Conformation and activity of mannose-and N-acetylgalactosamine- specific lectins from Vicia villosa seeds

Biochimie (1993) 75, 949-954 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

949

Conformation and activity of mannose- and N-acetylgalactosaminespecific lectins from Vicia viUosa seeds Z M S h e n a, W X S h i b, C S u n b, J T Y a n g a* aCardiovascular Research Institute, University of California, San Francisco, California 94143-0130, USA; bShanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China

(Received 12 February 1993; accepted 8 June 1993)

Summary - - The conformation of two Vicia villosa lectins specific for mannose and N-acetylgalactosamine, respectively, was studied by circular dichroism. Both showed a broad negative CD band around 220 nm and a positive one above 190 rim. CD data analysis indicated that they were rich in 13-sheet. However, they differed in conformational stability against extreme pH, at elevated temperature, and in guanidine hydrochloride and sodium dodecyl sulfate solutions. The unusual feature was that the conformation of Nacetylgalactosamine-specific lectin was virtually unaltered in 6 M guanidine hydrochloride and 7.5 mM surfactant. glycoprotein / lectin / conformation / circular dichroism / denaturation / hemagglutinating activity Introduction Lectins are multivalent carbohydrate-binding proteins. Recently considerable interest has been generated by using these proteins as potential probes for proteincarbohydrate interactions, and also for studying the structure of cell membrane during differentiation, development and transformation. To understand the molecular basis of these interactions, it is therefore imperative to first study the conformation and activity of lectins p e r se. Vicia villosa seeds contain two lectins with different sugar-binding specificity, immunogenicity and other physical properties such as isoelectric point and thermal stability (Qian, Shi and Sun, manuscript in preparation). VVLG and V V L M are GalNAc- and man-specific, respectively, and they showed no crossimmunogenicity. The binding of saccharide would not induce conformational changes in V V L G and V V L M (Qian et al, unpublished data). Herein we report the *Correspondence and reprints Abbreviations: VVLG, GalNAc-specific Vicia villosa lectin; VVLM, man-specific Vicia villosa lectin; GalNAc, Nacetylgalactosamine; con A, concanavalin A; Man, mannose; Gu HCI, guanidine hydrochloride; NaDodSO4 (or SDS), sodium dodecyl sulfate; CD, circular dichroism.

conformation of the two lectins and their slability against extreme pHs, at elevated temperatures aad in Gu HCI and NaDodSO4 solutions. The conformational changes were related to the corresponding ~emagglutinating activities. Materials and methods Materials Vicia villosa seeds were supplied by the Jiangsu Academy of Agriculture, China. VVLM and VVLG were isolated by affinity chromatography on Man-Sepharose (Qian, Shi and Sun, manuscript in preparation) and GalNAc-Sepharose columns, respectively. The VVLG preparation essentially followed the method of MacDonald et al [1] with minor modifications (galactosamine was used instead of GalNAc as the eluant). The purities of the two lectins were checked by disk polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, ManSepharose and GalNAc-Sepharose were prepared after March et al [2] and Sundberg and Porath [3]. Sepharose was purchased from Pharmacia, ultra-pure Gu HCI from Schwarz/Mann, and NaDodSO4 from Fluka. All other chemicals were of analytical grade. Water was double-distilled. Preparation of protein solution

VVLG and VVLM solutions from the affinity columns were lyophilized and dissolved in 0.01 M phosphate buffer (pH 7.0).

950 The protein concentrations were determined by the Jaenick method [4] in triplicate and averaged. For routine determinations, the absorption coefficients of A~. Icmat 280 n m = 12.9 for W L G and 16.4 for WLM were used.

Circular dichroism CD spectra were recorded on a Jasco J500A spectropolarimeter equipped with a DP-500 data processor for data acquisition and an IBM-PC for data analysis. The instrument was constantly purged under nitrogen flush. The optical cell containing the protein solution was placed in a water-jacketed cell holder through which constant-temperature water was circulated. Both the instrument and cells had been calibrated with a standard solutio~ of d-camphorsulfonic acid [5] The CD spectra were expressed as mean residue ellipticities, [0] in deg cm2/dmol, and calculated from [0] = (d x s x M)/ (c x 1), where d is the observed ellipticity (displacement in cm from the baseline), s the sensitivity in mdeg/cm, M the mean residue weight (110 for both lectins), c the protein concentration in mg/ml, and I the cell pathlength in mm. The CD data were analyzed by both the methods of Chang et al [6] and Provencher and GI6ckner [7]; the CD spectra of reference proteins were listed in Yang et al [81.

