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Photoaffinity labeling of an acorn barnacle lectin with a photoactivatable fluorescent reagent derivative of d -galactosamine
Developmentaland ComparativeImmunology,Vol. 16, pp. 1-8, 1992 Printed in the USA. All rights reserved.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc
PHOTOAFFINITY LABELING OF AN ACORN BARNACLE LECTIN WITH A PHOTOACTIVATABLE FLUORESCENT REAGENT DERIVATIVE OF D-GALACTOSAMINE Koji Muramoto and Hisao Kamiya School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan (Submitted January 1990; Accepted March 1990)
[]Abstract--A photoactivatable D-galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with 1-azide-5-naphthalene sulfonyl chloride (ANSCI). The derivative (GalN-ANS) inhibited the agglutination activity of an acorn barnacle lectin against rabbit erythrocytes to the same extent as D-galactosamine. We used GalN-ANS for photoaffinity labeling of the lectin. The photolabeled lectin was digested with prDnase and the digest was separated by reversed-phase high-performance liquid chromatography by monitoring fluorescence and uv absorption to isolate the peptide labeled with GaiN-ANS. Amino acid analyses of the labeled peptides revealed that GalN-ANS preferentially covalently labeled two regions in the carbohydrate recognition domain of the lectin. One of them was the highly conserved amino-acid sequence region throughout all calciumdependent animal lectins.
IqKeywords--Lectin; invertebrate lectin; acorn barnacle; Megabalanusrosa; Photoaffinity labeling; Amino acid sequence; Carbohydrate binding site.
Introduction Amino-acid sequences of several invertebrate lectins, isolated from the flesh fly (Sarcophaga peregrina) (1), the acorn barnacle (Megabalanus rosa) (2) and the sea urchin (Anthocidaric crassispina) (3), have been reported. In spite of the fact that the lectins have distinct molecAddress correspondence to Dr. K. Muramoto, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan.
ular weights and subunit structures, they have a domain which is homologous with the carbohydrate-recognition domain of the calcium-dependent (C-type) animal lectins (4). There are certain residues which are conserved in all of the carbohydrate-recognition domains, particularly around some of the cysteine residues and tryptophan residues. They may play important roles in carbohydrate recognition or binding; however, the carbohydrate binding site has yet to be identified within the amino-acid sequence by a different approach. Photoafflnity labeling is a useful tool in the identification of receptors and binding sites for various ligands and in locating functional sites of macromolecules (5,6). We have prepared and characterized a photoactivatable, heterobifunctional fluorescent reagent, l-azido-5-naphthalene sulfonyl chloride (ANS-C1) (7), and used the reagent to prepare photoreactive derivatives of an invertebrate lectin and a trypsin inhibitor (8). Upon exposure to ultraviolet light, the nonfluorescent photoreactive ligands bound to their binding proteins generated reactive nitrene i n t e r m e d i a t e s which could react with neighboring molecules to form fluorescent products. This fluorescence facilitated the detection of the photolabeled complexes on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis or high-performance liquid chromatography (HPLC). In this study, we prepared a photoactivatable o-galactosamine derivative
2
(GaIN-ANS) by coupling D-galactosamine and ANS-C1, and used GaIN-ANS for the photoaffinity labeling of a galactose-binding lectin (BRA-3) isolated from the acorn barnacle, Megabalanus rosa. The photolabeled BRA-3 was digested with pronase and the resulting photolabeled peptide fragments were isolated by reversed-phase HPLC by monitoring the fluorescence due to the GalN-ANS moiety, and were subjected to amino acid analysis to localize them within the amino-acid s e q u e n c e of BRA-3.
Materials and Methods
Animals Galactose-binding lectins, BRA-2 (Mr 140,000) and BRA-3 (Mr 64,000), were isolated from coelomic fluid of the acorn barnacle, Megabalanus rosa, as previously described (9). 1-Azido-5-naphthalene sulfonyl chloride (ANS-CI) was prepared by our method (7).
Preparation of GaIN-ANS D - G a l a c t o s a m i n e (500 mg, 2.78 mmol), dissolved in 20 mL of 50% acetone in 0.2 M sodium carbonate (pH 9.5) was reacted with ANS-CI (500 mg, 1.87 mmol) in 10 ml of acetone for 2 h at room temperature with stirring. The reaction mixture was concentrated by a rotary evaporator. The precipitate was collected by filtration, washed with distilled water, and dried to yield 570 mg (74% yield) of GaIN-ANS: mp 154-163°C (dec.). Anal. Calcd. for C16HlsN407S (410.4): C, 46.83; H, 4.42; N, 13.65. Found: C, 46.20; H, 4.49; N, 13.37. The A N S d e r i v a t i v e of D - g l u c o s a m i n e ( G I u N - A N S ) was p r e p a r e d b y the method used for GalN-ANS. The yield was 560 mg (73%): mp 173-175°C (dec.). Found: C, 46.43; H, 4.33; N, 13.67.
