A database analysis of jacalin-like lectins: sequence–structure–function relationships

Sujana Raval2, Sharan B. Gowda2, Desh D. Singh3 and Nagasuma R. Chandra1,2

2 Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012, India, and 3 Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India

Received on March 10, 2004; revised on August 19, 2004; accepted on August 20, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lectins are known to be important for many biological processes, due to their ability to recognize cell surface carbohydrates with high specificity. Plant lectins have been model systems to study protein–carbohydrate recognition, because individually they exhibit high sensitivity and as a group large diversity in recognizing carbohydrate structures. Although extensive studies have been carried out for legume lectins that have led to interesting insights into the sequence determinants of sugar recognition in them, frameworks with such specific correlations are not available for other plant lectin families. This study reports a large-scale data acquisition and extensive analysis of sequences and structures of ß-prism-I or jacalin-related lectins (JRLs) and shows that hypervariability in the binding site loops generates carbohydrate recognition diversity, a strategy analogous to that in legume lectins. Analyses of the size, conformation, and sequence variability in key regions reveal the existence of a common theme, encoded as a set of structural features over a common scaffold, in defining specificity. This study also points to the remarkable range of domain architectures, often arising out of gene duplication events in lectins of this family. The data analyzed here also indicate a spectacular variety of quaternary associations possible in this family of lectins that have implications for glycan recognition. These results thus provide sequence–structure–function correlations, an understanding of the molecular basis of carbohydrate recognition by ß-prism-I lectins, and also a rationale for engineering specific recognition capabilities in relevant molecules.

Key words: ß-prism-I fold / carbohydrate recognition / jacalin-related lectin / plant lectin / sequence analysis


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lectins, a well-known class of carbohydrate-binding proteins, are known to be important in a variety of biological processes, mediated through their carbohydrate specificities (Lis and Sharon, 1998Go; Vijayan and Chandra, 1999Go). Some of the well-characterized roles of lectins are in cell–cell communication, host–pathogen interactions, cancer metastasis, embryogenesis, and tissue development (Sharon and Lis, 1989Go). Detailed knowledge of the molecular mechanisms of carbohydrate recognition by lectins is therefore required not only to understand the prime events in various biological processes but also to translate them into applications in medicine and biotechnology. Plant lectins, particularly legume lectins, have been for decades model systems of choice to study the molecular basis of these recognition events because they are not only easy to purify but also exhibit a wide range of carbohydrate specificities, despite strong sequence conservation (Loris et al., 1998Go).

Plant lectins are broadly divided into five classes based on their subunit folds (Bettler et al., online data available at www.cermav.cnrs.fr/lectines). These are legume lectins, ß-prism-I lectins, monocot mannose-binding lectins, trefoil lectins, and hevein domain lectins. The best characterized of these are the legume lectins, the analyses of which has led to great insights into the structural basis for protein–carbohydrate recognition and has also provided a basis to explore many potential applications including disease diagnosis (Lis and Sharon, 1998Go; Sharma and Surolia, 1997Go). In recent years there has been good progress in the understanding of other lectin families as well. The ß-prism-I lectins, which are also commonly known as jacalin-related lectins (JRLs), are a classic example. Although only recently discovered, these lectins have become an important subject in plant biochemistry, physiology, and medicine (Aucouturier et al., 1989Go; Peumans et al., 2001Go; Tamma et al., 2003Go). It is of primary interest to carry out a systematic analysis of the available members of this family in an effort to elucidate their molecular mechanisms of action as well as to exploit their potential applications. Here we report a bioinformatics study of JRLs and present a description of various features leading to sequence–structure–function correlations among them.

JRLs derive their name from jacalin, the first member to be identified from the seeds of jackfruit (Bunn-Moreno and Campos-Neto, 1981Go). It has since triggered a lot of research in the area, which has resulted in the identification of many related lectins from different plants. Jacalin also happens to be the first member of the family to be studied by X-ray crystallography (Sankaranarayanan et al., 1996Go). The structure revealed a novel lectin fold, named the ß-prism-I fold. Lectins in this family have been found to exhibit a repertoire of functions, such as specific binding to the tumor associated T-antigen (Jeyaprakash et al., 2002Go), binding with specific regions of HIV and hence its inhibition (Tamma et al., 1996Go), potent and selective stimulation of distinct T cell functions (Lafont et al., 1997Go), and a unique ability to specifically recognize immunoglobulin A1 from human serum (Hashim et al., 2001Go). Many biomedical applications automatically spring out of these functions, some examples of which are detection of specific tumors and hence diagnosis of cancer (Sujathan et al., 1996Go), affinity chromatography of IgA1 (Booth et al., 1995Go), and selective immunostimulation (Lafont et al., 1996Go).

Each of the four subunits in jacalin is made of a major {alpha}-chain of 133 amino acids and a minor ß-chain of 20 amino acids. The crystal structure of jacalin indicated that each of its subunits exhibited a type I ß-prism fold, comprised of three Greek keys (four-stranded ß-sheets) contributed by both the chains (Sankaranarayanan et al., 1996Go). The crystal structures of other lectins in this family, artocarpin from Artocarpus integrifolia (Pratap et al., 2002Go), Helianthus tuberosus lectin (heltuba; Bourne et al., 1999Go), Maclura pomifera agglutinin (MPA; Lee et al., 1998Go), and Calystegia sepium lectin (calsepa; Bourne et al., 2004Go) determined subsequently, confirm this fold to be characteristic of the family, although significant differences in quaternary associations were observed. These crystal structures also indicate one carbohydrate-binding site per subunit. Residues forming the binding site emerge from different loops at one end of the prism. Based on the known sugar specificities, lectins in this family can be broadly divided into two classes: (1) the galactose-specific lectins and (2) the mannose/glucose-specific lectins (Peumans et al., 2001Go). Some characteristics important for carbohydrate specificity have also been discerned from these structures.

