Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M414336200 on January 28, 2005

J. Biol. Chem., Vol. 280, Issue 14, 13250-13255, April 8, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/14/13250    most recent
M414336200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nordlund, H. R.
Right arrow Articles by Kulomaa, M. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nordlund, H. R.
Right arrow Articles by Kulomaa, M. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Novel Avidin-like Protein from a Root Nodule Symbiotic Bacterium, Bradyrhizobium japonicum*

Henri R. Nordlund{ddagger}§, Vesa P. Hytönen{ddagger}, Olli H. Laitinen¶, and Markku S. Kulomaa{ddagger}||

From the {ddagger}Department of Biological and Environmental Science, NanoScience Center, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland and Department of Molecular Medicine, A. I. Virtanen Institute, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland

Received for publication, December 21, 2004 , and in revised form, January 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bradyrhizobium japonicum is an important nitrogenfixing symbiotic bacterium, which can form root nodules on soybeans. These bacteria have a gene encoding a putative avidin- and streptavidin-like protein, which bears an amino acid sequence identity of only about 30% over the core regions with both of them. We produced this protein in Escherichia coli both as the full-length wild type and as a C-terminally truncated core form and showed that it is indeed a high affinity biotin-binding protein that resembles (strept)avidin structurally and functionally. Because of the considerable dissimilarity in the amino acid sequence, however, it is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin did not show significant cross-reactivity with it. Therefore this new avidin, named bradavidin, facilitates medical treatments such as targeted drug delivery, gene therapy, and imaging by offering an alternative tool for use if (strept)avidin cannot be used, because of a deleterious patient immune response for example. In addition to its medical value, bradavidin can be used both in other applications of avidin-biotin technology and as a source of new ideas when creating engineered (strept)avidin forms by changing or combining the desired parts, interface patterns, or specific residues within the avidin protein family. Moreover, the unexpected discovery of bradavidin indicates that the group of new and undiscovered bacterial avidin-like proteins may be both more diverse and more common than hitherto thought.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several avidin proteins have been found in bird, reptile, and amphibian species (1–3). Those of the bird avidins that have been characterized are relatively similar, although displaying some differences in stability and immunological cross-reactivity when compared with those of chicken avidin (3, 4). In the chicken, the avidin gene forms a gene family together with the avidin-related genes (AVR) (5). The AVR proteins have recently been produced as recombinant proteins. Their characterization and comparison with each other and with avidin reveals some differences in the properties of stability, glycosylation, and biotin binding, although the primary amino acid sequences are rather well conserved (6, 7). Several Streptomyces strains were studied four decades ago, and the bacterial analog for avidin, streptavidin, was found in a strain that was given the name Streptomyces avidinii (8). Inspired by these studies, other streptomycetes have since been studied. So far, two new streptavidins have been found in Streptomyces venezuelae and have been named, accordingly, streptavidin v1 and v2. These new forms were found to be almost identical with streptavidin, displaying only one (v1) or five (v2) amino acid substitutions in the core region, which has no observed significance for either the structure or the function of these proteins (9).

Medical applications of avidin-biotin technology (10, 11) include, for example, gene therapy (12, 13), imaging (14), and targeted drug delivery (15, 16). In traditional one-step radioimmunotherapy, a therapeutic radioactive material is directly linked to a tumor-specific antibody (17–19). To improve the low target/non-target ratio, a drawback with this methodology, several improved protocols for delivering tumor cell-targeted radiation, which usually include more steps, have been developed (20). One of the most promising methods is three-step pretargeting radioimmunotherapy, which includes the following treatment steps: (i) a biotinylated antibody specific for the target tumor cells; (ii) chicken avidin (fast pharmacokinetic clearance) as a clearing agent to remove endogenous biotin and the excess free circulating biotinylated antibodies from the first step followed by streptavidin (slow pharmacokinetic clearance), which is mainly responsible for avidinylation of the tumor cells; and (iii) biotinylated radioactive material, which binds tightly to the free binding sites of the tetravalent (strept)avidin molecules immobilized by the biotinylated antibodies (21–23). In addition to chicken avidin and streptavidin, the existing, rather thoroughly characterized avidin protein pool for medical purposes includes poultry avidins, of which duck, goose, and ostrich avidin (4) in particular have been shown in vitro to be potential alternatives for patients who have strong immunological response toward (strept)avidin because of usually repeated treatments. Some of the AVR proteins (6) might also prove usable instead of or before (strept)avidin in sequential pretargeting radioimmunotherapy treatments, if they turn out to be immunologically different enough in vivo and show no significant cross-reactivity with the antibodies elicited in the possible preceding steps. Furthermore, differences in pharmacokinetics and other properties because of, for example, varied glycosylation patterns and protein pI (24), can be exploited when selecting avidins for specific applications. However, as all these biotin-binding proteins are xenoproteins and there is no avidin analog of human origin, they are likely to be antigenic and therefore cannot be used effectively on successive occasions with the same patient. Therefore, an immediate need exists for new and dissimilar avidins, such as the characterized bradavidin and others discussed in this report.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Production and Purification of Bradavidin—The gene coding for bradavidin (DDBJ AP005955.1) was amplified by PCR using Bradyrhizobium japonicum genomic DNA as a template and was extended using stepwise elongation of sequence (SES)-PCR (25) to include attL recombination sites at both ends (26). Two constructs were generated: the full-length wild type (138 amino acid residues) and a C-terminally truncated core form (118 amino acid residues). Both constructs also contained their innate signal peptides (25 amino acid residues). These constructs were then transferred to pBVboostFG vector1 using the site-specific recombination-based Gateway method (Invitrogen). The resulting expression vectors were confirmed to be as designed by DNA sequencing.

