- Email: [email protected]
Complete Amino Acid Sequence of the B Chain of Mistletoe Lectin I
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
246, 596–601 (1998)
RC988670
Complete Amino Acid Sequence of the B Chain of Mistletoe Lectin I Montserrat Huguet Soler, Stanka Stoeva, and Wolfgang Voelter1 Abteilung fu¨r Physikalische Biochemie des Physiologisch-chemischen Instituts der Universita¨t Tu¨bingen, Germany
Received April 14, 1998
The primary structure of the B chain of mistletoe lectin I, the component of a commercially available extract from Viscum album exhibiting immunomodulatory capacity, was established based on amino acid sequence analysis of the protein and peptides derived from its enzymatic digestion. It is composed of 264 residues, including seven cysteine residues and three N-linked carbohydrate chains. The amino acid sequence of MLB shows a high homology with those from other structurally related galactoside-specific lectins such as ricin and abrin with 169 and 146 identities, respectively. These results are of crucial importance in order to understand the biological activity of ML-I. q 1998 Academic Press Key Words: mistletoe lectin I; Immunomodulation; galactose-binding activity; amino acid sequence; Viscum album.
Proprietary extracts of mistletoe (Viscum album) are widely applied in the treatment of human cancer because of the evidence that it exhibits immunomodulatory capacity (1, 2). Among many other components, crude mistletoe extract has been reported to contain three classes of lectins with different molecular weights and different carbohydrate specificities (ML-I, ML-II and ML-III) (3, 4). The immunomodulatory potency of this extract has been attributed to the existence of the major mistletoe lectin (ML-I) (5). Several investigations proved that regular administration of the optimal dosis of ML-I (1 ng/kg body weight) results in a statistically significant increase of the number of, both, natural killer (NK) cells and helper T-lymphocytes (6-8), which are generally believed to be involved in antitumor effects. 1 Corresponding author: Prof. Dr. Dr. h.c. Wolfgang Voelter, Abteilung fu¨r Physikalische Biochemie des Physiologisch-chemischen Instituts der Universita¨t Tu¨bingen, Hoppe-Seyler-Str. 4, D-72076 Tu¨bingen, Germany. Fax: /49-7071-293348. E-mail: stanka.stoeva@ uni-tuebingen.de. Abbreviations used: ML-I, mistletoe lectin I; MLA, A chain of mistletoe lectin I; MLB, B chain of mistletoe lectin I; MALDI-MS, matrixassisted laser desorption ionization mass spectrometry.
0006-291X/98 $25.00
Moreover, ML-I administration yields a notable increment of granulocytes’ phagocytic activity and an enhanced expression of receptors for interleukin (IL)-2 and B-cells (8-12). Controlled increases of these non-specific defence mechanisms may induce clinically beneficial immunomodulation in the treatment of cancer (13). ML-I consists of two different subunits, the cytotoxic A chain (MLA) and the galactose-binding B chain (MLB), linked by a disulfide bond (14). MLA (29 kDa) inhibits protein synthesis (15-17) at the ribosomal level by the same mechanism as the ricin A chain (18). MLA enters the cell by endocytosis after binding of the subunit MLB (34 kDa) to cell surface glycoconjugates containing terminal galactose residues. It has been observed that cellbinding of ML-I is followed by modulation of intracellular processes such as Ca2/ mobilization and protein and phospholipid phosphorylation (19), which are absolutely necessary for protein endocytosis (20, 21). The finding that the carbohydrate-binding subunit (MLB) alone is capable to stimulate in vitro cytokine release (1) supports the assumption that the carbohydrate-binding activity of ML-I may be related with its immunomodulatory potency. Despite of the present lack of knowledge about the effector mechanism(s) involved in this biological activity, ML-I could recognize and bind to certain glycoconjugates with terminal galactose residues on the surface of immunologically active cells and these specific lectin-carbohydrate interactions could be responsable for eliciting such an immunoresponse (22). Presumably, the ability of ML-I to activate non-specific defense mechanisms in animals can be compared with the crucial role of lectins in the defence of plants against phytopathogens. Actually, the importance of lectins as cell recognition molecules in a wide range of biological systems (23) and in particular in the control of pathological processes (24) is known since many years. Two examples which support evidence for the proposed function of lectins in the protection of plants against fungal, bacterial and viral pathogens during germination and early growth of the seedlings are: 1) the binding of lectins to various fungi and their ability to inhibit fungal growth and germination
596
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(25) and 2) the presence of lectins at the potential site of invasion by infectious agents (26). In order to better understand the mechanisms of action of ML-I, knowledge about the precise biochemical structure of this active substance is essential. As the first step for the structural characterization of ML-I, we recently reported the complete primary structure of its cytotoxic subunit MLA (27). In this paper we present the amino acid sequence of the galactose-binding chain MLB, which completes the full characterization of the primary structure of this lectin. 2. MATERIALS AND METHODS 2.1. Protein isolation. ML-I was isolated from the mistletoe extract (Eurixor, medisculab Arzneimittel GmbH, Fellbach, Germany) as described (27). Disulfide bridges linking both chains could be cleaved by incubation of the immobilized lectin on the gel material with 5% b-mercaptoethanol (v/v) at room temperature overnight. After reduction, MLB was eluted from the column with 0.2 M lactose solution (2.0 ml/min) (28). The fractions containing protein were pooled, dialyzed against deionized water and lyophilized. 2.2. Protein identification. SDS-polyacrylamide gel electrophoresis of the isolated lectin was carried through with a PhastGel system (Pharmacia) (29). 2.3. Amino acid analyis. MLB was hydrolyzed in vacuo at 1107C in 6 M HCl for 24 h. The amino acid composition was determined with an Eppendorf/Biotronik LC 3000 amino acid analyzer. 2.4. Enzymatic cleavage. MLB was dissolved in 100 mM Tris-HCl buffer (pH 8.5) with up to 1 M urea and 10% (v/v) acetonitrile for solubilization and digested with trypsin (sequencing grade, Boehringer Mannheim) at 377C for 4 h at an enzyme:protein ratio of 1:30 (w/w). Cleavage with endoproteinase Asp-N (sequencing grade, Boehringer Mannheim, Germany) was performed at an enzyme:protein ratio of 1:1000 (w/w) in 50 mM sodium phosphate (pH 8.0) containing 2 M urea at 377C for 18 h. Digestion with chymotrypsin (sequencing grade, Boehringer Mannheim) was carried out at an enzyme:protein ratio of 1:20 (w/w) in 100 mM Tris-HCl buffer (pH 7.8) at 377C for 15 min. After enzymatic digestion, the reaction mixture was acidified up to pH 4.8 with 0.1 M HCl and centrifugated. Peptide fragments were isolated from the mixture as described bellow. 2.5. Peptide purification. Peptides were separated by HPLC (Biotronik,Maintal) on a Nucleosil C18 column (4 mm 1 250 mm) and eluted at a flow rate of 1 ml/min, using a linear gradient of acetonitrile containing 0.15% (v/v) TFA. The UV absorbance was monitored at 214 nm. 2.6. Reduction and alkilation of the protein. Fractions containing cystine residues (1-10 mg) were dissolved in 50 ml 6 M guanidineHCl buffered with 0.25 M Tris (pH 8.5) and subjected to reduction with 2.5 mL 10% b-mercaptoethanol (v/v) in the dark for 2h at room temperature. Modification of cysteine residues was performed by adding 2 mL 4-vinylpyridine to the mixture and incubating at the same conditions. After incubation, samples were immediately desalted by HPLC as indicated above. 2.7. Amino acid sequence determination. The amino acid sequences of the peptides were determined by automated Edman degradation on an Applied Biosystems sequencer (model 473A), equipped with an on-line phenylthiohydantoin amino acid analyzer. Peptides (50-200 pmol) were dissolved in 0.1% (v/v) TFA and loaded onto polybrene coated-filters. 2.8. Mass spectrometry. Mass spectral analyses were obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry with a Kratos equipment (Shimadzu, Europe). Peptides (10-50
FIG. 1. Amino acid sequence of MLB and sequencing strategy. T, C and D indicate peptides analysed from trypsin, chymotrypsin and Asp-N digestions, respectively. Only peptides useful for sequence and overlap determination are shown.
