Chickens are known to produce several different proteins which bind biotin in a non-covalent fashion. One of them is avidin, which is expressed by oviduct cells upon progesterone induction and is then transferred to the egg-white where it constitutes a minor fraction of the total protein content of the egg-white [1]. Independently of progesterone, avidin expression is also induced by an inflammation response in almost all of the studied chicken tissues [2]. Another biotin-binder, called literally biotin-binding protein (BBP), is presumably induced by estrogen [3] and secreted from the liver into chicken plasma [4]. From plasma, the BBP is thought to be deposited in egg-yolk [5]. In addition, Seshagiri and Adiga found another egg-white BBP, distinct from avidin, the biochemical characteristics of which resemble those reported for yolk BBP [6].

The affinities that avidin and yolk BBP exhibit toward biotin are extremely high, the dissociation constant being femtomolar for avidin [1] and picomolar for BBP [7]. According to the published data, the yolk BBP serves as a biotin reserve for the developing embryo and hence it is saturated with the vitamin [5]. In contrast, avidin in egg-white is mainly found as an apoprotein and it is assumed to function as an antimicrobial agent that harvests free biotin from its environment [1, 2]. Because of its high affinity to biotin, avidin has long been used as a separation, labelling and targeting tool in various bioscience fields [8].

The yolk BBP has been further characterised to consist of two different forms, BBP-I and BBP-II [3, 9]. It has been proposed that BBP-I is the primary gene product (67 kDa) that is converted by proteolytic cleavage to BBP-II (19 kDa) [4]. The biological function of BBP-I is believed to be a general biotin transporter in plasma, whereas the actual deposition role in egg-yolk is reserved for BBP-II [3]. BBP-II is a tetrameric protein, like avidin, and is composed of subunits homologous to each other. BBP-I has been thought to be a pseudotetramer containing four binding domains in a polypeptide chain and its gene should, therefore, contain four subsequent repeats encoding for similar peptide sequences [4, 9]. The egg-white BBP was also reported to exist in a large form similar to yolk BBP-I [6]. Interestingly, some egg-laying species, such as turkeys and alligators, showed only one type of BBP in the yolk of their eggs [10].

Despite their similar function, some biochemical properties of BBPs and avidin are different. The pI of the chicken yolk BBP (not defined which form) was reported to be 4.6 [7] in contrast to avidin which has a highly basic pI (≈ 10.4) [1]. BBP-I exhibits higher thermostability, being active at 60°C, whereas BBP-II is denatured at temperatures above 40°C [10]. Bush and White III have published the N-terminal amino acid sequences for both chicken yolk BBP forms, which are highly similar to each other and also resemble the avidin N-terminal sequence [4, 10]. BBP-I has been proposed to be a glycoprotein [7] whereas BBP-II is shown to be nonglycosylated [10]. Avidin is known to contain one N-linked carbohydrate moiety per subunit [1]. Differences in the radiobiotin exchange rates between these two BBP forms have also been observed: BBP-I showed slower exchange than BBP-II [10].

The published N-terminal sequences, the similar overall sizes of the proteins and the tetrameric appearance of BBP-II as well as the reported high biotin-binding affinities suggest that the BBPs could be related to avidin. Structurally and functionally, avidin is considered to be a member of the larger protein superfamily called calycins [11]. These proteins form a large and divergent family of relatively small extracellular proteins which typically bind small hydrophobic ligands. Two particular groups of calycin protein family, the lipocalins and avidins, are β-barrels composed of eight antiparallel β-strands. The ligand is bound inside the protein at one end of the β-barrel [12]. An interesting feature among the lipocalins and avidins is a structural signature wherein a conserved basic amino acid residue, close to the last β-strand, packs over a specific tryptophan residue on the first β-strand and forms hydrogen bonds with the short 3₁₀ helix prior to the first β-strand [11].

Because the genes, genomic locations, cDNAs or full amino acid sequences of chicken BBPs are not known, it is impossible to evaluate their true relationship to avidin or, in a broader sense, to the calycin protein superfamily. New hope to solve this enigma aroused when the first draft of the chicken genome was published in March 2004 [13, 14]. In addition to the genome project, a comprehensive collection of chicken cDNAs is also in progress [15].

In the current study we searched these databases in order to find the cDNAs and genes for BBPs. Indeed, we found two independent cDNAs whose translated amino acid sequences fitted well to the published N-terminal sequences of BBPs. The genomic fragments corresponding to these cDNAs were also identified and analysed. They showed features similar to those of the avidin gene family members. Interestingly, one of these BBP gene candidates is located together with the avidin gene family in the chicken chromosome Z [16]. In addition, more evidence supporting the previous hypothesis of the high recombination frequency in the avidin gene family is gathered. One of the two putative BBPs was found to significantly resemble avidin, showing a theoretical molecular mass and pI close to those of avidin, whereas the other showed theoretical characteristics fitting more closely to those published for BBPs. Almost all amino acids important for biotin binding in avidin [17] are conserved in both of these supposed BBPs. Neither of the found cDNAs/genes, however, encodes a protein composed of four similar domains as expected for the isolated pseudotetrameric BBP-I [3]. Instead, the encoded proteins show calculated molecular masses corresponding to one BBP domain per polypeptide. In silico analysis of these genes as well as modelled structures of the putative BBP proteins are presented.

The circumstantial evidence has indicated that chicken yolk BBPs may be structurally related to avidin and other members of the avidin family. Therefore we were eager to scan the chicken genome data to evaluate the correctness of this hypothesis. The database queries revealed two independent hits showing high similarity to the published N-terminal sequences of yolk BBPs I and II [4] and, indeed, a potential kinship between BBPs and avidin gene family members was revealed.

The avidin gene belongs to the gene family that has several other members called AVRs (avidin related genes). Previously, seven different AVR-genes have been cloned [16, 20] and the chromosomal location of this gene family has been tracked down to a relatively short region in the telomeric region q21 of the chicken sex chromosome Z [16]. It seems that the number of AVR genes varies between individual chickens and even between cells within the same chicken [30]. The deposited genome data, analysed in the present study, support this observation demonstrating a novel assembly of 3 AVR genes together in the same cluster with the avidin gene. Interestingly, the two AVR genes found in the chicken genome database seem to be novel variants of the formerly cloned AVRs, which also support the previous hypothesis of the high recombination frequency within the avidin gene family [30, 31].

Our observations link the BBPs to the avidin family for the first time, at the cDNA and gene level. There are many independent features indicating this. Firstly, the found cDNAs encode proteins that are evidently homologous to avidin. Secondly, the genomic location of the BBP-A gene close to the avidin gene family supports their relationship. Finally, the exon/intron structures of the BBP genes and avidin family genes are similar to each other.

According to the phylogenetic relationships and genome locations of the genes, one scenario for the BBP/ avidin evolution is as follow: an initial duplication may have occurred leading to the origin of BBP-B and the precursor of the BBP-A/ avidin family, followed by a further duplication leading to the origin of BBP-A and the precursor of the avidin family. This could have been followed by the formation of AVD and an AVR gene and finally the duplication of the latter in several avidin-related genes.

According to molecular modelling, BBP-A and B proteins both showed features that make them suitable for biotin binding. The biotin-binding contact amino acids of avidin [17] were almost perfectly conserved in both BBP sequences. In addition, good conservation of the inner part of the β-barrel in both BBPs is also important for the function of the ligand binding cavity. If we assume that one or both of these putative BBPs represent yolk BBPs I and/or II, other sequence differences should explain their observed weaker dissociation constant for biotin [7]. Similar observation has been done for AVRs which have only a few differences in their biotin-binding residues when compared to avidin, but still exhibit remarkable differences in their biotin-binding affinities [27].

It is probable that both BBP-A and BBP-B form similar tetrameric quaternary structures as avidin. The basis for this assumption is the fact that the conservation of the presumed interface residue patterns were highly similar to those of AVRs, which are also known to form extremely stable tetramers [27, 28]. The 1–2 interface, in particular, in which mutations have previously been shown to be extremely important to the stability properties of both avidin and streptavidin [32–34], was perfectly conserved in both BBPs. Hence the existing changes were concentrated on the 1–3 and 1–4 interfaces which are known to tolerate substitutions in avidin and AVRs [27–29]. The yolk BBPs have, however, been reported to be clearly less heat-stable than avidin [10] and, therefore either the sequence differences in the interfaces may explain this difference or the tertiary structure of the BBP barrel may be weaker than that of avidin. Overall, interfaces in the BBP models suggested tetramer formation, since the putative interface regions were hydrophobic. Furthermore, differences at the subunit interfaces of the BBP models, when compared to those of avidin, were at least partially complementary. For example the Thr-80-Val mutation at the 1–4 interface of BBP-A seemed to be in a highly hydrophobic environment.

The most striking feature that distinguished BBP-B from avidin and BBP-A was its extraneous, approximately 20 amino acid residue-long, C-terminal extension. According to modelling, this stretch could form an α-helix and the cysteine residue at the end of this stretch could form a disulphide bridge with another cysteine residue in β-strand 4 (Figure 5C). It is, however, hard to interpret the relevance of this predicted α-helix and the possible effect of the helix and the cysteine bridge to the structural and functional properties of BBP-B. One effect could be that it strengthens the structure of the protein. Interestingly, many members of the lipocalin protein family have similar C-terminal α-helical domains [11, 12]. Nonetheless, the exon/intron structure of the avidin gene family is different when compared to the lipocalin family [35, 36]. This suggests that even if the overall tertiary structures of these proteins are similar, the evolutionary distance between these lineages is overwhelmingly long, or alternatively these protein families have been developed independently. The manner in which BBP-B has acquired its C-terminal extension, found in lipocalins, remains therefore an enigma. Alternative models for this extension can be done as well; the distance between the beginning of C-terminal extension (Lys-123) of the subunit 1 and the free cysteine in loop 3 of the subunits 3 and subunit 4 is around 40 A (not shown). This is also approximately the length of the fully extended C-terminal extension (Lys-123-Cys-138). Based on these distances both 1–3 and 1–4 intersubunit disulphide-bridges are possible.

In the light of the current study, the story of the chicken BBP proteins gets blurred. As before, we have two possible candidates. However, these cDNAs/genes are able to encode proteins having molecular mass of 14–16.5 kDa, which correspond to only one BBP subunit instead of the four subsequent repeats hypothesized earlier [3, 9]. This means that either the database still lacks the genuine yolk-BBP gene or that some other phenomenon, like the abovementioned hypothetical disulphide bridges, must explain the previously reported molecular weight properties of BBP-I. Nonetheless, the fact that cDNAs for BBP-A and BBP-B were isolated from the chicken liver library [15], and that the putative proteins they encode have signal sequences, suggest that these cDNAs may really be the yolk BBP cDNAs, which have been reported to be secreted from the liver into the egg [4].

If we try to fit the characteristics of the found BBP candidates to those previously associated with BBPs, BBP-B looks more promising. It has similar low pI [7], its C-terminal extension makes its molecular mass closer to that determined for BBP-II [10] and it is most probably non-glycosylated as is BBP-II [10]. What is then the role of the BBP-A gene? Is it the mysterious BBP isolated from egg-white [6] or some unknown chicken BBP? It is evident that we need to continue the database queries and/or start cloning BBP genes to clarify this puzzle. In addition, we need to produce these found putative BBPs as recombinant proteins to investigate whether their properties are in agreement with the previous findings and the models of the present study. Furthermore, these new proteins can serve as a source for development of new tools for life sciences.

For example, it could be possible to construct a chimeric avidin-BBP-dimer [37] to adjust the ligand-binding properties of the resultant hybrid protein.

We have identified two putative genes and cDNAs for chicken egg-yolk biotin-binding proteins from NCBI database and chicken genome database. The genomic location and the structures of the found genes and the proteins they encode link clearly BBPs to the avidin family and, moreover, give an insight to the evolutionary history of this gene family. Our molecular modelling results support many preceding observations concerning the biochemical properties of BBPs but also impugn some of the previous hypothesis. Most importantly, the gene/cDNA structures provided no evidences of proteolytic processing of pseudotetramers to tetramers that has been presented as a possible maturation process for BBPs, i.e. conversion of BBP-I to BBP-II.