Denaturation For thermal denaturation the range of temperature used varied from 25°C to 65°C for VVLM and to 70°C for VVLG. At each chosen temperature, [0] at 210 nm was monitored until the reading no longer changed with time. The reversibility of denaturation was checked by air-cooling the solution back to room temperature. For pH study, the protein solution was adjusted to the desirable pHs by appropriate buffers (0.02 M): GIy-HCI for pH 2-3.5, sodium acetate for pH 4-5.5, sodium phosphate for pH 6-8, and Giy-NaOH for pH 8-12. For Gu HCi and NaDodSO4 studies, 8 M Gu HC! or 50 mM NaDodSO4 was added to a protein solution, respectively, to the desired concentrations of the protein and the denaturant.

Determination of disulJide bonds The number of cystine residues in the protein was determined by the method of Du and Zou [9].

former case the red cells would not be ruptured by NaDodSO4 after several dilutions (see Results).

Results Based on S D S - P A G E , our V V L G preparation gave one strong protein band with an Mr o f about 36 0 0 0 and one weak protein with an Mr o f about 34 000. Tollenfsen and Kornfeld [ 10] have reported three lectins from Vicia villosa seeds specific to GalNAc and designated them as B4, A4 and A2B2, w h e r e the subunits A and B have an Mr of 34 000 and 36 000, respectively. Thus, our preparation seemed to r e s e m b l e their mixture of subunits A and B. O u r V V L M preparation had ,,.n Mr o f 50 000; it had two subunits with an Mr o f :'a) 000 and 22 000, respectively. A notable difference in amino acid composition of the two lectins was that V V L G had five cystine residues and no cysteine ones, whereas V V L M had four free s u l f h y d r y l groups but no disulfide bonds.

Conformation The far-UV C D spectrum o f (t-helix has a characteristic double m i n i m u m at 222 and 208 nm and a positive m a x i m u m above 190 nm, w h e r e a s that of regular 13-sheet has a single m i n i m u m at 2 1 6 - 2 1 8 n m and a positive m a x i m u m near 1 9 3 - 1 9 7 n m [8]. Further, the magnitudes o f [0] for or-helix are several times larger than those o f the [l-sheet; thus, ix-helix, if present in a protein molecule, often d o m i n a t e s the spectrum over other conformations. V V L G and V V L M had rather similar far-UV C D spectra (fig 1), which showed a broad, negative band around 220 nm, no distinct m i n i m u m or shoulder below 210 nm, and a positive band above 190 nm. (There was a subtle difference between the two spectra; the s p e c t r u m of V V L M was red-shifted by a few n a m o m e t e r s from that of V V L G . )

Assay of activity The hemagglutinating activity of lectins was semi-quantitatively assayed at room temperature on V-well micro-hemagglutinating titer plates by a series of double dilutions of the lectin solution with phosphate-buffered saline solution (PBS; 0.075 M phosphate/0.075 M NaCI, pH 7.4). To the first hole was added 25 lal of 1 mg/ml lectin and diluted with 25 lal of phosphate-buffered saline solution. Then 25 l.tl of the solution was taken from the first hole to the second hole and mixed with 25 l.tl of the saline-buffer again. Similarly, the third hole was double-diluted and filled in the fourth hole, and the fourth hole into the fifth hole, etc. Finally, all the holes were treated with 2% (v/v) rabbit erythrocyte in PBS. Hemagglutination was recorded after incubation for 1 h at 25°C. The reciprocal of the highest dilution of the lectin that produced visible hemagglutination was taken as the titer. NaDodSO4 (above 1.25 mM) or concentrated Gu HCI (6 M) would rupture the erythrocytes. However, in the latter case after double dilution, ie in 3 M Gu HCI, the red cells were not broken down. Likewise, in the

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Fig 1. CD spectra of Vicia villosa lectins in phosphate buffer (pH 7) at 25°C. Curves: 1, mannose-specific (VVLM); 2, N-acetylgalactosamine-specific (VVLG).

