BACKGROUND
large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for evolution of different structural variants within a protein family  <cit> .
residues present in these loops are, on average, expected to be involved in fewer intra-molecular interactions  <cit>  and the resulting lowered constraint on side-chain identity makes loop regions candidates for rapid sequence divergence and evolution.
these loops can result in novel structural variants eventually leading to the acquisition of novel functions and/or protein domains.
sialidases or neuraminidases  are a family of glycohydrolytic enzymes that remove terminal sialic acid residues from various sialo-derivatives, such as glycoproteins, glycolipids and oligosaccharides  <cit> .
in mammals four enzymes have been identified: the lysosomal sialidase neu <dig>  the soluble or cytosolic sialidase neu <dig>  the membrane-associated sialidases neu <dig> and neu4; each one with different substrate specificity  <cit> .
the typical structure of sialidases is a β-propeller composed by four anti-parallel β-sheets organized in  <dig> blades which compose a compact and stable structure due to hydrogen bonds between β-sheets  <cit> .
such a structure also results in several loops connecting the various β-sheets and not involved in the catalytic process.
indeed, evidences collected so far from sialidases characterized in mammals  <cit> , zebrafish  <cit> , bacteria  <cit>  and viruses  <cit>  as well as from trypanosomal trans-sialidase  <cit>  have already demonstrated the presence of large and highly variable loops regions between β-sheets.
taken together these evidences suggest that the sialidase protein family could effectively be studied to get deeper insights on the rapid evolving loop hypothesis mentioned above.
following this intriguing scenario, we focused on the state of sialidase genes in another lineage in which genomic data has recently become available: avians and particularly chicken.
chicken has been used as animal model in several research fields  <cit>  and the study of bird genome is seen as a key to better understand vertebrate evolution.
efforts in avian genomics have led to the complete genome sequence of chicken in  <dig>  <cit> , and of turkey   <cit>  and zebra finch  in  <dig>  <cit> .
the analysis of the sialidase gene family in birds led us to the identification of a neu <dig> protein with a long amino acid repeat region that appears to be specific for chicken and strictly related avian species, such as turkey.
here we describe the peculiar features of this region in gallus gallus neu <dig>  our data support the occurrence of rapid evolving loops in the sialidase protein family.
RESULTS
identification of sialidase genes in gallus gallus
tblastn and blat searches conducted on chicken sequences using the four human sialidase proteins  as queries resulted in four chicken genes representing the putative orthologs of human neu1- <dig> genes.
the bioinformatic approach allowed us to recover the entire coding sequence of all chicken sialidases but neu <dig>  for which only one est sequence is present in public sequence databases and no genomic sequence is available.
the percentage of identity between human sialidases and the putative orthologous proteins in gallus gallus, as obtained from blastp comparison, together with the localization of the corresponding genes are given in table  <dig>  the unusual dimension of gg neu <dig> protein prompted us to a more detailed characterization of this member of the gene family.
search in the entrez gene database for sequences annotated as neu <dig> in gallus gallus led to the identification of a mrna  encoding a protein with high sequence identity to the middle and c-terminal region of human neu <dig>  but lacking the n-terminal portion.
the identification of  <dig> chicken ests showing significant similarity to human neu <dig> in a tblastn search allowed us to extend on the 5' side the xm_ <dig>  sequence and to assemble a cdna contig of  <dig> bp .
this sequence contains a  <dig> bp orf encoding a  <dig> amino acids protein with a calculated molecular mass of  <dig>  kda.
a blastp search versus a non-redundant protein sequence database revealed that this novel protein shows the highest sequence identity to neu <dig> of different vertebrates including homo sapiens  and mus musculus .
according to the chicken gene nomenclature committee guidelines, the gene was named neu <dig>
essential data of the  <dig> putative orthologs of human sialidase genes identified in gallus gallus based on a bioinformatic approach
n.d. = not determined; * = protein sequence translated from xr_ <dig> after mrna sequence correction based on comparison with ests sequences
characterization of the neu <dig> gene in gallus gallus
the gg neu <dig> gene is located in a  <dig> bp region on chicken chromosome  <dig> and, like its human counterpart, it is likely organized in  <dig> exons .