With this background, we carried out a systematic database analysis extending to several jacalin-like molecules to derive common minimum principles characterizing (1) the jacalin fold and (2) the features generating carbohydrate-recognition capability as well as the determinants of specificity. The vast number of sequences, a significant amount of biochemical data, as well as a few crystal structures reported enable a simultaneous analysis of all known members of the family to develop a broader perspective of the functionalities as well as potential uses of these lectins.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The data set
A standard protein–protein BLAST identified 167 homologs of the {alpha}-chain of jacalin in the nonredundant sequence database. Analysis of their distribution across various life forms (Figure 1), carried out using the Blink tool at NCBI, suggests that close homologs of this sequence is highly specific to plants because of the 167 BLAST hits, all emerge from 16 unique plants but none from archaea, bacteria, fungi, viruses, metazoa, or other eukaryotes. Of these 167 hits from the nonredundant (NR) database, 118 belonged to A. thaliana alone, whereas the other 49 arise from 15 other plants. Because this is the only plant for which so many JRLs have been identified (probably because of the availability of its full sequence), we considered it inappropriate to include all of these in the data set because it would then overtly bias the data. A detailed analysis of these 118 genes (and at least 173 possible JRL domains) present in this genome, however, requires a separate study owing to the large numbers observed. However, to represent the presence of JRLs in this plant, one JRL (accession number NP_849691, Table I) that bore the greatest similarity to the {alpha}-chain of jacalin was chosen to be included in this data set.



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Fig. 1. A taxonomical view of the distribution of JRLs in various plant families. The plant names from which lectin structures have been experimentally determined are indicated in bold. The tree has been prepared based on the JRLs identified in this study along with taxonomical information obtained from the NCBI databases.

 

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Table I. List of NR JRLs identified from the sequence database, using the {alpha}-chain of jacalin as a search template

 
Parsing the alignments followed by nonredundancy checks, as described in Materials and methods, resulted in identifying 33 sequences derived from 16 different plants that are identified by their accession numbers and listed in Table I. The short listed sequences showed enormous variations in their lengths, ranging from 121 amino acids to 988 amino acids (Table I). On closer examination of the BLAST results, it was observed that many of them contained internal repeats, the repeating unit corresponding to a JRL domain. Thus a total of 58 individual domains identified from these 33 sequences originating from 16 different plants are listed in Table I. The overall similarities in the sequences listed here with that of jacalin, leading to their inclusion in the family of JRLs is reflected in this table.

Multiple alignment and phylogenetic analysis
A multiple alignment carried out with the sequences of the domains identified reflects the overall similarities (Figure 2). The multiple alignment indicates that the second half of the sequence is more conserved in the different members of the family than the first half (Figure 2). A consensus profile derived for the alignment, with the help of the PRATT profile creation tool, indeed confirms this. The profile listed using the PROSITE syntax thus reads, F-x(2,3)-N-x(3,5)-G-x-[FHY]-G-x(3,4)[AGNQST]-x(3,4)-L-x(3,8)-G-F-x(0,1)-G-x(2)-G-x(2)-[ILV]-x(2)-[FILV]-[DGS], ranging from residues 86 to 128 of the jacalin {alpha}-chain. Mapping the profile onto the structure reveals that the conserved residues lie predominantly in the first and the third sheet regions of the ß-prism (Figure 2, also Figure 3).



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Fig. 2. Multiple alignment of 58 JRLs identified in this study, computed using CLUSTALW and rendered using BOXSHADE. The dark shaded regions indicate amino acids that are highly conserved, and the light gray shaded regions indicate amino acids that exhibit conservative substitutions. The four loop regions IL and BL1 to BL3 are boxed (see text). The top panel indicates the sequential arrangement of ß-strands in the structure, belonging to the three sheets 1, 2, and 3, with reference to the sequences in alignment. Interface regions involved in quaternary associations in the known structures are also indicated as QI-A and B (see text).

 


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Fig. 3. (A) Overall structure of a subunit of jacalin and other JRLs showing the ß-prism-I fold. BL1 to BL3 indicate the three carbohydrate binding loops, IL the influencing loop, QI the region involved in quaternary association (interface set A, see text), and PTM the region referring to the cleavage site due to a posttranslational modification. Also labeled are the three ß-sheets. (B) C{alpha} trace of a subunit of jacalin indicating residues contributing to the consensus sequence profile. Side chain atoms in the profile are shown by ball-and-stick representation. Conserved glycines are indicated as spheres. (C) C{alpha} trace of the subunit of jacalin showing exposed hydrophobic residues in one of the interfaces between subunits of jacalin, that have a solvent accessibility of >30 Å2.

 
Starting from the multiple alignment, a phylogenetic analysis was also carried out to gain a better understanding of the interrelatedness of the various sequences. The phylogenetic trees, constructed using three widely used algorithms (based on the unweighted pair group method with arithmetic mean, the neighbor joining; and the minimum evolution methods, as implemented in the MEGA suite of programs), are shown in Figure 4. All trees indicate similar branching patterns, grouping all sequences into three major clusters, named cluster 1, cluster 2, and cluster 3. The first cluster has an identifiable subcluster (marked 1A in Figure 4), which is made of the lectins from monocotyledonous plants, such as cereals (rice, wheat, barley) and banana. The other part of cluster 1 is made of lectins from asterids along with Fabaceae and Fagaceae (taxonomical classification in Figure 1). Cluster 2 can easily be further divided into subgroups. One distinct subgroup (2A) consists of all Moraceae family lectins, and the other contains domains from the chosen A. thaliana JRL and some domains from Oryza sativa and Castanea crenata. Cluster 3 contains lectins derived only from Brassica napus and Brassica rapa.