951 The magnitudes of these bands were small as compared with those of o~-helix ([0]s being about - 4 0 000 for the negative bands and +80 000 for the positive band). These findings suggest that both VVLG and VVLM may be rich in 13-sheet with little or no o~helix. This conclusion was supported by the data analysis by the Provencher-G16ckner method [7], which gave estimates of 5% o~-he!ix, 61% 13-sheet and 20% 13-turn for VVLG. and no t~-helix, 69% 13-sheet and 14% 13-turn for VVLM. Similar results were obtained from the estimates by the method of Chang et al [6]. The near-UV CD spectra of both lectins showed several positive bands between 265 and 310 nm (fig 1). VVLG had a broad band around 285 nm and another one at 275 nm, which may be attributed to Tyr residues, and a shoulder near 295 nm due to Trp residues, whereas VVLM had three bands at 270, 287 and 295 nm. Unlike the far-UV CD of the secondary structure, the near-UV CD spectrum of a protein reflects the contributions of aromatic groups and disulfide bonds. Thus, it depends not only on the number and kind of aromatic and cystinyl residues, but also on their local conformation in the protein molecule. Lack of the latter makes it impossible to predict or calculate the spectrum due to these residues, unlike the estimation of an o¢-helix or a 13-sheet based on the far-UV CD spectrum.

1,2). On the alkaline side, both lectins began to unfold at pH 12 and the spectra resembled the spectrum of an unordered form with a negative band near 200 nm (fig 4A, curve 3). -2.0

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Based on [0] at 210 nm, the conformation of both VVLG and VVLM seemed to be little changed between pH 3 and 10 (data not shown). As the pH was lowered from 7 to 2 the CD spectrum of VVLM remained virtually unchanged (fig 4A, curves 1, 2), but the negative band of VVLG was shifted from a broad minimum near 220 nm to about 210 nm together with an increase in the negative eUipticities (fig 4B, curves

80

Fig 2. Time-dependence of thermal denaturation of VVLM and VVLG based on CD at 210 nm. A. VVLM curves: 1, 55°; 2, 60°; 3, 65°C. B. VVLG curves: 1, 60°; 2, 65°; 3, 70°C.

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Fig 4. CD spectra of (A) VVLM and (B) VVLG at different pHs. Curves: 1, pH 7; 2, pH 2; 3, pH 12.

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Fig 6. CD spectra of (A) V VLM and (B) VVLG in s, _qum dodecyl sulfate solutions. Concentrations of NaDodSO4" 1, 0 M; 2, 1 mM; 3, 2.5 mM; 4, 5 mM; 5, 7.5 mM.

Guanydine hydrochloride denaturation The surprising feature of the Gu HCI effect on the two lectins was that the conformation, based on CD spectrum, of VVLG changed slightly even in 6 M Gu HC1 (fig 5B) unlike most proteins which will completely unfold under similar conditions. On the other hand, VVLM behaved 'normally'; this protein appeared to be stable up to 2 M Gu HCI (fig 5A, curves 1, 2), but in 4 and 6 M Gu HCI the CD spectra were converted to the spectrum typical of an unordered form with a negative band toward 200 nm (curves 3, 4).

Sodium dodecyl sulfate denaturation NaDodSO4 is a most potent denaturant; it will alter the protein conformation even with as little as 1 mM surfactant. Unlike Gu HCI denaturation, addition of NaDodSO4 to a protein solution can often enhance the amount of (x-helix at the expense of 13-sheet [ 11]. This was also true for VVLM (fig 6A). The CD spectrum 16

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began to change with 1 mM NaDodSO4 (cf curves 1 and 2). In 5 mM NaDodSO4 it showed a minimum near 208 nm with a shoulder near 222 nm. This was accompanied by a marked increase in the magnitude of CD. In 7.5 mM NaDodSO4 the double minimum became more distinct and the positive band blueshifted toward 190 nm. These features suggest the presence of an (x-helix. As in Gu HCI solution, the striking finding of VVLG in NaDodSO4 solution was the lack of significant changes in the CD spectrum and thereby the confonnation of this lectin in this potent denaturating solution (fig 6B), suggesting that VVLG did not unfold very much in NaDodSO4 solution and rewind into (x-helix.