we also analyzed the genomic region surrounding neu <dig> in chicken and in man .
conservation of synteny was observed between the two species with the exception of ppme <dig>  ucp <dig>  dnajb <dig> and paaf <dig> genes which in homo sapiens are located more distantly from neu <dig>  than in their chicken counterparts.
as expected, conserved synteny around the neu <dig> locus is observed also in zebra finch .
pcr amplification on chicken gdna  resulted in two different products, supporting the presence of two allelic isoforms of the gene which we named neu3*l  and neu3*s  , the latter encoding a polypeptide lacking  <dig> amino acids belonging to a tandem repeat region described in detail below.
as the neu3*l form was the one with a greatest est support , all further investigations were conducted on this variant.
the presence of the two neu <dig> polymorphic variants in the chicken population was investigated by pcr amplification of genomic dna extracted from  <dig> different individuals.
our results demonstrated the presence of chickens homozygous for both alleles, as well as individuals that are heterozygous.
an example of each genotype is shown in figure 1c.
rt-pcr amplification with exon-spanning primers performed as described in methods revealed detectable transcripts in all tissue samples, in agreement with expression information available in biogps for the neu <dig> gene in human and mouse tissues, with both allelic variants expressed at a comparable level .
neu <dig> protein analysis in gallus gallus and in other avian species
a multiple sequence alignment of gg neu <dig> with human neu <dig> and neu <dig>  as well as with neu <dig> from other vertebrates is shown in figure  <dig>  computational analysis of gg neu <dig> performed with motifscan and pfam revealed the presence of the typical sialidase signatures: a rip motif at residues 43- <dig>  and three canonical asp boxes  at residues 149- <dig>  222- <dig>  274- <dig>  the protein is  <dig> amino acid long, with a predicted molecular weight of  <dig>  kda, a theoretical pi of  <dig>  and a gravy value of - <dig>  .
the high level of sequence identity to human neu <dig> , whose 3d structure has been solved  <cit>  allowed the identification of amino acids possibly involved in the formation of the active site.
all  <dig> residues involved in the coordination of the competitive inhibitor dana in the active site of hs neu <dig> resulted conserved in gg neu <dig>  interestingly, the multiple sequence alignment revealed the presence in gg neu <dig> of a polypeptide region  absent in all other neu <dig> sialidases characterized so far, and responsible for the higher molecular weight of this enzyme of avian origin .
the analysis of gg neu <dig> sequence with the radar software showed that the region between positions  <dig> and  <dig> contains  <dig> repeat units , starting with the mostly conserved sequence tspngdtq and repeated in a complex pattern.
the complete organization of the repeat region within the neu <dig> polypeptide is shown in figure  <dig>  while the single repeat units are compared in figure 3a.
in gg neu <dig> this region is composed by two blocks of repeats , separated by a stretch of  <dig> unrelated residues .
rep <dig> is preceded by another stretch of  <dig> unrelated residues .
there are  <dig> almost identical motifs  of  <dig> residues,  <dig> motifs  of identical size but slightly different from those previously mentioned, and  <dig> less similar motifs of  <dig> and  <dig> residues .
although rep1- <dig> are peculiar of gg neu <dig>  the last 2/ <dig> of rep <dig> result conserved also in neu <dig> proteins from other vertebrates as well as in mammalian neu <dig>  and neu <dig> .
to investigate the conservation of the repeat region among avian species we identified the putative neu <dig> gene both in turkey, meleagris gallopavo , and zebra finch, taeniopygia guttata .
figure  <dig> shows a portion of the mg neu <dig> polypeptide, identified as described in the methods section, together with the putative tg neu <dig> amino acid sequence, retrieved from the ncbi database.
turkey neu <dig> presents the tandem repeat region whose length corresponds to the one found in gg neu3*s chicken allele.
interestingly this region is absent in the tg neu <dig> polypeptyde, while the rest of the protein shows a high degree of sequence conservation .
as shown in figure 3b, the chicken and turkey protein expansions are located in a region that is hypervariable in neu <dig> proteins of mammals, birds and teleosts.
this hypervariable region is included between two highly conserved amino acid blocks and, while gg neu <dig> shows the largest insertion , its length increases from lower vertebrates  to mammals neu3s .