Escherichia coli BL-21(AI) cells (Invitrogen) were used for protein expression as described previously (27). The recombinant proteins were isolated from bacterial cell extracts by affinity chromatography on 2-iminobiotin agarose column (27). Eluted proteins were analyzed by SDS-PAGE and subsequent Coomassie staining of the gels. Protein concentrations were determined using the calculated extinction coefficient 39 380 M–1 cm–1 for both bradavidins at 280 nm (28).

Primary Structure Analysis—Pairwise sequence alignments were done using the Needle program from the European Molecular Biology Open Software Suite (EMBOSS), and the ClustalW program was used to generate the multiple sequence alignment (29). The theoretic biochemical properties were determined using the ProtParam program (28). The putative signal peptide cleavage site was determined by the SignalP 3.0 program (30).

Analysis of Function and Properties—The dissociation rate constant of [3H]biotin (Amersham Biosciences) from the bradavidins and avidin was measured at various temperatures as described in detail previously (31). The dissociation rate of a fluorescent biotin conjugate (ArcDia BF560TM-biotin) was measured, as described previously (27), at 50 °C.

The purified proteins were analyzed by gel filtration using a Shimadzu HPLC instrument equipped with a Superdex 200 HR 10/30 column (Amersham Biosciences) with 50 mM sodium carbonate buffer (pH 11) with 150 mM NaCl as the liquid phase. The column was calibrated using a marker mixture (thyroglobulin, IgG, ovalbumin, myoglobin, vitamin B12; Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin (Roche Diagnostics) as molecular mass standards.

The thermal stability characteristics of the proteins were studied using an SDS-PAGE-based method as described previously in detail (32).

Antibody Recognition—Serum samples from cancer patients exposed to avidin and streptavidin were used to compare the immunological properties of the avidins. The serum samples as well as the negative control sera from persons not exposed to (strept)avidin were obtained from the Division of Nuclear Medicine, European Institute of Oncology, Milan, Italy. The analysis was performed similarly as described previously (4). ImmobilizerTM Amino plates (Nalge Nunc Int.) were coated with the proteins under study (10 µg/ml) in 100 mM sodium phosphate, pH 7.5, agitated for 1 h at room temperature, and blocked with PBS-T2 (PBS plus Tween 20, 0.05% v/v). The serum samples were diluted 1:100 in PBS-T and incubated in the wells for 1 h at 37 °C. After washing three times with PBS-T, polyvalent anti-human immunoglobulin alkaline phosphatase conjugate (Sigma) was used as a secondary antibody (dilution 1:6000; 1 h, 37 °C) followed by six washes with PBS-T. Finally, p-nitrophenyl phosphate (1 mg/ml, Sigma) was used as a substrate molecule, and a plate reader was used to measure the absorbance at 405 nm.

Proteins were further compared using polyclonal rabbit antibodies produced against avidin (University of Oulu, Finland) and streptavidin (a generous gift from Edward A. Bayer, Weissman Institute, Jerusalem, Israel). Proteins were first attached to ImmobilizerTM Amino plates as described above and blocked with PBS-T. Antibodies were diluted 1:2000 to PBS-T and applied to the protein-coated plates (1 h, 37 °C). After washing with PBS-T, goat anti-rabbit IgG alkaline phosphatase (Bio-Rad Laboratories) diluted 1:2000 in PBS-T was used as a secondary antibody (1 h, 37 °C), and the signal was measured as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production and Purification of Bradavidin—Two forms of bradavidin, the full-length wild type and a C-terminally truncated core (analogous to streptavidin core), were produced in E. coli with their innate signal peptides. After one-step purification on 2-iminobiotin agarose column, the eluted proteins appeared to be pure and virtually homogenous, as only one band/lane of the expected size was observed on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels.