pmol) were dissolved in 0.1% (v/v) TFA/H2O. a-Cyano-4-hydroxycinnamic acid was employed as matrix solution.
3. RESULTS The complete amino acid sequence of the B chain of ML-I with 264 amino acid residues was determined by cleavage of the protein with trypsin, endoproteinase Asp-N and chymotrypsin, respectively. Fig. 1 summarizes the sequencing results from the three sets of peptides. It includes only those peptides necessary to prove each position unambiguously (approximately one third of the peptides analyzed). Peptides from each digestion were separated by RP-HPLC and characterized by sequence and mass spectrometric analysis. All peptides could be sequenced to the end. The amino acid composition obtained by acid hydrolysis of the protein is in
597
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Amino Acid Composition of Intact MLB Amino acid
Residues
Asp Asn Thr Ser Glu Gln Pro Gly Ala 1/2 Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp
39.2 (19) (20) 22.1 (22) 20.3 (21) 20.2 (6) (14) 9.6 (10) 26.1 (25) 15.0 (15) n.d. (7) 19.3 (19) 4.7 (5) 17.3 (18) 17.0 (17) 6.3 (7) 5.1 (5) 4.1 (1) 8.7 (9) 15.3 (15) n.d. (9)
Total
(264)
Note. Numbers in parentheses are the residues determined after sequence analysis. n.d., not determined.
complete agreement with the amino acid sequence determined (Table 1). However, the molecular weight of MLB calculated on the basis of sequence data (28,950 Da) is about 5,000 Da less than the apparent molecular weight of the purified lectin estimated by SDS-PAGE (34,000 Da) (30). We attribute the difference between these two molecular weight values to the existence of three sugar chains covalently bound to the protein, as described below. The N-terminal sequence analysis of intact MLB up to position 20 led to an unambiguous identification of only some amino acid residues, since several positions were not clear. The amino acid sequence of MLB was mainly obtained following the isolation and identification of an almost complete set of tryptic peptides. Digestion with trypsin yielded 19 peptides, from which most of the sequence could be established. Alignment of sequence data determined from this cleavage was achieved by comparison with sequences of other homologous galactose-binding lectins, such as ricin and abrin (31, 32). Specific bond cleavages after the residues Arg21 and Lys170 were not detected. Digestion of MLB with endoproteinase Asp-N yielded peptides resulting only from the cleavage of the protein at the terminal region. This fact could be probably due to its high folding level. Although a core fraction could be precipitated by titration with HCl after enzymatic hydrolysis, all attempts to further cleave this isolated fraction were unsuccessful. Despite the lack of new sequence results, the sequence of D4 confirmed the alignments of T5 and
T6 and the sequences of D17, T18, T19 and T20, respectively. The presence of the fragments D17 and D18 indicates that the enzyme preparation used cleaved on the amino side of Ala240 and Thr253, representing atypical cleavage sites for endoproteinase Asp-N. Further sequence information could be obtained from the analysis of peptides generated by chymotrypsin. After digestion, the reaction mixture was separated in two approximately equal portions. One portion was fractionated directly by RP-HPLC. The other one was first subjected to reduction and alkylation with 4-vinylpyridine. The analysis of both sets of peptides provided the complete primary structure of MLB and it achieved the lacking overlaps between some tryptic fragments. In addition to the amino acid sequence of MLB, the sequence data obtained provide evidence for the existence of three potencial N-glycosylation sites, which could be confirmed by MALDI-MS analysis. Three different sequences could be identified as glycosylation patterns. These are Asn61-Gly62-Ser63, Asn96-Gly97Thr98 and Asn136-Asp137-Thr138, respectively. Information about the molecular weight of the carbohydrate moieties, covalently bound to the lectin, could be obtained by means of mass spectrometry. The data are summarized in Table 2, resulting from the mass difference between the observed mass for the fraction containing the glycosylation site and the calculated mass from the peptide sequence. The third N-glycosylation site (Asn136-Asp137-Thr138) appears to present two possible bound carbohydrate moieties, since two fragments, yielding the same amino acid sequence, but different mass spectral data, could be isolated. These results lead to the conclusion that the overall carbohydrate content of MLB is about 12% (w/w). The above experiments enable us to identify some heterogeneities along the amino acid sequence of MLB as well. Analyzed sequence data show 12 conservative substitutions, most of them located in the C-terminal region of the protein (Table 3). Detected replacements involve generally two structurally not related amino acid residues at the same position. However, while some substitutions suppose the replacement of one single amino acid residue (Glu56 r Asn56or Asn231 r Ser231), other ones involve several amino acid residues (Asn231-Gly232-Leu233 r Lys231-Gly232-Pro233). These results demonstrate that
TABLE 2
Glycosylation Sites and Detected Molecular Masses of N-Linked Carbohydrate Moieties of MLB Glycosylation site
Detected mass (Da)
Asn61-Gly62-Ser63 Asn96-Gly97-Thr98 Asn136-Asp137-Thr138 Asn136-Asp137-Thr138
1172.1 1378.9 1220.5 1382.4
598
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 3
Detected Substitutions among the Amino Acid Sequence of the Different Isoforms of MLB Asn18 r Ser18 Gly56 r Asn56 Gly157 r Gln157 Ser195 r Val195 Gly224 r Tyr224 Asn231 r Ser231 or Thr231 Asn231-Gly232-Leu233 r Lys231-Gly232-Pro233 Gly232-Leu233-Ala234-Met235 r r Ser232-Leu233-Met234-Val23