K. Muramoto and H. Kamiya
Agglutinating Activity The binding affinity of amino-sugar derivatives was estimated by an agglutinating-inhibition assay. BRA was dissolved in 50 mM Tris-HC1 (pH 7.5) containing 0.85% NaCI and l0 mM CaCl 2 (buffer A) at the concentration of 100 p,g/ mL. The solution (25 ~L) was mixed with a p p r o p r i a t e c o n c e n t r a t i o n s of GalN-ANS or GluN-ANS in 25 IxL of 25% dimethylsulfoxide (DMSO) in buffer A. DMSO used to dissolve the ANS derivatives was confirmed to have no effect on the agglutinating activity of BRA at this concentration prior to the experiment. Estimation of agglutinating activity was carried out by the addition of 50 IxL of 2% rabbit erythrocyte suspension in buffer A to the mixture of BRA and photoactivatable sugar derivatives in a multiwell titer plate.
Photolabeling of BRA with GaIN-ANS BRA (250 ~g) was incubated with 5 mM GaIN-ANS in 100 ~L of 50 mM TrisHCI (pH 7.5) containing 10 mM CaCI 2 and 6.25% DMSO in the dark at room temperature for 30 min. In the control experiment, o-galactose was added to the mixture to a final concentration of 0.1 M. The mixture was photolyzed at 0°C for 1 min using a Blak-Ray UV lamp emitting its principal radiation at 366 nm at a distance of 10 cm. Following the addition of 900 IxL of acetone, the mixture was centrifuged for 5 min at 12,000 x g. The pellet was solubilized with 250 ILL of SDS buffer, and divided into two portions. One of them was treated with 5% 13-mercaptoethanol. Twenty p~L of each sample were electrophoresed on 12.5% SDS-polyacrylamide gel by the method of Laemmli (10). After electrophoresis, the gel was removed from the glass plate and dehydrated with methanol to enhance the fluorescence intensity of the product (8). After photography, the gel
Photolabeling of lectin
3
"(o o>
gel filtration on a Sephadex G-25 column (2 x 25 cm) in 50 mM NH4HCO3. A fraction of the photolabeled protein (GalNH OH -IANS-BRA-3) was lyophilized. GAINI ANS-BRA-3 was reduced and carboxaNH2 N3 midomethylated; 2 mg of the protein was GalNI"I2 ANS-CI dissolved in 0.5 mL of 0.5 M Tris-HCl (pH 8.5) containing 6 M guanidine-HCl and 2.5 mM EDTA, and was then reduced with dithiothreitol (4 mg, 50 mM) at room temperature for 1 h. The reI d u c e d p r o t e i n was a l k y l a t e d w i t h monoiodoacetamide (10.2 mg) at room temperature for 20 min in the dark. The S-carboxamidomethylated (CAM) proGaIN-ANS N ~ tein was desalted by gel filtration on a N3 Sephadex G-25 column (2 x 25 cm) in 50 Figure 1. Reactionschemefor the preparation mM NH4HCO 3 and lyophilized. of GalN-ANS. One mg of CAM-GalN-ANS-BRA-3 was digested with pronase (Streptomywas stained for protein with Coomassie ces griseus, Boehringer Mannheim) at an brilliant blue R-250 and rephotographed. enzyme to substrate ratio of 1:50 (w/w) in 1 mL of 0. I M NH4HCO 3 (pH 8.0) at 37°C for 16 h. The digest was separated Isolation of Photolabeled by reversed-phase H P L C on a TSK Peptide Fragments ODS-120T column (5 p,m, 4.6 x 250 mm, Ten mg of BRA-3 was incubated with Toso) using a linear gradient of acetoni5 mM GalN-ANS in 2 mL of 50 mM Tris- trile from 10 to 20% in 0.1% trifluoroaceHCI (pH 8.0) containing 10 mM CaCI2 tic acid at a flow rate of 1.0 mL/min for and 6.25% DMSO for 30 min at room 60 min. The effluent was monitored with temperature. The mixture was photo- an 820-FP spectrofluorometer (Japan lyzed at 0°C for 1 min and subjected to Spectroscopic Co., excitation, 360 nm;
26}
241
221 Hemagg I]ut i nati ng]t i ter 221
0 mM GIuN-ANS
241
/ z
,///j ~/"/,
I
~
/
BRA-2 ~
~
2.5m 5.0mM~
M
~
261
.