Primary Structure Analysis—Pairwise sequence alignment for mature core regions of avidin and bradavidin revealed that 29.2% of the amino acids are identical and 39.2% similar, whereas with streptavidin these values are 30.2 and 41.7%, respectively. Interestingly, when avidin and streptavidin were compared equivalently, the values obtained, 31.9 and 45.2%, were only slightly higher. Multiple sequence alignment of streptavidin, bradavidin, and avidin (Fig. 1) revealed that most of the conserved residues are directly involved in biotin binding in avidin and streptavidin or are structurally important characteristics in the avidin protein family (33, 34). Over the plausible biotin-binding residues bradavidin bears a slightly closer resemblance to streptavidin than avidin. Bradavidin has two cysteine residues which, according to the known avidin structure (34), could form an intramonomeric disulfide bridge spatially different from that in chicken avidin, whereas streptavidin is devoid of cysteines. In line with this understanding, we observed only monomeric forms in the SDS-PAGE samples boiled in sample buffer without the reducing agent {beta}-mercaptoethanol (Fig. 2), indicating that bradavidin does not have intermonomeric disulfide bridges analogous to those present in engineered avidin forms (35, 36).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1.
Multiple sequence alignment of the core forms of strept(avidin), brad(avidin), and chicken (avidin). The arrows indicate the location of the successive {beta}-strands according to the structure of chicken avidin. The cysteine residues in chicken avidin (Cys-4 and Cys-84), which form an intramonomeric disulfide bridge, are shown as bold letters. Similarly the cysteine residues (Cys-39 and Cys-69) in bradavidin are shown in bold; these too could form an intramonomeric disulfide bridge although not spatially equivalent to that of chicken avidin. The conserved amino acids are marked below the sequences by an asterisk and strong amino acid group similarity by a colon, whereas weaker group similarity is indicated by a single dot. Biotin-binding residues of avidin and streptavidin are underlined (34).

 



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 2.
Non-reducing but denaturing SDS-PAGE analysis. Wild-type bradavidin is indicated by wt and the C-terminally truncated form by core. The unit of molecular mass markers, indicated by M, is kDa.

 
Analysis of Function and Properties—Ligand-binding properties were studied both by a [3H]biotin assay and a fluorescent biotin conjugate assay. According to the results (Table I, Fig. 3), the faster dissociation rate measured from bradavidin indicates weaker affinity toward radiobiotin than that found in avidin and streptavidin. However, bradavidin showed a clearly slower rate of fluorescent biotin displacement than did avidin, thus proving to be almost as extreme a biotin conjugate binder as streptavidin (37).


View this table:
[in this window]
[in a new window]
  		TABLE I
Protein characteristics

The apparent molecular mass is indicated and followed by the theoretical mass in parentheses. Transition temperature (Tr) indicates the temperature in which half of the protein is tetrameric and half monomeric in the absence (first value) and presence (second value) of biotin. In addition to the measured dissociation rate constant (kd), the release percentage of the fluorescent biotin conjugate in 1 h in the presence of excess free biotin is indicated. The calculated isoelectric point (pI) and the number of cysteine residues/monomer are also indicated.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3.
Biotin dissociation analysis. A, the [3H]biotin dissociation rate constant was measured at different temperatures. The values for streptavidin are from Klumb et al. (31). The scale of the y axis is logarithmic. B, release of fluorescent biotin conjugate from avidins was studied as a function of time in the presence of excess D-biotin at 50 °C.

 
Gel filtration chromatography showed that bradavidin is a homogenous tetramer, and both forms appeared as a single symmetrical and sharp peak on the chromatograms. SDS-PAGE stability analysis confirmed the tetrameric appearance. These oligomeric assemblies showed comparable stability with that of avidin and streptavidin (Table I).

Antibody Recognition—Immunological cross-reactivity of bradavidin with human and rabbit serum antibodies, elicited toward avidin and streptavidin, was analyzed by an enzyme-linked immunosorbent assay. The avidin protein under inspection was first immobilized on the plate followed by the antiserum and alkaline-phosphatase-conjugated secondary antibody (Fig. 4). Samples from cancer patients exposed to avidin and streptavidin recognized avidin and, even more clearly, streptavidin. This may stem not only from the number and extent of medical treatments but also from the fact that streptavidin is more antigenic than avidin (38, 39). None of the patient sera showed a significant response toward bradavidin, which clearly indicates that this protein is largely devoid of common epitopes with (strept)avidin. In addition to human samples, polyclonal rabbit antibodies recognized only the protein toward which they had been elicited in the first place.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4.
Immunological cross-reactivity assay. Patients labeled A through E were subjected to pretargeting radioimmunotherapy treatment using both avidin and streptavidin, whereas the donors of the negative control sera N1 and N2 were not exposed to avidin or streptavidin. Polyclonal rabbit antibodies toward streptavidin (SA) and avidin (AVD) were also tested for cross-reactivity.

 
Bradavidin was also probed by polyclonal rabbit antibodies against (strept)avidin on Western blots, with only the positive (strept)avidin controls being detected after immunostaining. Preceding that step, when the nitrocellulose filter was stained with Ponceau S dye, wild-type bradavidin was clearly visible at the expected location, whereas the bradavidin core appeared to be virtually absent from the blot at this stage (data not shown). This behavior may result from the rather low pI of the core form (Table I), as has also been suspected, for example, in the case of the acidic natural rubber latex allergen Hev b5 (40).