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Fig. 4. Phylogenetic tree (left) produced from the multiple alignment using minimum evolution algorithm and differences in sequence as a basis for the phylogeny model with bootstrapping to validate the branching pattern, as implemented in MEGA. The curved arrows indicate the branching break points showing three distinct clusters, marked 1, 2, and 3. The identifiable group within cluster 1 is labeled 1A. Similarly a distinct group within cluster 2 is labeled 2A, within which an identifiable subcluster 2A1 is labeled and also indicated by a shaded leaf (illustrating the clustering of known galactose-specific lectins). The radial trees (right) A, B, and C refer to trees produced using the same data but with different methods (A: neighbor joining, B: unweighted pair group method with arithmetic mean, and C: minimum evolution, using p-distance models), and indicate similar branching patterns. Subcluster 2A1 is shaded in these as well, and the cluster numbers 1 to 3 are also indicated.

 
Subunit structures
A secondary structure prediction carried out for those sequences where structural information is not available indicates them to contain predominantly ß-sheets, consistent with the all-ß class observed in the crystal structures (full results available online at http://144.16.93.115/lectins/jacalin/sec_struct). Besides, pairwise sequence similarities of each of the JRL domains with one or the other of the five crystal structures in the family are in the range of 60–80% (values with jacalin are listed in Table I). Viewing the predicted secondary structures in context of the individual pairwise alignments as well as the multiple alignment (Figure 2) clearly revealed that the secondary structures also align very well with those observed in the crystal structures. These observations strongly indicate that each sequence adopts a ß-prism-I fold in each of the JRL domains. Experimentally determined crystal structures are available for five of the sequences listed in Table I. Using these as templates, molecular models of all the other sequences/domains were built individually using homology modeling. The coordinates of the individual models are available online at http://144.16.93.115/lectins/jacalin/models.

Analysis of quaternary associations
Examination of the quaternary associations exhibited by different crystal structures indicate that there are three different types known so far, although the five crystal structures exhibit very high similarities in their individual subunits. The three different quaternary types can be attributed to interactions involving three different interface sets: (1) interface set A involving residues 102–109, 131–133 from the A chain and 11–15 from the B chain (numbers correspond to 1JAC) as seen in the tetramers of jacalin, MPA, and artocarpin; (2) interface set B involving residues 19–25 and 51–60 (residue numbers from 1OUW) as seen in the dimer of calsepa; and (3) interface set C involving residues 7–11, 119–124, and 20–26 (residue numbers from 1C3K), as seen in the octamer of heltuba, the first two segments significantly overlapping with the interface set A and the third segment partly overlapping with the interface set B (Figure 5). Thus jacalin, artocarpin, and MPA, which can be described as dimers of dimers (Sankaranarayanan et al., 1996Go), have their first dimerization interface on the back of the first sheet, forming extensive interactions between the first sheets in the two subunits (Figure 5). Calsepa shows an interaction surface on a face opposite to that in jacalin (equivalent to residues 3–8 of jacalin), leading to a dimer achieved through a completely different mode. Heltuba, on the other hand, has two interaction surfaces distributed on opposite faces of the prism, which enable it to associate with two distinct modes of the dimer of the jacalin type as well as the dimer of the calsepa type (albeit with altered intersubunit orientations), thus leading to a closed octameric arrangement.



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Fig. 5. Ribbon representation showing the known quaternary arrangements in jacalin (including artocarpin and MPA), heltuba, and calsepa. Jacalin, artocarpin, and MPA are tetramers, calsepa is a dimer, and heltuba exhibits an octameric arrangement. Subunits are colored black and gray alternately. The interface sets A, B, and C corresponding to those in jacalin (also artocarpin and MPA), calsepa, and heltuba, respectively, are labeled.

 
Presence of hydrophobic residues that are exposed to the outside on a subunit would lead to a strong requirement of having to bury these residues to achieve stability, thus dictating the mode of quaternary associations. As an example, the exposed hydrophobic surface in a subunit of jacalin, that gets buried on quaternary associations is shown in Figure 3C. The corresponding residues in calsepa have hydrophilic side chains, but hydrophobic residues are present in it on the opposite face, whereas heltuba shows hydrophobic residues corresponding to both faces, thus indicating that differences in quaternary types can be discerned at the sequence level, too.

Furthermore, the surfaces generated by the homology models were analyzed for the presence of exposed hydrophobic residues on their surfaces. Such residues are listed in Table II, and those corresponding to either the A or the B (extended to encompass the remaining part of interface set C) interface sets are differently highlighted. A large number of JRLs contained some exposed residues corresponding to interface set A, indicating the possibility of associations through this segment as in jacalin. Some JRLs, such as those in banana and mulberry, contained exposed hydrophobic residues predominantly in this segment. Some others, such as those from wheat and Convolvulous species, contained a clearly identifiable cluster (as determined by visual inspection to identify their presence on the same face of the prism) corresponding to interface set B, indicating the possibility of associations, as in calsepa. Some other JRLs, such as those from rice and Brassica species, showed such residues on both surfaces, corresponding to interface set C, indicating the possibility of interactions as in heltuba. Yet some other JRLs show exposed clusters distributed on other segments, suggesting other modes of quaternary association.