l
BRA-3
Figure2. Inhibitionof agglutinatingactivityof BRAsagainstrabbiterythrocytesbyphotoactivatable amino-sugarderivatives,
4
K. Muramoto and H. Kamiya
emission, 480 nm) and with a 875-UV UV/VIS detector (Japan Spectroscopic Co.) at 225 nm. Peptide fractions giving fluorescence were collected and lyophilized after rechromatography by shal-
low gradient elution. Peptides were taken in glass tubes (3 x 60 mm) and hydrolyzed with 80 ~L of 6 M HCI at 110°C for 22 h. Amino-acid analysis was performed as described previously (2).
A-b
A-a
123
1
2
3
B-b
B-a
3 2 K-1 6 K--
!!
~ 71 ii ¸¸¸
2
3
:
~ 1
2
3
1
Figure 3. SDS-12.5% polyacrylamide gel electrophoresis of BRA-3 photolabeled with GalN-ANS. (1) BRA-3 incubated with GalN-ANS, unphotolyzed; (2) BRA-3 incubated with GalN-ANS, photolyzed for 1 min; (3) BRA-3 incubated with GalN-ANS in the presence of 0.1 M o-galactose, photolyzed for 1 min; (A) fluorescent band patterns; (B) Coomassie blue staining patterns; (a) -13-mercaptoethanol; (b) + [~-mercaptoethanol.
Photolabeling of lectin
5
Results and Discussion A photoactivatable galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with ANS-CI (Fig. 1). The photoreactivity of the derivative was tested by changes in the absorption spectra with increasing photolytic time (data not shown). The binding affinities of GalN-ANS and GIuN-ANS to BRAs were assessed by agglutinating-inhibition assay with rabbit erythrocytes. We have shown that the binding of
BRAs labeled with fluorescein isothiocynate to rabbit erythrocyte ghosts was inhibited by several simple sugars including D-galactose and D-galactosamine (9). As shown in Fig. 2, 5 mM GalNANS considerably inhibited BRA agglutination of rabbit erythrocytes, whereas GluN-ANS showed only a marginal effect on agglutination. This result indicates that GalN-ANS has the same degree of affinity to BRAs as D-galactosamine, and is potentially useful for photoaffinity labeling of carbohydrate binding sites of BRAs.
2O s
L_
~0 oc
< D
C ¢-
D
L_ 0
,-r
0
20
40 60 Time(rain) Figure 4. Reversed-phase HPLC separation of photolabeled peptides resulting from pronase digestion of S-carboxamidomethylated BRA-3 photolabeled with GalN-ANS. Chromatographic conditions are described in the text. Top: elution profile detected at 225 nm. Bottom: elution profile detected by fluorescence (Ex: 360 nm; Em: 480 nm).