Comparison of New Potential Avidins—A multiple sequence alignment of many known avidin-like proteins (6, 8, 41) and some new candidates suggested biotin-binding capability for the open reading frames from Xanthomonas campestris (Gen-BankTM AE012315 [GenBank] .1), Rhizobium etli (GenBankTM U80928 [GenBank] .4), B. japonicum (another candidate in addition to bradavidin, DDBJ AP005940.1), Burkholderia pseudomallei (EMBL BX571965 [GenBank] .1), and Burkholderia mallei (GenBankTM CP000010 [GenBank] .1), referred to as Xant, Rhiz, Brad2, Burk_pseudomallei, and Burk_mallei, respectively (Fig. 5). The majority of the putative biotin-binding residues, according to the avidin (34) and streptavidin (33) structures, were conserved, albeit the overall sequence similarity was rather low (Fig. 5). Conserved structural characteristics of the avidin fold (42) were also observed in the sequences, which further supports our assumptions. However, at present we have more sequence data and experimental knowledge of these proteins at our disposal than Flower (42) had when he originally determined the structural signature for avidin. Therefore we may be able to both extend and circumscribe the essential characteristics of the avidin fingerprint.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 5.
Multiple sequence alignment of known and candidate avidin-like proteins. The new candidate sequences are shown below bradavidin. They were obtained by TBlastn using the bradavidin sequence as the query. Avidin-related proteins (AVR) are included because they have been characterized previously as high affinity biotin-binding proteins among avidin and streptavidin (6). The N- and C-terminal signals and extensions are included in this alignment. The conserved amino acids are marked below the sequences by an asterisk, strong amino acid group similarity is indicated by a colon, and weaker group similarity is indicated by a single dot. Biotin-binding residues of avidin and streptavidin are underlined (34). The proper avidin search string for data base queries can be obtained by selecting or emphasizing most of the positions marked below the sequences by an asterisk and a colon. Moreover, this knowledge can be used as the basis for cDNA and genomic library probe design. In addition, by appropriately limiting the distance between the fixed positions, allowing only small fluctuation between these essential avidin characteristics, most of the prospective research results have a strong potential to be avidins. An example of such an avidin string is: WXN(E/Q/N/D)XGSX(M/L/F)X(I/V)X7,12GX(F/Y)X17,36(F/W)XVX(F/W)X3,10(S/A)X(T/S)X(W/F)XGX5m, 14 (M/I/F/L)XXX(W/Y)X16,21(D/N)XF. In this string, X denotes any amino acid residue, alternatives for a certain position are shown in parentheses, and the subscripted numbers indicate the lower and upper limit, respectively, for the length of the X-stretch in question.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Biotin is an essential cofactor in many vital biochemical reactions (43, 44). Therefore it is understandable that (strept)avidin can work as a broad-range antimicrobial agent by forming a biotin-free zone or protective barrier around an organism or, for example, an egg possessing the biotin-binding protein (41). The biological role of bradavidin could also be protective, as it proved to be a high affinity biotin-binding protein. If the B. japonicum-containing root nodules on soybeans are found to express, possibly upon injury or infection, and contain or secrete at least a small amount of bradavidin among other defense proteins and compounds, the plant could be resistant toward many invaders. These could include harmful soil microbes, insects, and even higher animals. Experiments on transgenic corn have shown that the expression of avidin in the plant has an enormous impact on the majority of insect pests, particularly at certain developmental stage of the larvae (45, 46). However, the biological role suggested here for bradavidin is only hypothetical, and a bradavidin knock-out, B. japonicum, could give valuable information on this matter. In addition it would be interesting to exchange the bradavidin gene in B. japonicum with a different and even engineered avidin form displaying, for example, better ligand affinity, enhanced stability, or some other special property (such as the peculiar DNA-protein interaction or the lack of such trait, described recently for chicken avidin (47)).

Although avidin and streptavidin are structurally and functionally similar, their pharmacokinetic characteristics differ radically (24, 48, 49). Both glycosylation and high pI are thought to cause the rapid clearance of avidin from the blood. It has been found that glycosylation causes avidin accumulation in the liver, and the high pI is responsible for the avidin accumulation in the kidneys (50). Streptavidin, which has a significantly longer plasma half-life compared with avidin, is known to accumulate in the kidneys (48), possibly via integrin-mediated cell adhesion dependent on an RGD-like domain (51). A streptavidin mutant, in which this RGD-like stretch was modified, showed markedly reduced cell adhesion (52). Further study of bradavidin is essential to reveal its pharmacokinetic properties and possible divergences from those of (strept)avidin, before it can be appropriately and effectively used in medical applications.

Several attempts have been made to modify (strept)avidin in order to change its accumulation, clearance, and immunological properties in vivo. Paganelli and co-workers (38) were able to lower avidin accumulation in the kidneys and liver by attaching polyethylene glycol groups to avidin. Furthermore, these polyethylene glycol-containing avidins were found to be less immunogenic. Also epitope-modified recombinant streptavidins, carrying point mutations, with markedly improved immunological properties have been generated (53). In another study, deglycosylated and chemically neutralized avidin was found to be superior when compared with wild-type avidin in brain delivery (54). The better penetration was supposed to be due to the extended circulation time. Analogously, when galactose moieties are chemically attached to streptavidin, its blood clearance is accelerated (24). Yao et al. (55) in turn made another peculiar observation; they demonstrated that avidin itself accumulates efficiently in lectin-expressing tumors, whereas streptavidin and chemically neutralized avidin do not exhibit this kind of behavior. Altogether, these findings underline the idea that bradavidin should be characterized thoroughly in order to bring this novel and immunologically promising tool into use.