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Table II. List of hydrophobic residues having solvent accessible surface areas >30 Å2 in the 58 different JRL domains

 
Table II also shows that the multi-JRL proteins also exhibit exposed hydrophobic residues on their surfaces. It is possible for such surfaces to be involved in interactions between different domains to result in higher-order associations, resembling quaternary structures. A preliminary analysis of the subunit models built (by juxtaposing the different repeating units in different orientations using interactive graphics) for the sequences containing repeats (multiple copies of JRL domains) indeed indicates the feasibility of the repeating domains functioning as independent subunits of the quaternary structures of the single-domain structures. Thus, for example, the lectin from Castanea crenata (CCA), in its structure may resemble the dimer observed in calsepa, where as that from B. napus containing four copies of the JRL domain in its sequence could well resemble the tetramer seen in jacalin and artocarpin. The distances between the C-terminus of the first subunit with the N-terminus of the second subunit range from 9 to 25 Å for different types of dimeric units in the known structures, a distance that can be easily bridged by the linker residues between the domains present in these sequences, suggesting the feasibility of such quaternary associations. Some of the multi-JRL proteins such as those from B. napus show remarkable differences in the distribution of exposed hydrophobic residues in each of their domains. For example, the first domain of T08081 has hydrophobic residues corresponding only to interface set A, whereas the third and the fourth domains of the same protein have a number of hydrophobic residues corresponding to interface set B as well, indicating the possibility of newer types of interactions among them.

Structural features of carbohydrate recognition
Posttranslational modification
Apart from the known cases of posttranslational cleavage leading to a major {alpha} chain and a minor ß chain in galactose-specific JRLs (Houles-Astoul et al., 2002Go), such as jacalin (Sankaranarayanan et al., 1996Go), MPA (Lee et al., 1998Go), and the galactose variety from Morus nigra (Rouge et al., 2003Go), the multiple alignment carried out here indicates that some lectins of B. napus also contain a free amino-terminal group, aligning in structure with the N-terminal glycine of jacalin and other galactose-specific lectins (Figure 2). The sugar specificity of the domains in this case is not individually known, but they are generally annotated as mannose-specific and myrosinase-binding lectins.

A majority of JRLs do not exhibit such cleavage and function as single protomers. Even in these cases, the region corresponding to the cleavage site in jacalin aligns with a glycine containing loop connecting two ß-strands. Structural superpositions carried out here indicate that the glycine(s) in this loop are oriented toward the sugar and interact by forming hydrogen bonds through their peptidyl nitrogens. The architecture of the binding site in the crystal structures suggests that either a break in the loop resulting in a free amino group as in jacalin or a glycine containing loop as in artocarpin (Figure 6) are key features in creating the appropriate contours to form the binding pocket, irrespective of the specificity. A visual inspection of the homology models built for other lectins also reflect this property (the coordinates for the models are available online at http://144.16.93.115/lectins/jacalin/models). Replacing this glycine with a larger residue would lead to steric clashes with the sugar atoms in all the JRLs and hence abolish binding to a significant extent.



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Fig. 6. Stereo diagrams of the monosaccharide binding sites in (A) jacalin and (B) artocarpin representing galactose and mannose binding, respectively. The two structures have been superposed and artocarpin merely translated downward for clarity. The sugars are shown as ball-and-stick models. Broken lines indicate hydrogen bonds. Residues lying within a sphere of 5 Å of any atom of the sugars are shown in both cases. Residues forming the contours to enable sugar binding are indicated by surface representations in both.

 
The hydrophobic lid
The residues from three different loops are commonly attributed to be part of the carbohydrate-binding region in jacalin and homologous structures (Jeyaprakash et al., 2003Go; Sankaranarayanan et al., 1996Go). These loops corresponding to residues 47–50, 75–78, and 121–125 in jacalin are labeled binding loop 1 (BL1), BL2, and BL3 in the multiple alignment (Figure 2) and also in the schematic representation shown in Figure 3A. The conservation of sequence is very poor in BL1 and BL2 but quite high in three out of five positions in BL3, as indicated in Table III and can also be inferred from Figure 2. Yet some important interactions are observed between the residues in BL1 and BL2 with the bound carbohydrate in individual structures suggesting their role in generating specificity. For example, Phe47 of BL1 in jacalin and MPA makes several hydrophobic interactions with galactose, whereas in artocrapin, there is a deletion in the corresponding position resulting in nonparticipation of this loop in carbohydrate binding. The situation in banana and heltuba lectins is similar to that in artocarpin and, in fact, even more accentuated by a bigger deletion in this region. In calsepa, CCA and lectins of Convolvulous species, there is an insertion in this region leading to a very different conformation of the loop, pointing away from the binding site, again resulting in nonparticipation in sugar binding. Thus it appears that hydrophobic and aromatic residues in BL1 as in jacalin, MPA, and MornigaG are important for the formation of a binding site with a strong preference to galactose. The loop in all these JRLs can in fact be extended to six residues (starting from residue 45 in jacalin). The mannose counterpart in Morus nigra also has a six-residue loop but contains two prolines in it, which we predict will lead to a very different conformation as compared with its galactose counterpart, pointing away from the binding site. Examination of the sequence preferences in all the other mannose-specific lectins also show the same trend with many of them containing multiple glycines and prolines in this region, again indicating the importance of the conformation of this loop for sugar specificity. The overall dispositions of the loops in the crystal structures are shown in Figure 7.


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Table III. Features important for carbohydrate recognition in different JRLs

 


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Fig. 7. Schematic representation of the disposition of the loop regions involved in carbohydrate binding in JRLs whose structures have been superposed using all C{alpha} atoms. The three loops directly involved in sugar binding are marked as BL1, BL2, and BL3, whereas the loop influencing binding indirectly is labeled IL. The sugar is shown as a CPK object. The segments in a darker shade, correspond to the ones in jacalin and MPA. PTM refers to the cleavage site that results in a free amino terminal in jacalin and MPA.