6
K. Muramoto and H. Kamiya
labeling depends on the relative positioning of an activated nitrene and its counterpart. To localize the binding sites of GAINANS within the known amino-acid sequence of BRA-3, GalN-ANS-BRA-3 was digested to small fragments with pronase (Fig. 4). BRA-3 is composed of four identical subunits of 138 amino acids. Two interchain disulfide bonds cross-link to form dimers at the carboxyl-terminal segment of the subunit (2). Four major peaks having fluorescence were obtained and subjected to amino-acid analysis. Peak A, eluted at the void volume region, did not contain peptide. The amino-acid compositions and positions of the peptides are summarized in Table 1. All of the photolabeled peptides were localized within the carbohydrate recognition domain. In particular, peptide B corresponded to positions 64-77 that include the highly conserved sequence region throughout C-type animal lectins (4) (Fig. 5). This result substantiates the hypothesis that this region is responsible for carbohydrate binding. Peaks C and D could be localized in the same region (positions 35--40) adjacent to the intradisulfide bonds. It may be possible that carbohydrate recognition domain folds to a compact Table 1. Amino Acid Compositions of shape in order to bring these regions Photolabeled Peptide Fragments. close together. Some deviations in the Amino amino-acid c o m p o s i t i o n s might be acids B C D caused by covalent attachment of the ANS moiety to these amino-acid resiAsp 3.0 (3) ~ Glu 0.8 (1) dues. Specification of the modified Thr 0.9 (1) a m i n o - a c i d r e s i d u e s has not b e e n Gly 2.1 (2) 1.1 (1) 1.1 (1) achieved in this study. Ala 0.9 (1) 0.9 (1) Pro 0.6 (1) 0.9 (1) The results of experiments presented Val 1.2 (1) 1.3 (1) 0.6 (1) in this study suggest that GalN-ANS is a lie 2.0 (2) useful photoaffinity probe for an inverteLeu 0.4 (2) Trp ND b (1) brate lectin. Photoactivation of this comHis 0.7 (1) 0.5 (1) 1.0 (1) pound resulted in its covalent incorporaTyr 1.0 (1) 0.7 (1) 0.8 (1) tion into the lectin, as evidenced by Position 64-77 34-40 35-40 SDS-polyacrylamide gel electrophoresis. This incorporation was partially blocked a The numbers in parentheses~are from the sequence. b Not determined. in the presence of D-galactose. This phoBRA-3 was incubated with GAINANS and photolyzed in the presence and absence of 0.1 M D-galactose. SDSpolyacrylamide-gel electrophoretic patterns of the samples are shown in Fig. 3. As expected, BRA-3 incubated with GalN-ANS showed no fluorescent band without photolysis (A-a-l, A-b-l). Coomassie blue staining patterns show that the BRA-3 subunit dimer (32 kDa) cross-linked by disulfide bonds was converted to the monomer (16 kDa) by reduction with 13-mercaptoethanol (Fig. 3). When the incubation mixture was photolyzed, BRA-3 showed a fluorescent band corresponding to the stained band (A-a2, A-b-2). The reason for the unusually broad band in the absence of 13-mercaptoethanol has not been determined. Upon photolysis in the presence of 0.1 M D-galactose, only a weak fluorescent band was observed (A-a-3, A-b-3). This suggests that the photolabeling was inhibited in the presence of a competitive inhibitor. No specific photolabeling was observed with BRA-2 (data not shown). This result, probably due to the structural difference between the binding sites of BRA-2 and BRA-3 (11), is not surprising because the success of photoaffinity
Photolabeling of lectin
7
t
Figure 5. Schematic representation of the positions of the photolabeled peptides within the aminoacid sequence of the subunit of BRA-3. The numbering begins from the amino terminus.
toprobe offers a novel feature for exploring the structure o f lectins. Thus, the photolabeled peptide which is derived from photolabeled complex can be monitored by its fluorescence during the process o f isolation. F u r t h e r m o r e , GaINA N S is small enough to maximize the likelihood of covalent attachment in the
immediate neighborhood of the carbohydrate binding site.
Acknowledgments--This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a Grantin-Aid from the Fisheries Agency, Japan.
References 1. Takahashi, H.; Komano, H.; Kawaguchi, N.; Kitamura, N.; Nakanishi, S.; Natori, S. Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of
the body wall. J. Biol. Chem. 260:1222812233; 1985. 2. Muramoto, K.; Kamiya, H. The amino-acid sequence of a lectin of the acorn barnacle
8
3.
4.
5. 6.
7.
K. Muramoto and H. Kamiya
Megabalanus rosa. Biochim. Biophys. Acta 874:285-295; 1986. Giga, Y.; Ikai, A.; Takahashi, K. The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. J. Biol. Chem. 262:6197--6203; 1987. Drickamer, K. Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem. 263:9557-9560; 1988. Bayley, H.; Knowley, J. R. Photoaffinity labeling. Methods Enzymol. 46:69-114; 1977. Chowdhry, V.; Westheimer, F. H. PhotoatIinity labeling of biological systems. Ann. Rev. Biochem. 48:293-325; 1979. Muramoto, K.; Kamiya, H. Preparation and characterization of photoactivable heterobi-
8.
9.