The biotin-binding properties of bradavidin were shown in the present study to bear more resemblance to streptavidin than avidin. Bradavidin displayed the fastest dissociation rate when radiobiotin was used in the analysis. Avidin showed clearly the slowest dissociation rate, whereas the value for streptavidin fell between these two (Fig. 3A). Moreover, when the ligand was a fluorescent biotin conjugate, avidin was clearly the fastest in dissociation, and streptavidin and bradavidin were nearly identical, showing a very slow release in the assay (Fig. 3B). This finding is in line with a previous study (37) in which streptavidin was proven to be a better biotin conjugate binder than avidin. This property, also characteristic of bradavidin, is interesting and renders it a good tool in applications, because the biotin in use is usually a conjugate, and thus good affinity is essential. The structural differences in the loop between {beta}-strands 3 and 4 are thought to explain this divergent binding ability of avidin and streptavidin (33, 34, 37). In bradavidin this loop is extraordinary, because it contains a cysteine residue that could form a disulfide bridge with the cysteine residue on the structurally neighboring loop between {beta}-strands 5 and 6. This interesting motif, potentially on top of the entrance of the binding site, could have some effect on the binding parameters described above. It could also explain the fundamental reasons behind them, such as the divergent association rate, which we intend to study in greater detail together with the possible crystal structure of bradavidin.

The existence of the bradavidin gene in the genome of a root nodule symbiotic bacterium, B. japonicum, may be just the first example of other genes producing functionally similar proteins in other plant-related bacteria. It is possible that further study of the root nodules of species other than the soybean will reveal a variety of such proteins. According to our 16 S ribosomal RNA gene comparisons, the strains producing the different streptavidins (S. avidinii and S. venezuelae) described so far share about 97% sequence identity, indicating close evolutionary relationship. On the contrary, B. japonicum is clearly not a close relative of these bacteria, because its 16 S rRNA gene bears only about 74 and 77% sequence identity with those of S. avidinii and S. venezuelae, respectively. A straightforward assumption would be that some of the possible new avidins from symbiotic bacteria could closely resemble bradavidin, although completely different forms might also be found. Therefore, in addition to sequence data base queries based on whole sequence similarity, an effective string could be obtained from an extensive multiple sequence alignment of different avidins and related biotin-binding proteins (Fig. 5). By using such an avidin fingerprint string, containing only certain functionally and structurally necessary amino acid stretches and patterns, new avidins could be tracked down from virtually any life form once the sequence data becomes available. This string could also be utilized when designing probes for cDNA or genomic library screening, emphasizing the conserved spots, when the actual sequence is unknown.

The comparison of some potential avidin-like sequences with those of the avidin and avidin-related sequences shown in the multiple sequence alignment (Fig. 5) revealed intriguing details. The conserved Trp-10 on the first {beta}-strand (avidin numbering for amino acids and {beta}-strands) is invariably preceded by Gly-8 or Ser-8, with the exception of bradavidin, which has Trp in that position. This indicates the possibility of other acceptable substitutions in this position. On the bottom of the biotin-binding site is an important ligand contact residue, Tyr-33 (33, 34), which is conserved, excluding the putative avidin from B. japonicum (Brad2), which bears the Tyr to Phe substitution. In previous point mutagenesis studies of streptavidin (31) and avidin (56), the analogous mutation resulted in a 5- and 13-fold increase, respectively, in the dissociation rate constants of the ligands studied. It is possible, therefore, that this putative Brad2 protein may exhibit high biotin affinity despite an aberrant residue in this particular position. The loops connecting {beta}-strands 3 and 4 in avidin and streptavidin (Fig. 1) are different from each other. The role of this loop is to form certain hydrogen bonds and other contacts with biotin. It seems that the putative avidins of different origin may also form similar interactions, although this ability cannot be definitively demonstrated without a three-dimensional structure with the ligand. Moreover, Trp-70 and Trp-97 form part of the hydrophobic cavity of the ligand-binding site in avidin (34). The importance of the equivalent residues in streptavidin for biotin binding has also been experimentally shown by Stayton and co-workers (57), who mutated these residues to alanine and phenylalanine and observed a significant decrease in affinity in the case of the alanine mutants. However, in the case of the phenylalanine substitutions the decreases in the observed affinities were only mediocre. We assume, therefore, that the W97Y difference present in the putative Burk_pseudomallei avidin would not radically diminish the biotin affinity of this protein. In the present study we found that bradavidin is a high affinity biotin-binding protein, although it has Phe at position 70, whereas avidin has Trp at the same position. This further supports the idea that conservation of the complementarity of the binding site and the ligand structure is essential for high affinity (33, 34). The most radical differences in the multiple sequence alignment are found in the loop around Trp-110, which plays a central role in ligand binding (33, 34, 57–59). We can only suggest that the loop structures containing the proline residues in Rhiz and Brad2 sequences (Fig. 5) might be able to form contacts comparable, or at least compensatory, to those formed by the tryptophan in avidin and the others of the studied sequences.