 
Aromatic stacking
The loop BL2 houses Tyr78 in jacalin, which stacks with the ring of galactose. The stacking interaction is present in MPA as well. This study indicates that in fact, 13 out of 58 domains have a tyrosine or phenylalanine and in a few cases histidine, corresponding to Tyr78 of jacalin (Figure 2, Table III), all capable of stacking with the sugar ring. These 13 structures include mannose-specific lectins such as those from Castanea, Parkia, and Ipomoea species, apart from the galactose-binding Moraceae lectins. Wheat and Arabidopsis lectins also contain this aromatic residue, but their specificities are not yet known.

BL3 and the conserved GXXXD motif
Simultaneous analysis of the various crystal structures (jacalin, artocarpin, heltuba, MPA, and calsepa), facilitated by structural superpositions, reveal that the conformation of BL1 and BL2 vary significantly in these structures, whereas the conformation of BL3 remains constant (Figure 7). The same trend was observed in the homology models of the other JRL domains as well. Apart from the conserved conformation, there is also a much higher degree of sequence conservation in this region (Table III). A glycine and an aspartic acid (corresponding to residues 121 and 125 in jacalin) separated by a three-residue spacer, are seen to be predominantly conserved in the known JRL domains, as seen in Figure 2. The spatial position of this loop suggests that it is actually involved in forming the binding pocket. The interactions of the conserved glycine and aspartic acid with the sugar are also invariant, irrespective of the carbohydrate specificity, thus pointing to the conclusion that this feature is a key determinant of generating carbohydrate-recognition capability in general, which, in conjunction with the features from other loops, brings about the structural basis of specificity. A tryptophan in this loop is well conserved but not crucial for activity, because it is compensated either by other hydrophobic residues in the same loop or by hydrophobic residues from other loops. For example, Leu89 in BL2 and Leu139 in BL3 in artocarpin can play the same role as Trp123 in jacalin.

Deviations from the conserved GXXXD motif are observed in a few JRLs (Figure 2, Table III), such as some domains of B. napus, Hordeum vlugare, Oryza sativa, and Triticum aestivum, where a conservative substitution such as an asparagine or a glutamic acid is seen in the place of aspartic acid in most of them. Some of these also show a glycine and an aspartic acid, not directly aligned with the expected position but in the immediate neighborhood, which may be important for lectin activity. A histidine and an arginine are also found in some domains (from H. tuberosus, Hordeum vulgare). Two JRLs from T. aestivum and B. napus show extreme divergence with a hydrophobic residue in this position, although a glutamine is present within the loop in both. A substitution of the aspartic acid is also largely coupled with a substitution of the glycine, perhaps indicating directed evolution to alter primary sugar specificities.

The influencing loop
On a detailed analysis of protein–sugar contacts and the interaction networks of the participating residues (tables listing these are available online at http://144.16.93.115/lectins/jacalin/contacts), we observe that a fourth loop, an influencing loop (IL) can also influence the conformation of some of these residues and hence the specificity. This loop corresponding to residues 20–23 in jacalin and is found to be longer in the galactose-binding varieties than in the mannose ones (Table III). The residues in this loop interact with residues in BL1, BL2, and BL3 in jacalin (Asn 20 ND2 [IL]-Gly 50 O [BL1], Glu 22 O [IL]—Arg 82 NH2 [segment adjacent to BL2], Thr 23 O [IL]—Arg 82 NE [segment adjacent to BL2], Ala 24 [IL]—Val 75 [BL2; favorable van der Waals interaction], Ile 25 O [residue just outside IL]—Leu 124 [BL3] apart from many more hydrophobic interactions) and hence influence the orientation and conformation of these loop regions, thus indirectly influencing carbohydrate binding. In artocarpin and many other mannose-specific lectins, there is a deletion in this region leading to a small loop, resulting in eliminating the indirect influence on carbohydrate binding. In calsepa, a mannose-specific lectin, a longer loop corresponds to this region but has a conformation very different from that of jacalin, and its position does not allow interactions with the binding site loops, as judged from the distances between the corresponding residue sets.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Until recently, it was believed that JRLs were found only in a few genera of the family Moraceae, to which the jackfruit plant (A. integrifolia) belongs. In the past few years, however, it has become evident that related lectins are found in many other families as well (Peumans et al., 2000Go). The species distribution of the BLAST hits depicted in Figure 1 illustrates that this sequence is much more widespread than originally believed. Moreover the two plants whose genomes have been sequenced (complete sequence of A. thaliana and a complete draft of O. sativa) as listed in the GOLD database (Bernal et al., 2001Go), contain this type of lectins, suggesting that it may indeed be present in many others, which will be revealed as further genome sequences become available.

Sequence–structure correlations
The overall sequence of the JRLs appears to be well conserved across different plant species in which it is found, in fact leading to their recognition as JRLs, whose subunits bear the ß-prism-I fold, made of three ß-sheets arranged into a prism, with an ~ three fold, running parallel to the sheets. The three fold internal symmetry observed in the fold, however, is not apparent at the sequence level within each subunit, unlike in the case of mannose-specific bulb lectins, where both sequences and structures show the three fold repeats (Hester and Wright, 1996Go).

Sequence–structure mapping of members of this family indicate that the strand regions are highly conserved, especially in the first and the third sheets. On the other hand, the loop segments that connect the strands are highly variable in their lengths and sequences, consistent with their role in generating functional diversity. Furthermore, mapping the profile onto the structure reveals that the conserved residues lie predominantly in the first and the third sheet regions of the ß-prism (Figure 2, also Figure 3B). A twofold corollary stems out of this observation. The first is that the conserved sequence forming the profile in the second half of the sequence (made of the first and the third sheets in the structure) plus any sequence segment located appropriately to form the second ß-sheet can be regarded as a fingerprint of the ß-prism-I fold. The second is that the variable regions, which are predominantly in the first half of the sequence, are to a major extent responsible for the differences in detail and hence form causal factors in generating major diversity, both for carbohydrate specificity as well as for quaternary structure. The fact that these residues (from the loops and second ß-sheet forming the first half of the sequence) encompass IL, BL1, as well as the quaternary interface set B, is totally consistent with this argument.

While the members belonging to the Moraceae, Convolvulaceae, and the Musaceae families contain only a single copy of the domain, the members of Brassicaceae exhibit significant diversity with lectins ranging from a single copy to those containing six copies of the same JRL domain within a single sequence (Table I). On the other hand, some other sequences, such as those in O. sativa, contain one to few repeats of the JRL domain but also contain other domains. Sequence analysis indicates that one sequence of rice lectin (CAD40624) containing three copies of the JRL domain also contains a serine/threonine protein kinase domain (55% sequence similarity, e value: 9e–52), whereas yet another rice protein contains three copies of the JRL domain and a Lis-H containing cytoskeleton protein domain (50% sequence similarity, e-value: 2e–37). There have been earlier examples of coexistence of legume lectin domains with other domains, one of them with a jacalin-like fold in the insecticidal toxin (Chandra et al., 2001Go). These observations indicate the high propensity of association of this domain either with more of its own kind or with other domains, providing a framework to analyze their larger functional roles.

Available structures of JRLs indicate that the members are capable of adopting a wide range of quaternary associations. The crystal structure solutions of jacalin and artocarpin show tetrameric arrangements, whereas heltuba exhibits a remarkably different octameric association; the recently determined structure of calsepa shows yet another arrangement, that of a stable dimer (Figure 5). The presence of exposed hydrophobic residues spread over different regions in all the modeled JRLs, shown in Table II, indicate the remarkable diversity in quaternary associations that these structures may exhibit. Significant variations in quaternary associations have been observed earlier in the families of legume lectins (Prabu et al., 1999Go) and bulb lectins (Chandra et al., 1999Go) and have also been associated with differences in larger oligosaccharide binding, perhaps a strategy for generating diversity in function over a common subunit scaffold.

It turns out that all the currently available structures in the family belong to the sequences containing only a single copy of the jacalin-like domain in a protomer. The sequences coupled with the experimentally observed molecular weights for these lectins have provided clues about the nature and mode of possible protomer assemblies for some of the JRLs studied here. These predictions provide a framework to further probe the larger functions of the lectins. It must also be mentioned that the crystal structure of heltuba revealed the presence of an octamer, which appears to be capable of cross-linking N-glycans from different glycoproteins, as against a tetramer suggested by the biochemical studies (Bourne et al., 1999Go). It would therefore be of great interest to determine the structures of these proteins to get a clearer understanding of the precise nature of their higher-order structures. Despite the lack of thorough knowledge of these aspects, it is clear from the data described here that the lectins of this family exhibit a truly spectacular range of quaternary associations, despite high sequence and subunit structure conservation.

Carbohydrate binding: recognizing patterns that determine sugar specificity
Even though all known JRLs exhibit high sequence conservation in their overall sequences and hence the same subunit structure, marked variations are observed in their abilities to recognize various carbohydrates. The data available so far have led to their broad classification into galactose-specific and mannose-specific classes. Some of them, however, are not exclusive to galactose or mannose, but their primary specificities are consistent with the classification. For example, jacalin, a galactose-specific lectin, has been shown to bind to mannose as well, albeit with a lower affinity (Bourne et al., 2002Go).

The observation of three distinct clusters in the phylogenetic trees indicate that the sequences can be subclassified into different groups (Figure 4). Most of the plants that appear in cluster 1 are known to be mannose-specific from the various biochemical studies described in the literature. Lectins from B. napus present in cluster 3 are known to be specific to N-acetyl glucosamine and p-aminophenyl {alpha}-D-mannopyranoside and also capable of binding myrosinase (Taipalensuu et al., 1997Go). Thus cluster 1 seems to be predominantly mannose specific, and cluster 3 is specific to sugars containing glucose-mannose plus a high affinity to myrosinase. Cluster 2 seems to have subclassified lectins with galactose and mannose specificity into separate groups. The galactose-specific lectins are all grouped together into a subcluster (2A1) of the Moraceae lectins (2A). It appears from the tree as well as the detailed sequence analysis carried out that lectins from taxonomically related plants tend to have sequences closely related to each other in their overall compositions as compared with those from the taxonomically divergent ones. Yet within the taxonomical cluster (cluster 2), the known galactose-specific lectins in Moraceae group out into a subcluster from the mannose-specific ones from the same plants, indicating the ability of the phylogenetic tree to discriminate between the galactose- and the mannose-binding varieties.

Although lectins from rice are generally known to be mannose-specific, weak binding to galactose containing sugars is also reported (Poola et al., 1986Go). The phylogenetic tree suggests that some lectin domains of rice as also those from the chosen sequence of A. thaliana are closer to the Moraceae lectins than the cereal or the Brassicaceae lectin domains, respectively. It is possible that differences in carbohydrate specificity occur among the different rice lectins or even among different domains of a single lectin, which could perhaps get reflected as polyspecificity, observed in many biochemical experiments. The lectins from B. napus are also all simply automatically annotated as myrosinase-binding based on their overall sequence similarities (Geshi and Brandt, 1998Go). Such annotations, though useful in overall classifications, will not necessarily have the resolution of information required for understanding the finer functional features, such as carbohydrate specificities in this case, which can only be obtained by detailed sequence and structural studies.

A closer look at the multiple alignment in Figure 2 indicates that despite the immediately detectable overall similarities, fine differences in many regions of the polypeptide chain exist, particularly in the loop regions that form the binding sites. When viewed at the level of these individual segments, sequence variation patterns responsible for functional divergence stand out from those responsible for species divergence. The binding site loops of different lectins display variability not only in sequence but also in size. The fact that these regions house residues that are crucial for carbohydrate binding immediately suggests that sequence variability may be a strategy to generate carbohydrate recognition diversity. In legume lectins, a similar strategy has been identified, where the lengths of the last of the four loops dictate binding specificity (Sharma and Surolia, 1997Go). Furthermore, the density of insertions and deletions in these loop regions is high in JRLs, similar to that in legume lectins, as evident from the number of gaps in the multiple alignment in Figure 2.

The multiple alignment also indicates that the residues involved in various quaternary interactions are also among the variable segments. Although members of this family exhibit high variation in quaternary association, it is not likely to influence monosaccharide specificities. This is because the known crystal structures indicate that the residues involved in quaternary interactions do not make any direct contact with the bound monosaccharide. Differences in quaternary associations, however, could have major implications to the recognition of higher sugar structures, as seen in the case of bulb lectins (Chandra et al., 1999Go).

Mapping the variations observed in the loop segments from the multiple alignment onto the previously characterized structural features, required for carbohydrate binding, has led to the determination of conservation of each feature. Furthermore, two more features have been identified from this study to be important for defining specificity. From the available crystal structures, it is known that two main differences exist in the binding site residues between the galactose-specific and the mannose-specific JRLs. These relate to (1) posttranslational cleavage and (2) aromatic stacking with the sugar. In addition, the new sequence and structural features identified here are (3) the hydrophobic lid formed by BL1 and (4) presence of an IL that can be correlated with sugar-binding properties in these lectins. The study of all known JRL domains, analyzes each individual feature and provides insight about which of them determine specificities, versus which of them augment specificities in individual cases, as discussed next.

BL1
The crystal structures and the models suggest that residues in BL1 (encompassing the Phe47 in jacalin) in galactose-binding lectins form a hydrophobic lid to the binding pocket, the absence of which, as in the mannose-binding lectins, will allow binding of an oligosaccharide, extending at that end of the prism. The length and sequence of this loop is therefore important for monosaccharide specificity, and perhaps even more so for oligosaccharide specificities.

BL2
The presence of aromatic residues at this position in both galactose- and mannose-specific varieties suggest that the role of the aromatic residue in the second loop BL2, and hence the loop is mainly in enhancing binding in individual cases but is not crucial for determining monosaccharide specificity. The conformation of the loop in different proteins vary significantly, as do the loop sizes, and do not seem to suggest a correlation of the loop size with monosaccharide specificities. For example, the size and orientation of the loop in banana lectin and CCA resemble that of jacalin but not artocarpin. Although not critical for monosaccharide specificity, the spatial position and orientation of the loop suggests that it will strongly influence oligosaccharide binding. A recent crystallographic study involving artocarpin complexes in fact illustrates the influence of the variability of the BL2 loop region in generating diversity in oligosaccharide recognition (Jeyaprakash et al., 2004Go).

BL3
The loop BL3 appears to play a different role, that of determining lectin activity, by generating carbohydrate recognition capability, judged by (1) its invariant conformation and (2) housing the invariant aspartic acid involved in interactions with both the galactose as well as the mannose. The high sequence conservation in this loop leads to the derivation of a sequence motif GXXXD, where X is any amino acid. However, because the binding site in JRLs is formed by multiple segments not contiguous in the sequence, the signature, although a determinant of carbohydrate binding, does not reflect the carbohydrate specificity, unlike in bulb lectins, where a nine-residue sequence signature is related to their mannose-binding abilities (Ramachandraiah and Chandra, 2000Go).

IL
The length and conformation of the IL is also seen to discriminate between the two varieties, the longer one interacting with the three loops BL1–BL3 in galactose-binding JRLs,

Posttranslational cleavage
Posttranslational cleavage leading to a free N-terminus interacting with the sugar is observed in all galactose-specific JRLs and is not found in the known mannose-specific varieties. The first domains of some lectins of B. napus also contain a free amino-terminal group, aligning in position with the N-terminal glycine of jacalin and other galactose-specific lectins. It is also interesting to observe that the first domains of the three lectins of B. napus appear closer to the galactose-specific ones from Moraceae in the phylogenetic tree, away from the other lectins in the Brassicaceae cluster, suggesting that they could exhibit different specificities either at the mono- or at the oligosaccharide level. The sugar specificity of the domains in this case is not individually known, but they are generally annotated as mannose-specific and myrosinase-binding lectins. Although a free amino terminus will be accessible to the sugar in these domains, an aromatic residue analogous to Tyr78 in jacalin is not present to provide a possible stacking interaction. Moreover there is a remarkable variation in loop lengths in these domains as compared to those in the galactose-specific Moraceae lectins. These domains therefore may not exhibit the preference for galactose, despite having a free amino terminus at this site. The analysis presented provides a useful framework for the design of further experimental work to probe if a posttranslational modification is or is not the key in defining galactose specificity in these lectins.

The cleavage site loop
Despite the presence or absence of cleavage, the glycine-rich loop in the region is seen to provide contours to form a carbohydrate-recognition site and hence, in comibation with BL3, can be said to be the determinants of lectin activity in JRLs.

Thus the analysis indicates that it is a combination of factors that determine specificity, rather than a single discriminatory factor. Although the third loop BL3 and the free N-terminal glycine/glycine-rich loop form the contours of the binding site, the conformation of BL1 seems to determine specificity, whereas the interactions from BL2 enhance binding capabilities in individual cases and will heavily influence oligosaccharide binding. Thus features that are crucial versus features that merely enhance binding in individual cases can be understood. This knowledge also helps in predicting sugar specificity for lectins, for which experimental information is not yet available. Given the pace at which whole genome sequences are determined, the ability to predict primary ligand binding will be a valuable tool for understanding function on a large scale as well as to provide focus to design relevant experiments.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Derivation of the data set involved the following steps.

  1. Searching the NR sequence database available at NCBI for sequences resembling the {alpha}-chain of jacalin (accession number P18670, made of 133 amino acids) using BLASTp (Altschul et al., 1990Go). (The first analysis on 6 November 2003 was carried out using the NR database, which then contained 1,537,641 sequences. The analysis was repeated on 11 May 2004 using the NR database, which, although it then contained 1,798,171 sequences, did not show any additional homologs to jacalin.) Only the major {alpha}-subunit of jacalin was used as a search template to include proteins both with and without posttranslational modifications. Additional database searches using BLAST with different parameters as well as using FASTA (Pearson and Lipman, 1988Go) ensured that no borderline homologs were missed by the first BLAST.
  2. Parsing the search outputs to simultaneously filter for the length and the similarity criteria (sequences that bore at least 50% sequence similarity for at least 70% of the length of jacalin, with e-values of less than 0.01 were considered homologs).
  3. Searching for internal repeats and additional domains within each sequence using the CDD domain detection algorithm (Marchler-Bauer et al., 2002Go; lower cutoff for stringency was used here; e-values were relaxed up to 10 for additional domains, similarity and length cutoffs were 50% and 50%, respectively).
  4. Carrying out a two-step nonredundancy check. Filtering to ensure nonredundancy at the first step was carried out using a simple Perl script that parses the pairwise alignment outputs obtained by using Bestfit, a Smith and Waterman implementation in the GCG (Wisconsin) package, and iteratively eliminates the smaller of the 100% identical sequences in every pair from every plant. This was done to ensure that in the final data set no two sequences from a single plant were 100% identical. The second step involved carrying out multiple alignments for all sequences from each plant, individually, using CLUSTALW (Thompson et al., 1994Go) and parsing the outputs, again using a home-grown Perl script, to initially weed out the smaller of the sequences in every set that were at least 90% similar to any other in the set. However, if the weeded-out sequences differed from their closest homologs in any of the binding site segments, as judged by a visual inspection of the alignments, they were put back into the data set and retained as unique entries in this study so as not to miss out on any possible isolectins with a different functional significance. For this purpose, the four loops IL, BL1, BL2, and BL3 and three residues flanking each loop on either side were considered as binding site segments. This step was carried out to ensure that sequences with high overall similarities, but specific changes in the binding sites were not missed.

The individual alignments are available online at http://144.16.93.115/lectin/jacalin/alignments. All alignments—pairwise or multiple—have been carried out with full domain sequences, corresponding to that of the {alpha}-chain of jacalin. Structural alignments and superpositions have also been carried out with full domains.

Distribution across various life forms was analyzed using the Blink module along with the TaxBrowser tool available online at NCBI (www.ncbi.nlm.nih.gov/sutils/static//blinkhelp.html). The phylogenetic analyses were carried out using the MEGA suite of programs (Kumar et al., 2001Go). Sequence profiles were derived and analysed using PRATT and PROSITE (Bairoch and Boeckmann, 1992Go) tools.

The relevant protein structures were obtained from the Protein databank (Berman et al., 2000Go). Secondary structure prediction was carried out by using the PHD algorithm (Rost, 1996Go), through the NPSA server (http://npsa-pbil.ibcp.fr). Molecular models were built using Modeller 6.0 (Sali and Blundell, 1993Go) using homology modeling techniques. Structural equivalences were obtained using the Dali algorithm (Holm and Sander, 1995Go). Solvent-accessible surface areas were computed using the Lee and Richards (1971)Go algorithm, and residues Ala, Phe, Ile, Leu, Met, Pro, Trp, and Val were considered exposed if their surface areas were greater than 30 Å2. Intersubunit and protein–sugar interactions were computed using the CCP4 Contacts program (CCP4, 1994Go). The smaller subunits (of 18–20 residues) making up each subunits in the known galactose-specific lectins were appended to the major subunits and treated as one unit for this calculation, so as to ignore the intrasubunit interface.

Insight II software suite (Accelrys, www.accelrys.com) was used to visualize, overlay, and analyze various structures. Appropriate Perl scripts and C programs were written for various needs of file handling and extracting required information, as well as for appropriate representation. Structure-based sequence alignments were also carried out using home-grown Perl scripts that parsed the Dali outputs and produced appropriate sequence equivalences. Carbohydrate specificities were obtained from the literature and have been made available online at http://144.16.93.115/lectins/jacalin.


    Acknowledgements
 
We thank M. Vijayan and A. Surolia for constant encouragement and useful discussions. We are also grateful to M. Vijayan for critical reading of the manuscript. Financial support from DBT is gratefully acknowledged. Use of facilities at the Super Computer Education & Research Centre, Bioinformatics Centre, and Interactive Graphics facility supported by DBT is also acknowledged.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: nchandra{at}physics.iisc.ernet.in


    Abbreviations
 
BL, binding loop; calsepa, Calystegia sepium lectin; CCA, Castanea crenata agglutinin; heltuba, Helianthus tuberosus lectin; IL, influencing loop; JRLs, jacalin-related lectins; MPA, Maclura pomifera agglutinin; NR, nonredundant


    References
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 Discussion
 Materials and methods
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