10. 11.
functional fluorescent reagents. Agric. Biol. Chem. 48:2695-2699; 1984. Muramoto, K.; Kamiya, H. Preparation and characterization of a cleavable photoactivable heterobifunctional fluorescent reagent. Agric. Biol. Chem. 52:547-554; 1988. Muramoto, K.; Ogata, K.; Kamiya, H. Comparison of the multiple agglutinins of the acorn barnacle, Megabalanus rosa. Agric. Biol. Chem. 49:85-93; 1985. Laemmli, U. K. Cleavages of structural proteins during the assembly of the bacteriophage T4. Nature 227:680--685; 1970. Muramoto, K.; Kamiya, H. The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins. Biochim. Biophys. Acta 1039:42-51; 1990.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc
PHOTOAFFINITY LABELING OF AN ACORN BARNACLE LECTIN WITH A PHOTOACTIVATABLE FLUORESCENT REAGENT DERIVATIVE OF D-GALACTOSAMINE Koji Muramoto and Hisao Kamiya School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan (Submitted January 1990; Accepted March 1990)
[]Abstract--A photoactivatable D-galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with 1-azide-5-naphthalene sulfonyl chloride (ANSCI). The derivative (GalN-ANS) inhibited the agglutination activity of an acorn barnacle lectin against rabbit erythrocytes to the same extent as D-galactosamine. We used GalN-ANS for photoaffinity labeling of the lectin. The photolabeled lectin was digested with prDnase and the digest was separated by reversed-phase high-performance liquid chromatography by monitoring fluorescence and uv absorption to isolate the peptide labeled with GaiN-ANS. Amino acid analyses of the labeled peptides revealed that GalN-ANS preferentially covalently labeled two regions in the carbohydrate recognition domain of the lectin. One of them was the highly conserved amino-acid sequence region throughout all calciumdependent animal lectins.
IqKeywords--Lectin; invertebrate lectin; acorn barnacle; Megabalanusrosa; Photoaffinity labeling; Amino acid sequence; Carbohydrate binding site.
Introduction Amino-acid sequences of several invertebrate lectins, isolated from the flesh fly (Sarcophaga peregrina) (1), the acorn barnacle (Megabalanus rosa) (2) and the sea urchin (Anthocidaric crassispina) (3), have been reported. In spite of the fact that the lectins have distinct molecAddress correspondence to Dr. K. Muramoto, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan.
ular weights and subunit structures, they have a domain which is homologous with the carbohydrate-recognition domain of the calcium-dependent (C-type) animal lectins (4). There are certain residues which are conserved in all of the carbohydrate-recognition domains, particularly around some of the cysteine residues and tryptophan residues. They may play important roles in carbohydrate recognition or binding; however, the carbohydrate binding site has yet to be identified within the amino-acid sequence by a different approach. Photoafflnity labeling is a useful tool in the identification of receptors and binding sites for various ligands and in locating functional sites of macromolecules (5,6). We have prepared and characterized a photoactivatable, heterobifunctional fluorescent reagent, l-azido-5-naphthalene sulfonyl chloride (ANS-C1) (7), and used the reagent to prepare photoreactive derivatives of an invertebrate lectin and a trypsin inhibitor (8). Upon exposure to ultraviolet light, the nonfluorescent photoreactive ligands bound to their binding proteins generated reactive nitrene i n t e r m e d i a t e s which could react with neighboring molecules to form fluorescent products. This fluorescence facilitated the detection of the photolabeled complexes on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis or high-performance liquid chromatography (HPLC). In this study, we prepared a photoactivatable o-galactosamine derivative
2
(GaIN-ANS) by coupling D-galactosamine and ANS-C1, and used GaIN-ANS for the photoaffinity labeling of a galactose-binding lectin (BRA-3) isolated from the acorn barnacle, Megabalanus rosa. The photolabeled BRA-3 was digested with pronase and the resulting photolabeled peptide fragments were isolated by reversed-phase HPLC by monitoring the fluorescence due to the GalN-ANS moiety, and were subjected to amino acid analysis to localize them within the amino-acid s e q u e n c e of BRA-3.
Materials and Methods
Animals Galactose-binding lectins, BRA-2 (Mr 140,000) and BRA-3 (Mr 64,000), were isolated from coelomic fluid of the acorn barnacle, Megabalanus rosa, as previously described (9). 1-Azido-5-naphthalene sulfonyl chloride (ANS-CI) was prepared by our method (7).
Preparation of GaIN-ANS D - G a l a c t o s a m i n e (500 mg, 2.78 mmol), dissolved in 20 mL of 50% acetone in 0.2 M sodium carbonate (pH 9.5) was reacted with ANS-CI (500 mg, 1.87 mmol) in 10 ml of acetone for 2 h at room temperature with stirring. The reaction mixture was concentrated by a rotary evaporator. The precipitate was collected by filtration, washed with distilled water, and dried to yield 570 mg (74% yield) of GaIN-ANS: mp 154-163°C (dec.). Anal. Calcd. for C16HlsN407S (410.4): C, 46.83; H, 4.42; N, 13.65. Found: C, 46.20; H, 4.49; N, 13.37. The A N S d e r i v a t i v e of D - g l u c o s a m i n e ( G I u N - A N S ) was p r e p a r e d b y the method used for GalN-ANS. The yield was 560 mg (73%): mp 173-175°C (dec.). Found: C, 46.43; H, 4.33; N, 13.67.
K. Muramoto and H. Kamiya
Agglutinating Activity The binding affinity of amino-sugar derivatives was estimated by an agglutinating-inhibition assay. BRA was dissolved in 50 mM Tris-HC1 (pH 7.5) containing 0.85% NaCI and l0 mM CaCl 2 (buffer A) at the concentration of 100 p,g/ mL. The solution (25 ~L) was mixed with a p p r o p r i a t e c o n c e n t r a t i o n s of GalN-ANS or GluN-ANS in 25 IxL of 25% dimethylsulfoxide (DMSO) in buffer A. DMSO used to dissolve the ANS derivatives was confirmed to have no effect on the agglutinating activity of BRA at this concentration prior to the experiment. Estimation of agglutinating activity was carried out by the addition of 50 IxL of 2% rabbit erythrocyte suspension in buffer A to the mixture of BRA and photoactivatable sugar derivatives in a multiwell titer plate.
Photolabeling of BRA with GaIN-ANS BRA (250 ~g) was incubated with 5 mM GaIN-ANS in 100 ~L of 50 mM TrisHCI (pH 7.5) containing 10 mM CaCI 2 and 6.25% DMSO in the dark at room temperature for 30 min. In the control experiment, o-galactose was added to the mixture to a final concentration of 0.1 M. The mixture was photolyzed at 0°C for 1 min using a Blak-Ray UV lamp emitting its principal radiation at 366 nm at a distance of 10 cm. Following the addition of 900 IxL of acetone, the mixture was centrifuged for 5 min at 12,000 x g. The pellet was solubilized with 250 ILL of SDS buffer, and divided into two portions. One of them was treated with 5% 13-mercaptoethanol. Twenty p~L of each sample were electrophoresed on 12.5% SDS-polyacrylamide gel by the method of Laemmli (10). After electrophoresis, the gel was removed from the glass plate and dehydrated with methanol to enhance the fluorescence intensity of the product (8). After photography, the gel
Photolabeling of lectin
3
"(o o>
gel filtration on a Sephadex G-25 column (2 x 25 cm) in 50 mM NH4HCO3. A fraction of the photolabeled protein (GalNH OH -IANS-BRA-3) was lyophilized. GAINI ANS-BRA-3 was reduced and carboxaNH2 N3 midomethylated; 2 mg of the protein was GalNI"I2 ANS-CI dissolved in 0.5 mL of 0.5 M Tris-HCl (pH 8.5) containing 6 M guanidine-HCl and 2.5 mM EDTA, and was then reduced with dithiothreitol (4 mg, 50 mM) at room temperature for 1 h. The reI d u c e d p r o t e i n was a l k y l a t e d w i t h monoiodoacetamide (10.2 mg) at room temperature for 20 min in the dark. The S-carboxamidomethylated (CAM) proGaIN-ANS N ~ tein was desalted by gel filtration on a N3 Sephadex G-25 column (2 x 25 cm) in 50 Figure 1. Reactionschemefor the preparation mM NH4HCO 3 and lyophilized. of GalN-ANS. One mg of CAM-GalN-ANS-BRA-3 was digested with pronase (Streptomywas stained for protein with Coomassie ces griseus, Boehringer Mannheim) at an brilliant blue R-250 and rephotographed. enzyme to substrate ratio of 1:50 (w/w) in 1 mL of 0. I M NH4HCO 3 (pH 8.0) at 37°C for 16 h. The digest was separated Isolation of Photolabeled by reversed-phase H P L C on a TSK Peptide Fragments ODS-120T column (5 p,m, 4.6 x 250 mm, Ten mg of BRA-3 was incubated with Toso) using a linear gradient of acetoni5 mM GalN-ANS in 2 mL of 50 mM Tris- trile from 10 to 20% in 0.1% trifluoroaceHCI (pH 8.0) containing 10 mM CaCI2 tic acid at a flow rate of 1.0 mL/min for and 6.25% DMSO for 30 min at room 60 min. The effluent was monitored with temperature. The mixture was photo- an 820-FP spectrofluorometer (Japan lyzed at 0°C for 1 min and subjected to Spectroscopic Co., excitation, 360 nm;
26}
241
221 Hemagg I]ut i nati ng]t i ter 221
0 mM GIuN-ANS
241
/ z
,///j ~/"/,
I
~
/
BRA-2 ~
~
2.5m 5.0mM~
M
~
261
.