   FOOTNOTES
 
* This work was supported by ARK Therapeutics Oy, Kuopio, Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Institute of Medical Technology, FIN-33014 University of Tampere, Finland. Back

|| To whom correspondence should be addressed: Institute of Medical Technology, FIN-33014 University of Tampere, Finland. E-mail: markku.kulomaa{at}uta.fi.

1 Laitinen, O. H., Airenne, K. J., Hytönen, V. P., Peltomaa, E., Mähönen, A. J., Wirth, T., Lind, M. M., Mäkelä, K. A., Toivanen, P. I., Schenkwein, D., Heikura, T., Nordlund, H. R., Kulomaa, M. S., and Ylä-Herttuala, S. (2005) Nucleic Acids Res., in press. Back

2 The abbreviation used is: PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Irene Helkala, Jarno Hörhä, Tuomas Kulomaa, and Juha Määttä for outstanding assistance and Kirsi Liimatainen (from Finnzymes) for the ImmobilizerTM Amino plates. We are also grateful to Kazusa DNA Research Institute, in particular to Drs. Takakazu Kaneko and Satoshi Tabata, for kindly providing the B. japonicum USDA110 genomic DNA sample and to Dr Giovanni Paganelli (European Institute of Oncology) for providing the patient serum samples.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hertz, R., and Sebrell, W. H. (1942) Science 96, 257[Free Full Text]
  2. Jones, P. D., and Briggs, M. H. (1962) Life Sci. 11, 621–623
  3. Korpela, J. K., Kulomaa, M. S., Elo, H. A., and Tuohimaa, P. J. (1981) Experientia 37, 1065–1066[CrossRef][Medline] [Order article via Infotrieve]
  4. Hytönen, V. P., Laitinen, O. H., Grapputo, A., Kettunen, A., Savolainen, J., Kalkkinen, N., Marttila, A. T., Nordlund, H. R., Nyholm, T. K., Paganelli, G., and Kulomaa, M. S. (2003) Biochem. J. 372, 219–225[CrossRef][Medline] [Order article via Infotrieve]
  5. Ahlroth, M. K., Kola, E. H., Ewald, D., Masabanda, J., Sazanov, A., Fries, R., and Kulomaa, M. S. (2000) Anim. Genet 31, 367–375[CrossRef][Medline] [Order article via Infotrieve]
  6. Laitinen, O. H., Hytönen, V. P., Ahlroth, M. K., Pentikäinen, O. T., Gallagher, C., Nordlund, H. R., Ovod, V., Marttila, A. T., Porkka, E., Heino, S., Johnson, M. S., Airenne, K. J., and Kulomaa, M. S. (2002) Biochem. J. 363, 609–617[CrossRef][Medline] [Order article via Infotrieve]
  7. Hytönen, V. P., Nyholm, T. K., Pentikäinen, O. T., Vaarno, J., Porkka, E. J., Nordlund, H. R., Johnson, M. S., Slotte, J. P., Laitinen, O. H., and Kulomaa, M. S. (2004) J. Biol. Chem. 279, 9337–9343[Abstract/Free Full Text]
  8. Chaiet, L., and Wolf, F. J. (1964) Arch Biochem. Biophys. 106, 1–5[CrossRef][Medline] [Order article via Infotrieve]
  9. Bayer, E. A., Kulik, T., Adar, R., and Wilchek, M. (1995) Biochim. Biophys. Acta 1263, 60–66[Medline] [Order article via Infotrieve]
  10. Wilchek, M., and Bayer, E. (1990) Methods Enzymol. 184, 5–13[Medline] [Order article via Infotrieve]
  11. Wilchek, M., and Bayer, E. A. (1999) Biomol. Eng. 16, 1–4[CrossRef][Medline] [Order article via Infotrieve]
  12. Wojda, U., Goldsmith, P., and Miller, J. L. (1999) Bioconjugate Chem. 10, 1044–1050[CrossRef][Medline] [Order article via Infotrieve]
  13. Lehtolainen, P., Wirth, T., Taskinen, A. K., Lehenkari, P., Leppänen, O., Lappalainen, M., Pulkkanen, K., Marttila, A., Marjomäki, V., Airenne, K. J., Horton, M., Kulomaa, M. S., and Ylä-Herttuala, S. (2003) Gene Ther. 10, 2090–2097[CrossRef][Medline] [Order article via Infotrieve]
  14. Rosebrough, S. F. (1996) Q. J. Nucl. Med. 40, 234–251[Medline] [Order article via Infotrieve]
  15. Lehtolainen, P., Taskinen, A., Laukkanen, J., Airenne, K. J., Heino, S., Lappalainen, M., Ojala, K., Marjomäki, V., Martin, J. F., Kulomaa, M. S., and Ylä-Herttuala, S. (2002) J. Biol. Chem. 277, 8545–8550[Abstract/Free Full Text]
  16. Räty, J. K., Airenne, K. J., Marttila, A. T., Marjomäki, V., Hytönen, V. P., Lehtolainen, P., Laitinen, O. H., Mähönen, A. J., Kulomaa, M. S., and Ylä-Herttuala, S. (2004) Mol. Ther. 9, 282–291[Medline] [Order article via Infotrieve]