MLB, like MLA (27), occurs in several isoforms, supporting further evidence for the previously reported existence of different isolectins of ML-I (33). 4. DISCUSSION The major purpose of this work was to characterize the primary structure of MLB, the galactose-specific subunit of ML-I. The complete amino acid sequence was established by means of enzymatic digestion of the protein and Edman degradation of the generated fragments, following the strategy illustrated in Fig. 1. Determined amino acid sequences were confirmed by mass spectrometric analysis. Obtained sequence data show that MLB is composed of 264 residues including seven half-cystine residues. By comparison of the amino acid sequence of MLB with the primary and secondary structure of the ricin B chain (34), it can be assumed that the residue Cys5 links MLB to MLA by an intermolecular disulfide bridge, and that the six remaining residues build three intramolecular disulfide bridges. The presence of internal disulfide bridges in MLB has been already reported (3). A more extensive examination of the amino acid sequence of MLB predicts three potencial N-glycosylation sites. As glycosylation patterns we could identify the following sequences: Asn61-Gly62-Ser63, Asn96-Gly97Thr98 and Asn136-Asp137-Thr138 . Mass spectrometric analysis of the fragments enclosing putative N-glycosylation sequences confirmed the existence of N-linked carbohydrate chains at position 61, 96 and 136, respectively (Table 2). Our detected molecular masses for the carbohydrate chains covalently bound to MLB are in excellent agreement with those isolated from ML-I and calculated on the basis of the chemical structure (35). The presented data suggest that the carbohydrate chain linked to the residue Asn96 can also appear glycosylating the residue Asn136 . A similar heterogenity in the N-linked sugar structures of ricin D and abrin a has been earlier described (36, 37). The same glycosylation pattern Asn-Gly-Ser with an almost identical molecular weight for the bound carbohydrate chain has been recently reported on MLA (27). From these results it
can be deduced that the same carbohydrate moiety may presumably be present in, both, A and B chains. The relative high carbohydrate content of MLB (about 12% by weight) is likely to be related with the importance of N-linked oligosaccharides maintaining a biologically active conformation of the protein. It has been observed that the recombinant and N-glycosylated ricin B chain (RTB), expressed in Spodoptera frugiperda or in Xenopus oocytes, was, both, biologically active and stable (38, 39). These results contrast with recombinant RTB produced in Escherichia coli, which, although initially soluble and biologically active, was unstable and tended to aggregate rapidly (39, 40). Recombinant RTB, partially glycosylated, initially displays lectin activity, but it is also relatively unstable (41). The primary structure of MLB presented was compared with those of other structurally and biologically related lectins like ricin and abrin (31, 32). An interesting common feature of these lectins is that they all exhibit specificity for galactose residues. After performing sequence alignment, the amino acid sequence of MLB shows a high overall similarity to those of ricin-D (RTB) and abrin-a (ABB) with 169 and 146 identities, respectively (Fig. 2). The most rigorously conserved part of the molecule extends the first half of the protein, as the sequence comparison clearly reveals. Thus, it is worth mentioning that MLB contains a third N-glycosylation site (Asn136-Asp137-Thr138), which is not present in RTB and ABB, and one intramolecular disulfide bridge less due to the replacement of Cys r Arg at position 21 and Cys r Ser at position 40. Internal disulfide bridges are believed to stabilize the V-loops in RTB (42). A more direct approach towards the sequence alignment of these lectins shows that the residues involved in the sugar-binding sites of RTB are all conserved in the amino acid sequence of MLB. There are two galactose binding sites on RTB which have been reported to have similar (43) or different affinities (44) and to be capable of acting independently (45). Primary structure analysis (46) and X-ray crystallographic studies have shown that RTB is composed of two similar domains, I and II, each carrying one galactose binding site (47). Further refinement of the ricin crystal structure showed both domains to be composed of three related subdomains, a, b and g, each thought to be derived from an ancient galactose binding peptide (48, 49). However, only two of this subdomains, 1a and 2g, retain their ability to bind galactose. Both contain a conserved tripeptide with the sequence Asp-Val-Arg, not present in the other 4 subdomains (48), and key binding residues, hydrogen-bonded to the sugar moiety. Subdomain 1a of RTB involves amino acid residues Asp22, Gln35, Trp37, Asn46 and Gln47, and subdomain 2g residues Asp234, Ile246, Tyr248, Asn255 and Gln256 (50). All these residues are conserved at the corresponding positions of the amino acid sequence of MLB (Fig. 2). The above mentioned conserved tripeptide is also present in the presumably first sugar binding
599
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Comparison of amino acid sequences of some galactose binding lectins. MLB is the ML-I B chain. RTB is the ricin-D B chain (31) and ABB is the abrin-a B chain (32). Sequences are aligned to maximize their similarities. Conserved residues among these proteins are highlighted. N-glycosylation sites are boxed. Residues marked with an * are those that are involved in the galactose binding sites in the 1a and 2g subdomains of the protein.