l
BRA-3
Figure2. Inhibitionof agglutinatingactivityof BRAsagainstrabbiterythrocytesbyphotoactivatable amino-sugarderivatives,
4
K. Muramoto and H. Kamiya
emission, 480 nm) and with a 875-UV UV/VIS detector (Japan Spectroscopic Co.) at 225 nm. Peptide fractions giving fluorescence were collected and lyophilized after rechromatography by shal-
low gradient elution. Peptides were taken in glass tubes (3 x 60 mm) and hydrolyzed with 80 ~L of 6 M HCI at 110°C for 22 h. Amino-acid analysis was performed as described previously (2).
A-b
A-a
123
1
2
3
B-b
B-a
3 2 K-1 6 K--
!!
~ 71 ii ¸¸¸
2
3
:
~ 1
2
3
1
Figure 3. SDS-12.5% polyacrylamide gel electrophoresis of BRA-3 photolabeled with GalN-ANS. (1) BRA-3 incubated with GalN-ANS, unphotolyzed; (2) BRA-3 incubated with GalN-ANS, photolyzed for 1 min; (3) BRA-3 incubated with GalN-ANS in the presence of 0.1 M o-galactose, photolyzed for 1 min; (A) fluorescent band patterns; (B) Coomassie blue staining patterns; (a) -13-mercaptoethanol; (b) + [~-mercaptoethanol.
Photolabeling of lectin
5
Results and Discussion A photoactivatable galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with ANS-CI (Fig. 1). The photoreactivity of the derivative was tested by changes in the absorption spectra with increasing photolytic time (data not shown). The binding affinities of GalN-ANS and GIuN-ANS to BRAs were assessed by agglutinating-inhibition assay with rabbit erythrocytes. We have shown that the binding of
BRAs labeled with fluorescein isothiocynate to rabbit erythrocyte ghosts was inhibited by several simple sugars including D-galactose and D-galactosamine (9). As shown in Fig. 2, 5 mM GalNANS considerably inhibited BRA agglutination of rabbit erythrocytes, whereas GluN-ANS showed only a marginal effect on agglutination. This result indicates that GalN-ANS has the same degree of affinity to BRAs as D-galactosamine, and is potentially useful for photoaffinity labeling of carbohydrate binding sites of BRAs.
2O s
L_
~0 oc
< D
C ¢-
D
L_ 0
,-r
0
20
40 60 Time(rain) Figure 4. Reversed-phase HPLC separation of photolabeled peptides resulting from pronase digestion of S-carboxamidomethylated BRA-3 photolabeled with GalN-ANS. Chromatographic conditions are described in the text. Top: elution profile detected at 225 nm. Bottom: elution profile detected by fluorescence (Ex: 360 nm; Em: 480 nm).
6
K. Muramoto and H. Kamiya
labeling depends on the relative positioning of an activated nitrene and its counterpart. To localize the binding sites of GAINANS within the known amino-acid sequence of BRA-3, GalN-ANS-BRA-3 was digested to small fragments with pronase (Fig. 4). BRA-3 is composed of four identical subunits of 138 amino acids. Two interchain disulfide bonds cross-link to form dimers at the carboxyl-terminal segment of the subunit (2). Four major peaks having fluorescence were obtained and subjected to amino-acid analysis. Peak A, eluted at the void volume region, did not contain peptide. The amino-acid compositions and positions of the peptides are summarized in Table 1. All of the photolabeled peptides were localized within the carbohydrate recognition domain. In particular, peptide B corresponded to positions 64-77 that include the highly conserved sequence region throughout C-type animal lectins (4) (Fig. 5). This result substantiates the hypothesis that this region is responsible for carbohydrate binding. Peaks C and D could be localized in the same region (positions 35--40) adjacent to the intradisulfide bonds. It may be possible that carbohydrate recognition domain folds to a compact Table 1. Amino Acid Compositions of shape in order to bring these regions Photolabeled Peptide Fragments. close together. Some deviations in the Amino amino-acid c o m p o s i t i o n s might be acids B C D caused by covalent attachment of the ANS moiety to these amino-acid resiAsp 3.0 (3) ~ Glu 0.8 (1) dues. Specification of the modified Thr 0.9 (1) a m i n o - a c i d r e s i d u e s has not b e e n Gly 2.1 (2) 1.1 (1) 1.1 (1) achieved in this study. Ala 0.9 (1) 0.9 (1) Pro 0.6 (1) 0.9 (1) The results of experiments presented Val 1.2 (1) 1.3 (1) 0.6 (1) in this study suggest that GalN-ANS is a lie 2.0 (2) useful photoaffinity probe for an inverteLeu 0.4 (2) Trp ND b (1) brate lectin. Photoactivation of this comHis 0.7 (1) 0.5 (1) 1.0 (1) pound resulted in its covalent incorporaTyr 1.0 (1) 0.7 (1) 0.8 (1) tion into the lectin, as evidenced by Position 64-77 34-40 35-40 SDS-polyacrylamide gel electrophoresis. This incorporation was partially blocked a The numbers in parentheses~are from the sequence. b Not determined. in the presence of D-galactose. This phoBRA-3 was incubated with GAINANS and photolyzed in the presence and absence of 0.1 M D-galactose. SDSpolyacrylamide-gel electrophoretic patterns of the samples are shown in Fig. 3. As expected, BRA-3 incubated with GalN-ANS showed no fluorescent band without photolysis (A-a-l, A-b-l). Coomassie blue staining patterns show that the BRA-3 subunit dimer (32 kDa) cross-linked by disulfide bonds was converted to the monomer (16 kDa) by reduction with 13-mercaptoethanol (Fig. 3). When the incubation mixture was photolyzed, BRA-3 showed a fluorescent band corresponding to the stained band (A-a2, A-b-2). The reason for the unusually broad band in the absence of 13-mercaptoethanol has not been determined. Upon photolysis in the presence of 0.1 M D-galactose, only a weak fluorescent band was observed (A-a-3, A-b-3). This suggests that the photolabeling was inhibited in the presence of a competitive inhibitor. No specific photolabeling was observed with BRA-2 (data not shown). This result, probably due to the structural difference between the binding sites of BRA-2 and BRA-3 (11), is not surprising because the success of photoaffinity
Photolabeling of lectin
7
t
Figure 5. Schematic representation of the positions of the photolabeled peptides within the aminoacid sequence of the subunit of BRA-3. The numbering begins from the amino terminus.
toprobe offers a novel feature for exploring the structure o f lectins. Thus, the photolabeled peptide which is derived from photolabeled complex can be monitored by its fluorescence during the process o f isolation. F u r t h e r m o r e , GaINA N S is small enough to maximize the likelihood of covalent attachment in the
immediate neighborhood of the carbohydrate binding site.
Acknowledgments--This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a Grantin-Aid from the Fisheries Agency, Japan.
References 1. Takahashi, H.; Komano, H.; Kawaguchi, N.; Kitamura, N.; Nakanishi, S.; Natori, S. Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of
the body wall. J. Biol. Chem. 260:1222812233; 1985. 2. Muramoto, K.; Kamiya, H. The amino-acid sequence of a lectin of the acorn barnacle
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Megabalanus rosa. Biochim. Biophys. Acta 874:285-295; 1986. Giga, Y.; Ikai, A.; Takahashi, K. The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. J. Biol. Chem. 262:6197--6203; 1987. Drickamer, K. Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem. 263:9557-9560; 1988. Bayley, H.; Knowley, J. R. Photoaffinity labeling. Methods Enzymol. 46:69-114; 1977. Chowdhry, V.; Westheimer, F. H. PhotoatIinity labeling of biological systems. Ann. Rev. Biochem. 48:293-325; 1979. Muramoto, K.; Kamiya, H. Preparation and characterization of photoactivable heterobi-
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functional fluorescent reagents. Agric. Biol. Chem. 48:2695-2699; 1984. Muramoto, K.; Kamiya, H. Preparation and characterization of a cleavable photoactivable heterobifunctional fluorescent reagent. Agric. Biol. Chem. 52:547-554; 1988. Muramoto, K.; Ogata, K.; Kamiya, H. Comparison of the multiple agglutinins of the acorn barnacle, Megabalanus rosa. Agric. Biol. Chem. 49:85-93; 1985. Laemmli, U. K. Cleavages of structural proteins during the assembly of the bacteriophage T4. Nature 227:680--685; 1970. Muramoto, K.; Kamiya, H. The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins. Biochim. Biophys. Acta 1039:42-51; 1990.