  17. Klein, J. L., Nguyen, T. H., Laroque, P., Kopher, K. A., Williams, J. R., Wessels, B. W., Dillehay, L. E., Frincke, J., Order, S. E., and Leichner, P. K. (1989) Cancer Res. 49, 6383–6389[Abstract/Free Full Text]
  18. Beaumier, P. L., Venkatesan, P., Vanderheyden, J. L., Burgua, W. D., Kunz, L. L., Fritzberg, A. R., Abrams, P. G., and Morgan, A. C., Jr. (1991) Cancer Res. 51, 676–681[Abstract/Free Full Text]
  19. Knox, S. J., Goris, M. L., and Wessels, B. W. (1992) Radiother. Oncol. 23, 111–117[CrossRef][Medline] [Order article via Infotrieve]
  20. Boerman, O. C., van Schaijk, F. G., Oyen, W. J., and Corstens, F. H. (2003) J. Nucl. Med. 44, 400–411[Abstract/Free Full Text]
  21. Paganelli, G., Magnani, P., Zito, F., Villa, E., Sudati, F., Lopalco, L., Rossetti, C., Malcovati, M., Chiolerio, F., and Seccamani, E. (1991) Cancer Res. 51, 5960–5966[Abstract/Free Full Text]
  22. Paganelli, G., Grana, C., Chinol, M., Cremonesi, M., De Cicco, C., De Braud, F., Robertson, C., Zurrida, S., Casadio, C., Zoboli, S., Siccardi, A. G., and Veronesi, U. (1999) Eur. J. Nucl. Med. 26, 348–357[CrossRef][Medline] [Order article via Infotrieve]
  23. Grana, C., Chinol, M., Robertson, C., Mazzetta, C., Bartolomei, M., De Cicco, C., Fiorenza, M., Gatti, M., Caliceti, P., and Paganelli, G. (2002) Br. J. Cancer 86, 207–212[CrossRef][Medline] [Order article via Infotrieve]
  24. Rosebrough, S. F., and Hartley, D. F. (1996) J. Nucl. Med. 37, 1380–1384[Abstract/Free Full Text]
  25. Majumder, K. (1992) Gene (Amst.) 110, 89–94[CrossRef][Medline] [Order article via Infotrieve]
  26. Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) Genome Res. 10, 1788–1795[Abstract/Free Full Text]
  27. Hytönen, V. P., Laitinen, O. H., Airenne, T. T., Kidron, H., Meltola, N. J., Porkka, E., Hörhä, J., Paldanius, T., Määttä, J. A., Nordlund, H. R., Johnson, M. S., Salminen, T. A., Airenne, K. J., Ylä-Herttuala, S., and Kulomaa, M. S. (2004) Biochem. J. 384, 385–390[CrossRef][Medline] [Order article via Infotrieve]
  28. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319–326[CrossRef][Medline] [Order article via Infotrieve]
  29. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673–4680[Abstract/Free Full Text]
  30. Bendtsen, J. D., Nielsen, H., von Heijne, G., and Brunak, S. (2004) J. Mol. Biol. 340, 783–795[CrossRef][Medline] [Order article via Infotrieve]
  31. Klumb, L. A., Chu, V., and Stayton, P. S. (1998) Biochemistry 37, 7657–7663[CrossRef][Medline] [Order article via Infotrieve]
  32. Bayer, E. A., Ehrlich-Rogozinski, S., and Wilchek, M. (1996) Electrophoresis 17, 1319–1324[CrossRef][Medline] [Order article via Infotrieve]
  33. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., and Salemme, F. R. (1989) Science 243, 85–88[Abstract/Free Full Text]
  34. Livnah, O., Bayer, E. A., Wilchek, M., and Sussman, J. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5076–5080[Abstract/Free Full Text]
  35. Reznik, G. O., Vajda, S., Smith, C. L., Cantor, C. R., and Sano, T. (1996) Nat. Biotechnol. 14, 1007–1011[CrossRef][Medline] [Order article via Infotrieve]
  36. Nordlund, H. R., Laitinen, O. H., Uotila, S. T., Nyholm, T., Hytönen, V. P., Slotte, J. P., and Kulomaa, M. S. (2003) J. Biol. Chem. 278, 2479–2483[Abstract/Free Full Text]
  37. Pazy, Y., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2002) J. Biol. Chem. 277, 30892–30900[Abstract/Free Full Text]
  38. Chinol, M., Casalini, P., Maggiolo, M., Canevari, S., Omodeo, E. S., Caliceti, P., Veronese, F. M., Cremonesi, M., Chiolerio, F., Nardone, E., Siccardi, A. G., and Paganelli, G. (1998) Br. J. Cancer 78, 189–197[Medline] [Order article via Infotrieve]