gg neu <dig> protein structure prediction
prediction of gg neu <dig> structure , based on its alignment with human sialidase neu <dig> and performed using swiss-model, revealed, as expected, a high similarity with the neu <dig> structure .
the predicted structure of the protein is the six blades β-propeller typical of sialidase  <cit> , each blade being composed of four anti-parallel β-strands.
the repeat region is located at the opposite side of the catalytic crevice and emerges as a long loop connecting two β-strands of the fifth blade of the β-propeller.
this region is comprised between two highly conserved amino acid blocks, gcqgsvsf  and wlly  .
based on gg neu <dig> structural model , the highly conserved svvsf block  represents the first β-strand of the fifth blade of the β-propeller, followed by the expanded loop region containing the repeat units .
the next highly conserved region  is part of the seventh repeat unit organized in: i) wllyshp , as second β-strand of the fifth blade of the propeller; ii) tnrrhrr , as loop portion connecting the two anti-parallel β-strands and containing the essential catalytic residue arg- <dig> and; iii) dlg , as first portion of the third β-strand of the same blade.
the ab initio structure prediction on the repeat region peculiar of gg neu <dig> , performed with rosetta and refined using i-tasser, resulted in a long hairpin architecture with no particular secondary structure features .
several hydrogen bonds and hydrophobic interactions occur between the first  <dig> and the last  <dig> residues of this region .
the  <dig> putative β-sheets predicted in the repeat region by psipred are also indicated in figure 4d.
expression and characterization of gg neu <dig> enzyme
to demonstrate that the gg neu <dig> gene encodes for an active sialidase, the entire orf was subcloned in the mammalian expression vector pmt <dig> and the resulting construct was used to transiently transfect cos <dig> cells.
crude homogenates from cells transfected with pmt21-gg neu3- myc or pmt <dig> alone were tested for sialidase activity using the artificial substrate 4-mu-neuac.
transfection with pmt21-gg neu3-myc resulted in a  <dig> -fold increase in the enzymatic activity detected in the crude extract .
the enzyme showed a ph optimum at  <dig> , similarly to bovine, human and mouse neu <dig> enzymes  <cit>  .
fractionation by ultracentrifugation of crude extracts from transfected cos <dig> cells clearly demonstrated the association of gg neu <dig> with the particulate fraction, as about 85% of the activity detected in the homogenate was recovered in the pelleted material .
this is in agreement with data available for the human  <cit>  and mouse counterparts  <cit> .
in order to study gg neu <dig> association to detergent resistant membranes , crude extracts were treated with triton x- <dig> as described in methods.
immunoblotting analysis with anti-myc antibodies revealed a partial recovery of gg neu <dig> in drm fraction , identified by the presence of cav- <dig>  a specific marker for these membrane domains  <cit> .
this finding is in agreement with the results already obtained for human and mouse neu <dig>  <cit> .
analysis of the hydrophilic/hydrophobic features of gg neu <dig> by aqueous-to-detergent repartition after triton x- <dig> treatment revealed the almost complete segregation of gg neu <dig> in the detergent phase together with cav- <dig>  supporting a strong association with cellular membranes .
interestingly, this behavior differs from that of mm neu3-ha, tested as a control protein, which segregated in the aqueous phase  <cit> .
immunofluorescence localization was studied in cos <dig> cells transiently transfected with pmt21-gg neu3-myc at  <dig> h post transfection.
laser confocal microscopy analysis showed plasma membrane labelling together with intracellular tubular and vesicular structures, the latter being mainly concentrated in the juxtanuclear region of the cell .
these observations are in agreement with the localization of human and mouse neu <dig>  also performed in cos <dig> cells  <cit> .
co-localization experiments carried out using makers related to vesicular trafficking and protein recycling, such as tfr and eea <dig>  are illustrated in figure  <dig> .
co-localization of gg neu <dig> with tfr and eea <dig> indicate that, beside its localization at the plasma membrane, the protein is present also in early and recycling endosomal compartments, as already reported for mm neu <dig>  <cit> .