Test of renaturation Thermal denaturation of both VVLM and VVLG were irreversible. For instance, a VVLM solution at 65°C and a VVLG solution at 70°C were each kept for about 1 h until their CD levelled off (see fig 2). The CD spectra remained unchanged when both solutions were air-cooled to room temperature. The CD spectrum of VVLG in acidic solution could be restored to that at neutral pH (fig 4B) by raising the pH of the solution from 2 to 7 (the CD of VVLM at pH 2 and 7 closely resembled each other in figure 4A). On the other hand, alkaline denaturation at, say, pH 13 of both lectins was irreversible. The CD spectrum of VVLM remained unchanged upon lowering the pH of the solution to 7 and that of VVLG became slightly less negative than that shown in figure 4B (data not shown). Denaturation of VVLM by Gu HCI was irreversible; for instance, diluting a VVLM solution from 6 M to 1 M Gu HCI did not alter the CD spectrum (the conformation of VVLG presumably changed little in concentrated Gu HCI solution because its CD

953 spectrum was almost identical to that without the denaturant as can be seen in figure 5B). In NaDodSO4 solution the surfactant anions are known to strongly bind to the protein molecule [11]. Thus, VVLM in 5 mM NaDodSO4 or more was irreversibly denatured (fig 6A), whereas the conformation of VVLG was little affected by the presence of NaDodSO4 as judged from its CD spectra in this surfactant solution (fig 6B). Hemagglutinating activity

In general the hemagglutinating activity of VVLG and VVLM agreed with their conformational stability under different conditions of denaturation (table I). Both lectins essentially retained their activity at 55°C, which was completely lost at 70°C. The activity of VVLG and VVLM was virtually unchanged between pH 2 and 10. Both lectins were partially denatured at pH 12; however, the relative high activity at this pH may be partly due to renaturation during double dilution with phosphate buffered-saline solution (pH 7). In Gu Hcl or NaDodSO4 solutions VVLM w a s less stable than VVLG. For instance, VVLM completely lost its activity in 6 M Gu HCI, as most proteins do under similar conditions, but VVLG still retained some activity (its titer dropped from 2s to 26). Likewise, the activity of VVLM was totally lost in 10 mM NaDodSO4, but that of VVLG had its titer dropped from 28 to 27 and 24 in 7.5 mM and 10 mM NaDodSO4, respectively. NaDodSO4 was far more effective than Gu HC1 in the rupture of red cells. Starting with 10 mM NaDodSO4 (table I), the cells would be broken down until the detergent reached 0.63 mM or a titer of 24 and the hemagglutinating activity could be assayed if the titer was larger than 24. Likewise, starting with 7.5 mM NaDodSO4, the cells would be ruptured in 0.94 mM detergent or at a 23 titer.

Discussion Our objective was to determine the similarities and dissimilarities to conform the two Vicia villosa lectins.

In their native state both VVLG and VVLM seemed to be rich in [3-sheet with little [3-helix (fig 1). However, their conformational stabilities against denaturants were quite different. In particular, VVLG was quite stable even in 6 M Gu HCI or 7.5 mM NaDodSO4 (figs 5B, 6B). The lack of significant conformational changes in such potent denaturants is unusal and rare for proteins. That VVLG had five -S-Sbonds and VVLM had none may be partially accounted for by their marked difference in conformational stability. However, many disulfide-containing proteins are susceptible to denaturation by NaDodSO4 and concentrated Gu HCI solutions. Thus, the reason for the unusual stability of VVLG against denaturants such as Gu HCI and NaDodSO4 is still not known. All current methods of CD analysis of proteins do not guarantee a successful solution without X-ray diffraction results to check their reliability [12]. However, the CD spectra of both VVLG and VVLM resembled those of conA from jack bean [13], leucoagglutinin from kidney bean [14] and other lectins from Dolichos biflorus, Helix pomatia, Lotus tetragonolobus, Phaseolus vulgaris, Pisum sativum, Sophora japonica, and Ulrex europaeus I [15]. It is tempting to suggest that VVLG and VVLM belong to the same all-13 class as conA, whose X-ray diffraction analysis indicates 51% [3-sheet and 2% ix-helix [ 16]. Analysis of CD data by the Provencher and Gltickner method [7] gives 41% [3-sheet and 8% or-helix for conA, which is reasonably close to the X-ray results (the method of Chang et al [6] gave a good estimate for 13sheet but a too high estimate of tx-helix, which is unacceptable). Thus, our estimates of 60--70% ~-sheet and 0-5% tx.helix for VVLG and VVLM may not be too far from their actual values. Ultimately this conclusion must be proven or disproven by X-ray diffraction studies. Like those of conA and other lectins, the CD spectra of VVLG and VVLM differed from the spectrum of regular all-13 proteins such as immunoglobins and prealbumins in the red-shift of the broad negative band (cffig 1) by several nanometers. We suggest that