  39. Paganelli, G., Chinol, M., Maggiolo, M., Sidoli, A., Corti, A., Baroni, S., and Siccardi, A. G. (1997) Eur. J. Nucl. Med. 24, 350–351[CrossRef][Medline] [Order article via Infotrieve]
  40. Akasawa, A., Hsieh, L. S., Martin, B. M., Liu, T., and Lin, Y. (1996) J. Biol. Chem. 271, 25389–25393[Abstract/Free Full Text]
  41. Green, N. M. (1975) Adv. Protein Chem. 29, 85–133[Medline] [Order article via Infotrieve]
  42. Flower, D. R. (1993) FEBS Lett. 333, 99–102[CrossRef][Medline] [Order article via Infotrieve]
  43. Wood, H. G., and Barden, R. E. (1977) Annu. Rev. Biochem. 46, 385–413[CrossRef][Medline] [Order article via Infotrieve]
  44. Samols, D., Thornton, C. G., Murtif, V. L., Kumar, G. K., Haase, F. C., and Wood, H. G. (1988) J. Biol. Chem. 263, 6461–6464[Free Full Text]
  45. Morgan, T. D., Oppert, B., Czapla, T. H., and Kramer, K. J. (1993) Entomol. Exp. Appl. 69, 97–108[CrossRef]
  46. Kramer, K. J., Morgan, T. D., Throne, J. E., Dowell, F. E., Bailey, M., and Howard, J. A. (2000) Nat. Biotechnol. 18, 670–674[CrossRef][Medline] [Order article via Infotrieve]
  47. Morpurgo, M., Radu, A., Bayer, E. A., and Wilchek, M. (2004) J. Mol. Recognit. 17, 558–566[CrossRef][Medline] [Order article via Infotrieve]
  48. Schechter, B., Silberman, R., Arnon, R., and Wilchek, M. (1990) Eur. J. Biochem. 189, 327–331[Medline] [Order article via Infotrieve]
  49. Rosebrough, S. F. (1993) Nucl. Med. Biol. 20, 663–668[CrossRef][Medline] [Order article via Infotrieve]
  50. Yao, Z., Zhang, M., Sakahara, H., Nakamoto, Y., Higashi, T., Zhao, S., Sato, N., Arano, Y., and Konishi, J. (1999) J. Nucl. Med. 40, 479–483[Abstract/Free Full Text]
  51. Alon, R., Bayer, E. A., and Wilchek, M. (1993) Eur. J. Cell Biol. 60, 1–11[Medline] [Order article via Infotrieve]
  52. Murray, S., Maraveyas, A., Dougan, T., and Chu, A. C. (2002) Biochim. Biophys. Acta 1570, 81–88[Medline] [Order article via Infotrieve]
  53. Meyer, D. L., Schultz, J., Lin, Y., Henry, A., Sanderson, J., Jackson, J. M., Goshorn, S., Rees, A. R., and Graves, S. S. (2001) Protein Sci. 10, 491–503[CrossRef][Medline] [Order article via Infotrieve]
  54. Kang, Y.-S., and Pardridge, W. M. (1994) J. Pharmacol. Exp. Ther. 269, 344–350[Abstract/Free Full Text]
  55. Yao, Z., Zhang, M., Sakahara, H., Saga, T., Arano, Y., and Konishi, J. (1998) J. Natl. Cancer Inst. 90, 25–29[Abstract/Free Full Text]
  56. Marttila, A. T., Hytönen, V. P., Laitinen, O. H., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (2003) Biochem. J. 369, 249–254[CrossRef][Medline] [Order article via Infotrieve]
  57. Chilkoti, A., Tan, P. H., and Stayton, P. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1754–1758[Abstract/Free Full Text]
  58. Laitinen, O. H., Nordlund, H. R., Hytönen, V. P., Uotila, S. T., Marttila, A. T., Savolainen, J., Airenne, K. J., Livnah, O., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (2003) J. Biol. Chem. 278, 4010–4014[Abstract/Free Full Text]
  59. Laitinen, O. H., Airenne, K. J., Marttila, A. T., Kulik, T., Porkka, E., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (1999) FEBS Lett. 461, 52–58[CrossRef][Medline] [Order article via Infotrieve]
  60. Bruch, R. B., and White, H. B. (1982) Biochemistry 21, 5334–5341[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
P. Macor, C. Tripodo, S. Zorzet, E. Piovan, F. Bossi, R. Marzari, A. Amadori, and F. Tedesco
In vivo Targeting of Human Neutralizing Antibodies against CD55 and CD59 to Lymphoma Cells Increases the Antitumor Activity of Rituximab
Cancer Res., November 1, 2007; 67(21): 10556 - 10563.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/14/13250    most recent
M414336200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nordlund, H. R.
Right arrow Articles by Kulomaa, M. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nordlund, H. R.
Right arrow Articles by Kulomaa, M. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement