salicylate solution and rechromatographed (sample volume 2 ml) on Sepharose 6B using salicylate solution as eluent (other conditions as described above). Fractions were assayed for carbohydrate content (e), for protein (x) and for receptor activity (o).J,, position of Vo (dextran blue marker). Scale gives V,,/Vo values °. The bar marked L gives the position of the purified lectin eluting with Ve/Vo values 6 between 2.36 and 2.70 containing the lectin receptor were collected and concentrated in a dialysis tubing with poly(ethylene glycol) (Carbowax 6000) to 2 ml. The total receptor activity recovered after these procedures was determined to be 8640 units (Table 1) (Fraction II). Fraction II, containing 105.2/~g neutral carbohydrates,
Other methods Protein was determined by the Fluoram method mS, neutral carbohydrates (D-glucose standard) according to the method of Dubois et al. 9, hexuronic acid (Oglucuronic acid standard) according to Avigad 1°, lipids according to Hinsberg et al. 11, the 5'-nucleotidase activity according to Miiller et al. ]2. Molecular weight was determined by dodecylsulphate/polyacrylamide (10%) gel electrophoresis, with bovine serum albumin, aldolase, c¢-chymotrypsinogen and thyroglobulin as reference standards. The gels were stained with periodic acid/Schiff stain 13. Determination of the molecular weight by gel chromatography was carried out on Sepharose 6B which was calibrated with the same protein markers mentioned above.
Results The lectin receptor from Tethya lyncurium has been isolated and purified by gel chromatography and lectin chromatography. The final specific activity was found to be 2135 receptor units//~g carbohydrate. Physical characterization The purity of the lectin receptor preparation was checked on polyacrylamide gel electrophoresis containing sodium dodecylsulphate. Figure 2 shows that with the periodic acid/Schiff reaction the receptor contains only one band. From this finding we conclude that the lectin receptor was purified to homogeneity and consisted of glycosylated protein. The molecular weight determined by dodecylsulphate gel electrophoresis was found to be
Int. J. Biol. Macromol., 1980. Vol 2, October
303
Interaction o./'a sponge lectin with a cell-surface glycoprotein: W. E. G. Mfiller et al.
o n
n
[
l
I
0
[
[
I
I
i
05 R e l o t i v e mobil It3/
10
Figure 2 Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate, showing the lectin receptor. Gels were loaded with 50 /~1 of Fraction 1II and subjected to periodic acid/Schiff staining. The absorbance scans are shown. The peak represents an apparent molecular weight of 170 000, calculated relative to the migration of series of reference proteins (see Experimental)
004
003
~
÷0 0 0 5
8c OO2
S
t_
~0
<
c
g
o ol
c
C~ O 300
J
t
I
I 280
Wavelength
J
t
I
I 260
I
-0005
(nm)
Figure 3 Spectral analysis of lectin receptor, lectin and lectin lectin receptor complex: pure lectin preparation (25 /~g protein/ml) (•), Fraction III of the lectin receptor (1/zg protein/ml) (©) and lectin-lectin receptor complex ( x ) formed after incubation (20 min; 20°C) of the two components at the concentrations indicated above. Samples were dissolved in CMF. The difference spectrum (A) (change in absorbance) was calculated by subtraction of the absorbance of the complex from the sum of the absorbances of lectin and lectin receptor 170 000 (Figure 2). F r o m the elution pattern on Sepharose 6B (Ve/Vo=2.35) the apparent size of the receptor was 155000. Lectin receptor activity is 90% destroyed after heat treatment (3 min, 100°C).
values for the lectin and the lectin receptor has a maximum at 280 nm if the u.v. values from the lectinlectin receptor mixture are subtracted. This finding indicates that the lectin and the lectin receptor form a complex under quenching of the u.v. absorption of these chemical groups of the lectin and/or lectin receptor with a maximum around 280 nm. The kinetics of complex formation between lectin a~d lectin receptor are shown in Figure 4. The change in absorbance, which is a measure of complex formation, occurs immediately after mixing the two components and is terminated after 12 min incubation.
Discussion The D-galactose-specific lectin from the sponge Geodia cydonium z seems to have distinct functions both in the homologous biological model (control of the cell aggregation process; Ref 13) as well as in heterologous systems (to support the self-non-self mechanism: Ref. 1). In the present work using the heterologous system Geodia cydonium and Tethya lyncurium, the relation between the Geodia lectin and the Tethya cell membrane could be elucidated at a molecular level. Geodia lectin was found to react with a distinct receptor macromolecule from Tethya cell membrane. From its chemical composition the lectin receptor is a glycoprotein containing neutral carbohydrates as major components. The molecule has a molecular weight of 155 000 as determined by gel chromatography on Sepharose 6B; the higher value for the apparent molecular weight (170000) determined by dodecylsulphate electrophoresis is most likely due to the" high presence of carbohydrates in the receptor molecule, resulting in an anomalous migration 14. It is interesting that during complex formation between the lectin and the lectin receptor the u.v. absorption is quenched at a wavelength of 280 nm. At present we explain this result by assuming that aromatic amino acids (phenylalanine, tyrosine, tryptophan) which are characterized by a pronounced peak between 240 and 300 nm 15 are involved in the process of complex formation. Because complex formation is also independent of divalent ions (Ref 1, and this work) we have some evidence that the physico-chemical basis for the binding of the lectin to the
I
,7.
I
I
÷0.003
Chemical characterization Chemical analysis showed that the tectin receptor consists mainly of neutral carbohydrates (53.4~o dry weight); protein (35.4~o), lipid (6.5~o) and hexuronic acid (1.8~o) are present in lower concentrations.
Lectin-lectin receptor complex The spectrum of the lectin shows a sharp maximum at 274 nm while the spectrum of the lectin receptor has no pronounced peak within the range 300-250 nm (Figure 3). A mixture of the two components has an absorption spectrum with a maximum at 272 nm. The difference spectrum, calculated from the three spectra shows a characteristic pattern (Figure 3); the sum of the absorption
304
Int. J. Biol. Macromol., 1980, Vol 2, October
O,
I
0
I
I
m
1
I
I
10 Incubation t i m e (rain)
Figure 4 Kinetics of formation of the lectin-lectin receptor complex. The change in the difference spectrum with time between lectin and lectin receptor in separate cuvettes and an assay containing these components together was determined at 280 nm. Concentrations of the components were the same as described in Fi.qure 3. The incubation was performed at 20°C
20
Interaction of a sponge lectin with a cell-surface glycoprotein: W E. G. Miiller et al.
lectin receptor might be hydrophobic interactions. The involvement of this type of force in the adhesive behaviour of some biological membranes was first demonstrated experimentally in the system of cuvierian tubules of
5 6
Holothuria forskali“j 8
Acknowledgement This work was supported by a grant of the Stiftung Volkswagenwerk (35850; W.E.G.M.). References 1 2 3 4
Miiller, W. E. G., Zahn, R. K., Kurelec, B., Miiller, I., Vaith, P. and Uhlenbruck, G. Int. J. Biol. Macromol. 1980, 2, 297 Vaith, P., Uhlenbruck, G.. Miiller, W. E. G. and Holz, G. Deu. Comp. Immunol. 1979, 3, 399 Miiller, W. E. G. and Zahn, R. K. Exp. Cell. Res. 1973, 80, 95 Miiller, W. E. G.,Zahn, R. K., Kurelec, B., Miiller, I., Uhlenbruck, G. and Vaith, P. J. Biol. Chem. 1979, 254, 1280
9 10 11 I2 13 14 15 16
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