domain of MLB (Asp23-Val24-Arg25), but not in the second one (Asp236-Val237-Ala238). The replacement Arg r Ala at the third position might not result in significant alterations, since only Asp residues of the tripeptide sequence are described to stabilize the pocket formed by key binding residues in each subdomain, where galactose lies (50). All these results, together with the very high overall primary structure homology (about 65% identities) and the similar protein conformation observed for both MLB and RTB (51), provide further evidence that MLB and
RTB may probably have very similar polypeptide folding, and accordingly the carbohydrate binding sites could occur at similar positions. This findings correlate with the proposal that these lectins could originate from similar evolution processes, and thus an evolutionary relationship among these lectins could be established. In conclusion, the data presented here, as well as earlier data about the amino acid sequence of MLA (27), are of crucial importance in order to better understand the biological activities of ML-I and for the design
600
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of more effective therapeutic substances. Therefore, the hypothesis that the immunostimulating potency of MLB could be also modulated by the presence of the toxic MLA chain or/and other components of the mistletoe extracts should not be excluded. REFERENCES 1. Hajto, T., Hostanska, K., and Gabius, H.-J. (1989) Cancer Res. 49, 4803–4808. 2. Hajto, T., Hostanska, K., and Gabius, H.-J. (1990) Therapeutikon 4, 135–145. 3. Olsnes, S., Stirpe, F., Sandvig, K., and Pihl, A. (1982) J. Biol. Chem. 257, 13263–13270. 4. Franz, H., Ziska, P., and Kindt, A. (1981) Biochem. J. 195, 481– 484. 5. Beuth, J., Ko, H.-L., Tunggal, L., Buss, G., Jeljaszewicz, J., Steuer, M. K., and Pulverer, G. (1994) Arzneim.-Forsch./Drug Res. 44(II), 1255–1258. 6. Beuth, J., Ko, H. L., Tunggal, L., Gabius, H.-J., Steuer, M., Uhlenbruck, G., and Pulverer, G. (1993) Med. Welt 44, 217–220. 7. Beuth, J., Ko, H. L., Tunggal, L., Geisel, J., and Pulverer, G. (1993) Arzneim.-Forsch./Drug Res. 43(I), 166–169. 8. Beuth, J., Ko, H. L., Gabius, H.-J., Burrichter, H., Oette, K., and Pulverer, G. (1992) Clin. Investig. 70, 658–661. 9. Hajto, T., Hostanska, K., Frei, K., Rordorf, C., and Gabius, H.-J. (1990) Cancer Res. 50, 3322–3326. 10. Beuth, J., Ko, H.-L., and Pulverer, G. (1993) Dtsch. Zschr. Onkol. 25, 73–76. 11. Heiny, B.-M., and Beuth, J. (1994) Anticancer Res. 14, 1339– 1342. 12. Beuth, J., Stoffel, B., Ko, H.-L. Buss, G., and Pulverer, G. (1995) Arzneim.-Forsch./Drug Res. 45(I), 505–507. 13. Beuth, J., Ko, H.-L., Tunggal, L., Steuer, M. K., Geisel, J., Jeljaszewicz, J., and Pulverer, G. (1993) In Vivo 7, 407–410. 14. Ziska, P., Franz, H., and Kindt, A. (1978) Experientia 34, 123– 124. 15. Stirpe, F., Legg, R. F., Onyon, L. J., Ziska, P., and Franz, H. (1980) Biochem. J. 190, 843–845. 16. Stirpe, F., Sandvig, K. Olsnes, S., and Pihl, A. (1982) J. Biol. Chem. 257, 13271–13277. 17. Franz, H. (1985) in Lectins: Biology, Biochemistry, Clinical Biochemistry (Bøg-Hansen, T. C., and Breborowicz, J., Eds.), Vol. IV, pp. 463–472, Walter de Gruyter, Berlin. 18. Endo, Y., Tsurugi, K., and Franz, H. (1988) FEBS Lett. 231, 378–380. 19. Gabius, H.-J., Walzel, H., Joshi, S. S., Kruip, J., Kojima, S., Gerke, V., Kratzin, H., and Gabius, S. (1992) Anticancer Res. 12, 669–676. 20. Sanwig, K., and Olsnes, S. (1982) J. Biol. Chem. 257, 7495– 7503. 21. Sanwig, K., and Olsnes, S. (1982) J. Biol. Chem. 257, 7504– 7513. 22. Beuth, J., Ko, H. L., and Pulverer, G. (1994) Dtsch. Apoth. Zeitung 25, 17–28. 23. Sharon, N., and Lis, H. (1989) Science 246, 227–246.
24. Lis, H., and Sharon, N. (1986) Ann. Rev. Biochem. 55, 35–67. 25. Mirelman, D., Galun, E., Sharon, N., and Lotan, R. (1975) Nature 256, 414–416. 26. Mishkind, M., Raikhel, N. V., Palevitz, B. A., and Keegstra, K. (1982) J. Cell Biol. 92, 753–764. 27. Huguet Soler, M., Stoeva, S., Schwamborn, C., Wilhelm, S., Stiefel, T., and Voelter, W.(1996) FEBS Letters 399, 153–157. 28. Franz, H., Ziska, P., and Kindt, A. (1982) in Lectins: Biology, Biochemitsry, Clinical Biochemistry (Bøg-Hansen, T. C., Ed.) Vol. II, pp. 171–176, Walter de Gruyter, Berlin. 29. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406– 4412. 30. Luther, P., Theise, H., Chatterjee, B., Karduck, D., and Uhlenbruck, G. (1980) Int. J. Biochem. 11, 429–435. 31. Halling, K. C., Halling, A. C., Murray, E. E., Ladin, B. F., Houston, L. L., and Weaver, R. F. (1985) Nucl. Acids Res. 13, 8019– 8033. 32. Hung, C.-H., Lee, M.-C., Lee, T.-C., and Lin, J.-Y. (1993) J. Mol. Biol. 229, 263–267. 33. Gabius, S., Kaiser, K., and Gabius, H.-J. (1991) Dtsch. Zschr. Onkol. 5, 113–119. 34. Robertus, J. D., and Ready, M. P. (1984) J. Biol. Chem. 259, 13953–13956. 35. Debray, H., Wieruszeski, J.-M., Strecker, G., and Franz, H. (1992) Carbohydrate Res. 236, 135–143. 36. Kimura, Y., Hase, S., Kobayashi, Y., Kyogoku, Y., Ikenaka, T., and Funatsu, G. (1988) J. Biochem. 103, 944–949. 37. Kimura, Y., Hase, S., Kobayashi, Y., Ikenaka, T., and Funatsu, G. (1988) Biochim. Biophys. Acta 966, 150–159. 38. Frankel, A., Roberts, H., Afrin, L., Vesely, J., and Willingham, M. (1994) Biochem. J. 303, 787–794. 39. Richardson, P. T., Hussain, K., Wooland, H. R., Lord, J. M., and Roberts, L. M. (1991) Carbohydrate Res. 213, 19–25. 40. Hussain, K., Bowler, C., Roberts, L. M., and Lord, J. M. (1988) FEBS Letters 244, 383–387. 41. Wales, R., Richardson, P. T., Roberts, L. M., Wooland, H. R., and Lord, J. M. (1991) J. Biol. Chem. 266, 19172–19179. 42. Leszczynski, J. F., and Rose, G. D. (1986) Science 234, 849–855. 43. Houston, L. L., and Dooley, T. P. (1982) J. Biol. Chem. 257, 4147–4151. 44. Zentz, C., Fre´noy, J.-P., and Bourrillon, R. (1978) Biochim. Biophys. Acta 536, 18–26. 45. Wales, R., Richardson, P. T., Roberts, L. M., and Lord, J. M. (1992) Arch. Biochem. Bioph. 294, 291–296. 46. Roberts, L. M., Lamb, F. I., Pappin, D. J. C., and Lord, J. M. (1985) J. Biol. Chem. 260, 15682–15686. 47. Villafranca, J. E., and Robertus, J. D. (1981) J. Biol. Chem. 256, 554–556. 48. Rutenber, E., Ready, M., and Robertus, J. D. (1987) Nature 326, 624–626. 49. Montfort, W., Villafranca, J. E., Monzingo, A. F., Ernst, S. R., Katzin, B., Rutenber, E., Xuong, N. H., Hamlin, R., and Robertus, J. D. (1987) J. Biol. Chem. 262, 5398–5403. 50. Rutenber, E., and Robertus, J. D. (1991) Proteins: Structure, Function and Genetics 10, 260–269. 51. Bushueva, T. L., and Tonevitsky, A. G. (1988) FEBS Letters 229, 119–122.
601
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
246, 596–601 (1998)
RC988670
Complete Amino Acid Sequence of the B Chain of Mistletoe Lectin I Montserrat Huguet Soler, Stanka Stoeva, and Wolfgang Voelter1 Abteilung fu¨r Physikalische Biochemie des Physiologisch-chemischen Instituts der Universita¨t Tu¨bingen, Germany
Received April 14, 1998
The primary structure of the B chain of mistletoe lectin I, the component of a commercially available extract from Viscum album exhibiting immunomodulatory capacity, was established based on amino acid sequence analysis of the protein and peptides derived from its enzymatic digestion. It is composed of 264 residues, including seven cysteine residues and three N-linked carbohydrate chains. The amino acid sequence of MLB shows a high homology with those from other structurally related galactoside-specific lectins such as ricin and abrin with 169 and 146 identities, respectively. These results are of crucial importance in order to understand the biological activity of ML-I. q 1998 Academic Press Key Words: mistletoe lectin I; Immunomodulation; galactose-binding activity; amino acid sequence; Viscum album.
Proprietary extracts of mistletoe (Viscum album) are widely applied in the treatment of human cancer because of the evidence that it exhibits immunomodulatory capacity (1, 2). Among many other components, crude mistletoe extract has been reported to contain three classes of lectins with different molecular weights and different carbohydrate specificities (ML-I, ML-II and ML-III) (3, 4). The immunomodulatory potency of this extract has been attributed to the existence of the major mistletoe lectin (ML-I) (5). Several investigations proved that regular administration of the optimal dosis of ML-I (1 ng/kg body weight) results in a statistically significant increase of the number of, both, natural killer (NK) cells and helper T-lymphocytes (6-8), which are generally believed to be involved in antitumor effects. 1 Corresponding author: Prof. Dr. Dr. h.c. Wolfgang Voelter, Abteilung fu¨r Physikalische Biochemie des Physiologisch-chemischen Instituts der Universita¨t Tu¨bingen, Hoppe-Seyler-Str. 4, D-72076 Tu¨bingen, Germany. Fax: /49-7071-293348. E-mail: stanka.stoeva@ uni-tuebingen.de. Abbreviations used: ML-I, mistletoe lectin I; MLA, A chain of mistletoe lectin I; MLB, B chain of mistletoe lectin I; MALDI-MS, matrixassisted laser desorption ionization mass spectrometry.
0006-291X/98 $25.00
Moreover, ML-I administration yields a notable increment of granulocytes’ phagocytic activity and an enhanced expression of receptors for interleukin (IL)-2 and B-cells (8-12). Controlled increases of these non-specific defence mechanisms may induce clinically beneficial immunomodulation in the treatment of cancer (13). ML-I consists of two different subunits, the cytotoxic A chain (MLA) and the galactose-binding B chain (MLB), linked by a disulfide bond (14). MLA (29 kDa) inhibits protein synthesis (15-17) at the ribosomal level by the same mechanism as the ricin A chain (18). MLA enters the cell by endocytosis after binding of the subunit MLB (34 kDa) to cell surface glycoconjugates containing terminal galactose residues. It has been observed that cellbinding of ML-I is followed by modulation of intracellular processes such as Ca2/ mobilization and protein and phospholipid phosphorylation (19), which are absolutely necessary for protein endocytosis (20, 21). The finding that the carbohydrate-binding subunit (MLB) alone is capable to stimulate in vitro cytokine release (1) supports the assumption that the carbohydrate-binding activity of ML-I may be related with its immunomodulatory potency. Despite of the present lack of knowledge about the effector mechanism(s) involved in this biological activity, ML-I could recognize and bind to certain glycoconjugates with terminal galactose residues on the surface of immunologically active cells and these specific lectin-carbohydrate interactions could be responsable for eliciting such an immunoresponse (22). Presumably, the ability of ML-I to activate non-specific defense mechanisms in animals can be compared with the crucial role of lectins in the defence of plants against phytopathogens. Actually, the importance of lectins as cell recognition molecules in a wide range of biological systems (23) and in particular in the control of pathological processes (24) is known since many years. Two examples which support evidence for the proposed function of lectins in the protection of plants against fungal, bacterial and viral pathogens during germination and early growth of the seedlings are: 1) the binding of lectins to various fungi and their ability to inhibit fungal growth and germination
596
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(25) and 2) the presence of lectins at the potential site of invasion by infectious agents (26). In order to better understand the mechanisms of action of ML-I, knowledge about the precise biochemical structure of this active substance is essential. As the first step for the structural characterization of ML-I, we recently reported the complete primary structure of its cytotoxic subunit MLA (27). In this paper we present the amino acid sequence of the galactose-binding chain MLB, which completes the full characterization of the primary structure of this lectin. 2. MATERIALS AND METHODS 2.1. Protein isolation. ML-I was isolated from the mistletoe extract (Eurixor, medisculab Arzneimittel GmbH, Fellbach, Germany) as described (27). Disulfide bridges linking both chains could be cleaved by incubation of the immobilized lectin on the gel material with 5% b-mercaptoethanol (v/v) at room temperature overnight. After reduction, MLB was eluted from the column with 0.2 M lactose solution (2.0 ml/min) (28). The fractions containing protein were pooled, dialyzed against deionized water and lyophilized. 2.2. Protein identification. SDS-polyacrylamide gel electrophoresis of the isolated lectin was carried through with a PhastGel system (Pharmacia) (29). 2.3. Amino acid analyis. MLB was hydrolyzed in vacuo at 1107C in 6 M HCl for 24 h. The amino acid composition was determined with an Eppendorf/Biotronik LC 3000 amino acid analyzer. 2.4. Enzymatic cleavage. MLB was dissolved in 100 mM Tris-HCl buffer (pH 8.5) with up to 1 M urea and 10% (v/v) acetonitrile for solubilization and digested with trypsin (sequencing grade, Boehringer Mannheim) at 377C for 4 h at an enzyme:protein ratio of 1:30 (w/w). Cleavage with endoproteinase Asp-N (sequencing grade, Boehringer Mannheim, Germany) was performed at an enzyme:protein ratio of 1:1000 (w/w) in 50 mM sodium phosphate (pH 8.0) containing 2 M urea at 377C for 18 h. Digestion with chymotrypsin (sequencing grade, Boehringer Mannheim) was carried out at an enzyme:protein ratio of 1:20 (w/w) in 100 mM Tris-HCl buffer (pH 7.8) at 377C for 15 min. After enzymatic digestion, the reaction mixture was acidified up to pH 4.8 with 0.1 M HCl and centrifugated. Peptide fragments were isolated from the mixture as described bellow. 2.5. Peptide purification. Peptides were separated by HPLC (Biotronik,Maintal) on a Nucleosil C18 column (4 mm 1 250 mm) and eluted at a flow rate of 1 ml/min, using a linear gradient of acetonitrile containing 0.15% (v/v) TFA. The UV absorbance was monitored at 214 nm. 2.6. Reduction and alkilation of the protein. Fractions containing cystine residues (1-10 mg) were dissolved in 50 ml 6 M guanidineHCl buffered with 0.25 M Tris (pH 8.5) and subjected to reduction with 2.5 mL 10% b-mercaptoethanol (v/v) in the dark for 2h at room temperature. Modification of cysteine residues was performed by adding 2 mL 4-vinylpyridine to the mixture and incubating at the same conditions. After incubation, samples were immediately desalted by HPLC as indicated above. 2.7. Amino acid sequence determination. The amino acid sequences of the peptides were determined by automated Edman degradation on an Applied Biosystems sequencer (model 473A), equipped with an on-line phenylthiohydantoin amino acid analyzer. Peptides (50-200 pmol) were dissolved in 0.1% (v/v) TFA and loaded onto polybrene coated-filters. 2.8. Mass spectrometry. Mass spectral analyses were obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry with a Kratos equipment (Shimadzu, Europe). Peptides (10-50
FIG. 1. Amino acid sequence of MLB and sequencing strategy. T, C and D indicate peptides analysed from trypsin, chymotrypsin and Asp-N digestions, respectively. Only peptides useful for sequence and overlap determination are shown.
pmol) were dissolved in 0.1% (v/v) TFA/H2O. a-Cyano-4-hydroxycinnamic acid was employed as matrix solution.
3. RESULTS The complete amino acid sequence of the B chain of ML-I with 264 amino acid residues was determined by cleavage of the protein with trypsin, endoproteinase Asp-N and chymotrypsin, respectively. Fig. 1 summarizes the sequencing results from the three sets of peptides. It includes only those peptides necessary to prove each position unambiguously (approximately one third of the peptides analyzed). Peptides from each digestion were separated by RP-HPLC and characterized by sequence and mass spectrometric analysis. All peptides could be sequenced to the end. The amino acid composition obtained by acid hydrolysis of the protein is in
597
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Amino Acid Composition of Intact MLB Amino acid
Residues
Asp Asn Thr Ser Glu Gln Pro Gly Ala 1/2 Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp
39.2 (19) (20) 22.1 (22) 20.3 (21) 20.2 (6) (14) 9.6 (10) 26.1 (25) 15.0 (15) n.d. (7) 19.3 (19) 4.7 (5) 17.3 (18) 17.0 (17) 6.3 (7) 5.1 (5) 4.1 (1) 8.7 (9) 15.3 (15) n.d. (9)
Total
(264)
Note. Numbers in parentheses are the residues determined after sequence analysis. n.d., not determined.
complete agreement with the amino acid sequence determined (Table 1). However, the molecular weight of MLB calculated on the basis of sequence data (28,950 Da) is about 5,000 Da less than the apparent molecular weight of the purified lectin estimated by SDS-PAGE (34,000 Da) (30). We attribute the difference between these two molecular weight values to the existence of three sugar chains covalently bound to the protein, as described below. The N-terminal sequence analysis of intact MLB up to position 20 led to an unambiguous identification of only some amino acid residues, since several positions were not clear. The amino acid sequence of MLB was mainly obtained following the isolation and identification of an almost complete set of tryptic peptides. Digestion with trypsin yielded 19 peptides, from which most of the sequence could be established. Alignment of sequence data determined from this cleavage was achieved by comparison with sequences of other homologous galactose-binding lectins, such as ricin and abrin (31, 32). Specific bond cleavages after the residues Arg21 and Lys170 were not detected. Digestion of MLB with endoproteinase Asp-N yielded peptides resulting only from the cleavage of the protein at the terminal region. This fact could be probably due to its high folding level. Although a core fraction could be precipitated by titration with HCl after enzymatic hydrolysis, all attempts to further cleave this isolated fraction were unsuccessful. Despite the lack of new sequence results, the sequence of D4 confirmed the alignments of T5 and
T6 and the sequences of D17, T18, T19 and T20, respectively. The presence of the fragments D17 and D18 indicates that the enzyme preparation used cleaved on the amino side of Ala240 and Thr253, representing atypical cleavage sites for endoproteinase Asp-N. Further sequence information could be obtained from the analysis of peptides generated by chymotrypsin. After digestion, the reaction mixture was separated in two approximately equal portions. One portion was fractionated directly by RP-HPLC. The other one was first subjected to reduction and alkylation with 4-vinylpyridine. The analysis of both sets of peptides provided the complete primary structure of MLB and it achieved the lacking overlaps between some tryptic fragments. In addition to the amino acid sequence of MLB, the sequence data obtained provide evidence for the existence of three potencial N-glycosylation sites, which could be confirmed by MALDI-MS analysis. Three different sequences could be identified as glycosylation patterns. These are Asn61-Gly62-Ser63, Asn96-Gly97Thr98 and Asn136-Asp137-Thr138, respectively. Information about the molecular weight of the carbohydrate moieties, covalently bound to the lectin, could be obtained by means of mass spectrometry. The data are summarized in Table 2, resulting from the mass difference between the observed mass for the fraction containing the glycosylation site and the calculated mass from the peptide sequence. The third N-glycosylation site (Asn136-Asp137-Thr138) appears to present two possible bound carbohydrate moieties, since two fragments, yielding the same amino acid sequence, but different mass spectral data, could be isolated. These results lead to the conclusion that the overall carbohydrate content of MLB is about 12% (w/w). The above experiments enable us to identify some heterogeneities along the amino acid sequence of MLB as well. Analyzed sequence data show 12 conservative substitutions, most of them located in the C-terminal region of the protein (Table 3). Detected replacements involve generally two structurally not related amino acid residues at the same position. However, while some substitutions suppose the replacement of one single amino acid residue (Glu56 r Asn56or Asn231 r Ser231), other ones involve several amino acid residues (Asn231-Gly232-Leu233 r Lys231-Gly232-Pro233). These results demonstrate that
TABLE 2
Glycosylation Sites and Detected Molecular Masses of N-Linked Carbohydrate Moieties of MLB Glycosylation site
Detected mass (Da)
Asn61-Gly62-Ser63 Asn96-Gly97-Thr98 Asn136-Asp137-Thr138 Asn136-Asp137-Thr138
1172.1 1378.9 1220.5 1382.4
598
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 3
Detected Substitutions among the Amino Acid Sequence of the Different Isoforms of MLB Asn18 r Ser18 Gly56 r Asn56 Gly157 r Gln157 Ser195 r Val195 Gly224 r Tyr224 Asn231 r Ser231 or Thr231 Asn231-Gly232-Leu233 r Lys231-Gly232-Pro233 Gly232-Leu233-Ala234-Met235 r r Ser232-Leu233-Met234-Val23