Table I. Hemagglutinating activity of mannose- and N-acetylgalactosamine-specific Vicia villosa lectins as expressed in titersa T (°C)

WLG

25 55 60 65 70

28 28 27 27 0

WLM

27 27 26 25 0

pH

2 7 9 10 12

WLG

2s 2s 28 27 26

WLM

27 27 27 27 25

Gu HCI M

WLG

2 4 6

2s 27 26

VVLM

NaDodS04 VVLG

WLM

mM

27 26 0

1.0 2.5 5.0 7.5 10

28 28 28 27 24

27 27 26 23 0

aHemagglutination was assayed by serial double dilutions with phosphate-buffered saline solution (pH 7.4) after incubation for 1 h at 25°C.

954 the two lectins may contain some segments of 1311 conformation, which consist of highly distorted [3-sheets or very short, irregular ~-strands whose C D spectrum resembles that of unordered forms [17, 18]. Although V V L G and V V L M were not all-13II proteins, the presence of such conformation for some segments could account for the broadening of the C D minimum as seen in figure 1. In conclusion, mannose- and N-acetylgalactosamine-specific Vicia villosa lectins had very similar CD spectra; both proteins were rich in 13-sheet. However, they significantly differed in their conformational stability against denaturants. One salient feature was that the conformation of VVLG, but not V V L M , appeared to be quite stable even in 6 M guanidine hydrochloride and 7.5 mM sodium dodecyl sulfate.

Acknowledgments This work was supported by National Science Foundation of China grant 38970213 and US Public Health Service grant GM-10880-32.

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5 Chen GC, Yang JT (1977) Two-point calibration of circular dichrometer with d-10-camphorsulfonic acid. Anal Lett 10, 1195-1207 6 Chang CT, Wu CSC, Yang JT (1978) Circular dichroic analysis of protein conformation: inclusion of the 13-turns. Anal Biochem 91, 13-21 7 Provencher SW, Gl6cknerr J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20, 33-37 8 Yang JT, Wu CSC, Martinez HM (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol 130, 208-269 9 Du YC, Zou CL (1962) The spectrophotometric determination of disulfide groups in proteins. Acta Biochim Biophys Sinica 2, 100-109 10 Tollenfsen SE, Kornfeld R (1983) Isolation and characterization of lectins from Vicia villosa. J Biol Chem 258, 5165-5171 11 Wu CSC, Yang JT (1981) Sequence-dependent conformation of short polypeptides in a hydrophobic environment. Mol Cell Biochem 40, 109-122 12 Yang JT (1991) Commentary on "Protein secondary structure and circular dichroism: a practical guide" by Johnson WC Jr (1990) Proteins: Struct Fctn Genet 7, 205-214. ChemTracts - Biochem Mol Biol 1,484-490 13 Wang JM, Takeda A, Yang JT, Wu CSC (1992) Conformation of concanavalin A and its fragments in aqueous solution and organic solvent-water mixtures. J Protein Chem 11,157-164 14 Jirgensons B (1979) Circular dichroism and conformational transitions of leucoagglutinin. Biochim Biophys Acta 577, 307-313 15 Jirgensons B (1980) Circular dichroism study on structural reorganization of lectins by sodium dodecyi sulfate. Biochim Biophys Acta 623, 69-76 16 Reeke GN Jr, Becker JW, Edelman GM (1975) The covalent and three-dimensional structure of concanavalin A. J Bioi Chem 250, 1525-1547 17 Manavalan P, Johnson WC Jr (1983) Sensitivity of circular dichroism to protein tertiary structure class. Nature 305, 831-832 18 Wu J, Yang JT, Wu CSC (1992) 13II conformation of all-~ proteins can be distinguished from unordered form by circular dichroism. Anal Biochem 200, 359-364