MLB, like MLA (27), occurs in several isoforms, supporting further evidence for the previously reported existence of different isolectins of ML-I (33). 4. DISCUSSION The major purpose of this work was to characterize the primary structure of MLB, the galactose-specific subunit of ML-I. The complete amino acid sequence was established by means of enzymatic digestion of the protein and Edman degradation of the generated fragments, following the strategy illustrated in Fig. 1. Determined amino acid sequences were confirmed by mass spectrometric analysis. Obtained sequence data show that MLB is composed of 264 residues including seven half-cystine residues. By comparison of the amino acid sequence of MLB with the primary and secondary structure of the ricin B chain (34), it can be assumed that the residue Cys5 links MLB to MLA by an intermolecular disulfide bridge, and that the six remaining residues build three intramolecular disulfide bridges. The presence of internal disulfide bridges in MLB has been already reported (3). A more extensive examination of the amino acid sequence of MLB predicts three potencial N-glycosylation sites. As glycosylation patterns we could identify the following sequences: Asn61-Gly62-Ser63, Asn96-Gly97Thr98 and Asn136-Asp137-Thr138 . Mass spectrometric analysis of the fragments enclosing putative N-glycosylation sequences confirmed the existence of N-linked carbohydrate chains at position 61, 96 and 136, respectively (Table 2). Our detected molecular masses for the carbohydrate chains covalently bound to MLB are in excellent agreement with those isolated from ML-I and calculated on the basis of the chemical structure (35). The presented data suggest that the carbohydrate chain linked to the residue Asn96 can also appear glycosylating the residue Asn136 . A similar heterogenity in the N-linked sugar structures of ricin D and abrin a has been earlier described (36, 37). The same glycosylation pattern Asn-Gly-Ser with an almost identical molecular weight for the bound carbohydrate chain has been recently reported on MLA (27). From these results it
can be deduced that the same carbohydrate moiety may presumably be present in, both, A and B chains. The relative high carbohydrate content of MLB (about 12% by weight) is likely to be related with the importance of N-linked oligosaccharides maintaining a biologically active conformation of the protein. It has been observed that the recombinant and N-glycosylated ricin B chain (RTB), expressed in Spodoptera frugiperda or in Xenopus oocytes, was, both, biologically active and stable (38, 39). These results contrast with recombinant RTB produced in Escherichia coli, which, although initially soluble and biologically active, was unstable and tended to aggregate rapidly (39, 40). Recombinant RTB, partially glycosylated, initially displays lectin activity, but it is also relatively unstable (41). The primary structure of MLB presented was compared with those of other structurally and biologically related lectins like ricin and abrin (31, 32). An interesting common feature of these lectins is that they all exhibit specificity for galactose residues. After performing sequence alignment, the amino acid sequence of MLB shows a high overall similarity to those of ricin-D (RTB) and abrin-a (ABB) with 169 and 146 identities, respectively (Fig. 2). The most rigorously conserved part of the molecule extends the first half of the protein, as the sequence comparison clearly reveals. Thus, it is worth mentioning that MLB contains a third N-glycosylation site (Asn136-Asp137-Thr138), which is not present in RTB and ABB, and one intramolecular disulfide bridge less due to the replacement of Cys r Arg at position 21 and Cys r Ser at position 40. Internal disulfide bridges are believed to stabilize the V-loops in RTB (42). A more direct approach towards the sequence alignment of these lectins shows that the residues involved in the sugar-binding sites of RTB are all conserved in the amino acid sequence of MLB. There are two galactose binding sites on RTB which have been reported to have similar (43) or different affinities (44) and to be capable of acting independently (45). Primary structure analysis (46) and X-ray crystallographic studies have shown that RTB is composed of two similar domains, I and II, each carrying one galactose binding site (47). Further refinement of the ricin crystal structure showed both domains to be composed of three related subdomains, a, b and g, each thought to be derived from an ancient galactose binding peptide (48, 49). However, only two of this subdomains, 1a and 2g, retain their ability to bind galactose. Both contain a conserved tripeptide with the sequence Asp-Val-Arg, not present in the other 4 subdomains (48), and key binding residues, hydrogen-bonded to the sugar moiety. Subdomain 1a of RTB involves amino acid residues Asp22, Gln35, Trp37, Asn46 and Gln47, and subdomain 2g residues Asp234, Ile246, Tyr248, Asn255 and Gln256 (50). All these residues are conserved at the corresponding positions of the amino acid sequence of MLB (Fig. 2). The above mentioned conserved tripeptide is also present in the presumably first sugar binding
599
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Comparison of amino acid sequences of some galactose binding lectins. MLB is the ML-I B chain. RTB is the ricin-D B chain (31) and ABB is the abrin-a B chain (32). Sequences are aligned to maximize their similarities. Conserved residues among these proteins are highlighted. N-glycosylation sites are boxed. Residues marked with an * are those that are involved in the galactose binding sites in the 1a and 2g subdomains of the protein.
domain of MLB (Asp23-Val24-Arg25), but not in the second one (Asp236-Val237-Ala238). The replacement Arg r Ala at the third position might not result in significant alterations, since only Asp residues of the tripeptide sequence are described to stabilize the pocket formed by key binding residues in each subdomain, where galactose lies (50). All these results, together with the very high overall primary structure homology (about 65% identities) and the similar protein conformation observed for both MLB and RTB (51), provide further evidence that MLB and
RTB may probably have very similar polypeptide folding, and accordingly the carbohydrate binding sites could occur at similar positions. This findings correlate with the proposal that these lectins could originate from similar evolution processes, and thus an evolutionary relationship among these lectins could be established. In conclusion, the data presented here, as well as earlier data about the amino acid sequence of MLA (27), are of crucial importance in order to better understand the biological activities of ML-I and for the design
600
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC
Vol. 246, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of more effective therapeutic substances. Therefore, the hypothesis that the immunostimulating potency of MLB could be also modulated by the presence of the toxic MLA chain or/and other components of the mistletoe extracts should not be excluded. REFERENCES 1. Hajto, T., Hostanska, K., and Gabius, H.-J. (1989) Cancer Res. 49, 4803–4808. 2. Hajto, T., Hostanska, K., and Gabius, H.-J. (1990) Therapeutikon 4, 135–145. 3. Olsnes, S., Stirpe, F., Sandvig, K., and Pihl, A. (1982) J. Biol. Chem. 257, 13263–13270. 4. Franz, H., Ziska, P., and Kindt, A. (1981) Biochem. J. 195, 481– 484. 5. Beuth, J., Ko, H.-L., Tunggal, L., Buss, G., Jeljaszewicz, J., Steuer, M. K., and Pulverer, G. (1994) Arzneim.-Forsch./Drug Res. 44(II), 1255–1258. 6. Beuth, J., Ko, H. L., Tunggal, L., Gabius, H.-J., Steuer, M., Uhlenbruck, G., and Pulverer, G. (1993) Med. Welt 44, 217–220. 7. Beuth, J., Ko, H. L., Tunggal, L., Geisel, J., and Pulverer, G. (1993) Arzneim.-Forsch./Drug Res. 43(I), 166–169. 8. Beuth, J., Ko, H. L., Gabius, H.-J., Burrichter, H., Oette, K., and Pulverer, G. (1992) Clin. Investig. 70, 658–661. 9. Hajto, T., Hostanska, K., Frei, K., Rordorf, C., and Gabius, H.-J. (1990) Cancer Res. 50, 3322–3326. 10. Beuth, J., Ko, H.-L., and Pulverer, G. (1993) Dtsch. Zschr. Onkol. 25, 73–76. 11. Heiny, B.-M., and Beuth, J. (1994) Anticancer Res. 14, 1339– 1342. 12. Beuth, J., Stoffel, B., Ko, H.-L. Buss, G., and Pulverer, G. (1995) Arzneim.-Forsch./Drug Res. 45(I), 505–507. 13. Beuth, J., Ko, H.-L., Tunggal, L., Steuer, M. K., Geisel, J., Jeljaszewicz, J., and Pulverer, G. (1993) In Vivo 7, 407–410. 14. Ziska, P., Franz, H., and Kindt, A. (1978) Experientia 34, 123– 124. 15. Stirpe, F., Legg, R. F., Onyon, L. J., Ziska, P., and Franz, H. (1980) Biochem. J. 190, 843–845. 16. Stirpe, F., Sandvig, K. Olsnes, S., and Pihl, A. (1982) J. Biol. Chem. 257, 13271–13277. 17. Franz, H. (1985) in Lectins: Biology, Biochemistry, Clinical Biochemistry (Bøg-Hansen, T. C., and Breborowicz, J., Eds.), Vol. IV, pp. 463–472, Walter de Gruyter, Berlin. 18. Endo, Y., Tsurugi, K., and Franz, H. (1988) FEBS Lett. 231, 378–380. 19. Gabius, H.-J., Walzel, H., Joshi, S. S., Kruip, J., Kojima, S., Gerke, V., Kratzin, H., and Gabius, S. (1992) Anticancer Res. 12, 669–676. 20. Sanwig, K., and Olsnes, S. (1982) J. Biol. Chem. 257, 7495– 7503. 21. Sanwig, K., and Olsnes, S. (1982) J. Biol. Chem. 257, 7504– 7513. 22. Beuth, J., Ko, H. L., and Pulverer, G. (1994) Dtsch. Apoth. Zeitung 25, 17–28. 23. Sharon, N., and Lis, H. (1989) Science 246, 227–246.
24. Lis, H., and Sharon, N. (1986) Ann. Rev. Biochem. 55, 35–67. 25. Mirelman, D., Galun, E., Sharon, N., and Lotan, R. (1975) Nature 256, 414–416. 26. Mishkind, M., Raikhel, N. V., Palevitz, B. A., and Keegstra, K. (1982) J. Cell Biol. 92, 753–764. 27. Huguet Soler, M., Stoeva, S., Schwamborn, C., Wilhelm, S., Stiefel, T., and Voelter, W.(1996) FEBS Letters 399, 153–157. 28. Franz, H., Ziska, P., and Kindt, A. (1982) in Lectins: Biology, Biochemitsry, Clinical Biochemistry (Bøg-Hansen, T. C., Ed.) Vol. II, pp. 171–176, Walter de Gruyter, Berlin. 29. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406– 4412. 30. Luther, P., Theise, H., Chatterjee, B., Karduck, D., and Uhlenbruck, G. (1980) Int. J. Biochem. 11, 429–435. 31. Halling, K. C., Halling, A. C., Murray, E. E., Ladin, B. F., Houston, L. L., and Weaver, R. F. (1985) Nucl. Acids Res. 13, 8019– 8033. 32. Hung, C.-H., Lee, M.-C., Lee, T.-C., and Lin, J.-Y. (1993) J. Mol. Biol. 229, 263–267. 33. Gabius, S., Kaiser, K., and Gabius, H.-J. (1991) Dtsch. Zschr. Onkol. 5, 113–119. 34. Robertus, J. D., and Ready, M. P. (1984) J. Biol. Chem. 259, 13953–13956. 35. Debray, H., Wieruszeski, J.-M., Strecker, G., and Franz, H. (1992) Carbohydrate Res. 236, 135–143. 36. Kimura, Y., Hase, S., Kobayashi, Y., Kyogoku, Y., Ikenaka, T., and Funatsu, G. (1988) J. Biochem. 103, 944–949. 37. Kimura, Y., Hase, S., Kobayashi, Y., Ikenaka, T., and Funatsu, G. (1988) Biochim. Biophys. Acta 966, 150–159. 38. Frankel, A., Roberts, H., Afrin, L., Vesely, J., and Willingham, M. (1994) Biochem. J. 303, 787–794. 39. Richardson, P. T., Hussain, K., Wooland, H. R., Lord, J. M., and Roberts, L. M. (1991) Carbohydrate Res. 213, 19–25. 40. Hussain, K., Bowler, C., Roberts, L. M., and Lord, J. M. (1988) FEBS Letters 244, 383–387. 41. Wales, R., Richardson, P. T., Roberts, L. M., Wooland, H. R., and Lord, J. M. (1991) J. Biol. Chem. 266, 19172–19179. 42. Leszczynski, J. F., and Rose, G. D. (1986) Science 234, 849–855. 43. Houston, L. L., and Dooley, T. P. (1982) J. Biol. Chem. 257, 4147–4151. 44. Zentz, C., Fre´noy, J.-P., and Bourrillon, R. (1978) Biochim. Biophys. Acta 536, 18–26. 45. Wales, R., Richardson, P. T., Roberts, L. M., and Lord, J. M. (1992) Arch. Biochem. Bioph. 294, 291–296. 46. Roberts, L. M., Lamb, F. I., Pappin, D. J. C., and Lord, J. M. (1985) J. Biol. Chem. 260, 15682–15686. 47. Villafranca, J. E., and Robertus, J. D. (1981) J. Biol. Chem. 256, 554–556. 48. Rutenber, E., Ready, M., and Robertus, J. D. (1987) Nature 326, 624–626. 49. Montfort, W., Villafranca, J. E., Monzingo, A. F., Ernst, S. R., Katzin, B., Rutenber, E., Xuong, N. H., Hamlin, R., and Robertus, J. D. (1987) J. Biol. Chem. 262, 5398–5403. 50. Rutenber, E., and Robertus, J. D. (1991) Proteins: Structure, Function and Genetics 10, 260–269. 51. Bushueva, T. L., and Tonevitsky, A. G. (1988) FEBS Letters 229, 119–122.
601
AID
BBRC 8670
/
6953$$$101
05-12-98 19:17:19
bbrcg
AP: BBRC