gg neu <dig> mutant protein properties
in order to evaluate the impact of the repeat region on the general properties of gg neu <dig> we carried out mutagenesis of the original protein as described in methods.
this allowed us to generate a new protein of  <dig> amino acids  with a predicted mw of  <dig>  kda, lacking  <dig> of the  <dig> units contained in the repeat region described above.
in detail, we performed a  <dig> amino acids  deletion from thr- <dig> to gly- <dig>  leaving intact the seventh repeat unit and a  <dig> amino acids spacer .
the seventh repeat unit was maintained in the mutated construct since it contains fundamental structural elements and the enzyme catalytic residue arg- <dig> .
overall gg neu3-del and the wild-type protein showed superimposable results in the biochemical and cellular studies we performed.
indeed, the transfection with pmt21-gg neu3-del-myc resulted in a  <dig> -fold increase of the enzymatic activity detected in the crude extract  with a ph optimum at ph  <dig>  .
fractionation of crude extracts from transfected cos <dig> cells after ultracentrifugation showed that about 80% of the activity detected in the homogenate was recovered in the pelleted material .
the mutant protein repartition after treatments with triton x- <dig> and triton x- <dig> is also comparable with the results obtained with the wild-type protein .
finally, the subcellular localization in cos <dig> transfected cells showed no significant differences in the distribution of the wild-type and mutant protein .
discussion
the insertion of loop of various lengths and not involved in catalytic activity, between two structurally conserved motifs, has been already observed in the ras gtpases protein family  <cit>  and could represent a mechanism for protein evolution and creation of novel protein domain  <cit> .
a preliminary survey of sialidase enzymes in vertebrates and mammals revealed the presence of a loop region which shows high sequence divergence and variable length in vertebrate neu <dig>  and neu <dig> .
moreover as suggested by the sialidase characterization in danio rerio, which posses  <dig> orthologs of human neu <dig> gene but apparently no neu <dig> ortholog  <cit> , evolution of this protein family has been complex, probably following the increasing complexity of sialoconjugates from microorganisms to higher vertebrates  <cit> .
to get better insight into evolution of the sialidase protein family, we decided to extend our studies to other vertebrates, based on the presence of rapid diverging loops in this class of enzymes.
a bioinformatic analysis carried out on genomic and expressed sequences revealed that gallus gallus genome, similarly to mammals, contains four genes that, based on sequence identity and synteny analysis, encode the putative orthologs of neu <dig>  neu <dig>  neu <dig> and neu <dig> proteins.
the comparison of the primary structure of neu <dig> from gallus gallus with its human counterpart confirmed the presence in the former of an amino acid insertion larger than the one found in all neu <dig> sialidases characterized so far.
this large insertion may represent a rapid evolving loop and so a potentially important event for sialidase evolution.
we thus decided to focus our interest on this member of the gallus gallus sialidase family.
the gg neu <dig> gene is located on chicken chromosome  <dig> in a region of conserved synteny with chromosome 11q <dig>  harboring neu <dig> in homo sapiens.
while in both species the gene is flanked on one side by the kcne <dig>  pold <dig>  chrdl <dig>  xrra <dig> and spcs <dig> genes, an evolutionary rearrangement, probably an inversion of a  <dig> kb region delimited by neu <dig> and kcne <dig>  has separated gg neu <dig> from ppme <dig> in mammals.
in chicken the neu <dig> gene is apparently expressed in an ubiquitous manner, like its mammalian counterparts  <cit> .
primary structure analysis of the gg neu <dig> polypeptide revealed that, besides the high identity score with the human and mouse enzymes, all sialidase canonical amino acid signatures rip motif and asp boxes) are present and conserved in topologically equivalent positions.
the insertion identified in gg neu <dig> corresponds to a  <dig> amino acid region  mainly constituted by a peptide motif repeated in tandem and organized in six plus one highly conserved repeat sequences .
the analysis of  <dig> different chickens allowed us to identify a shorter allele of the gene characterized by the absence of a region encoding one of the repeats .
these results demonstrate the presence of two alleles of gallus gallus neu <dig> gene.
the repeat cluster that characterizes gg neu <dig> may derive from complex duplication events originated from rep <dig>  as its last portion  is conserved also in sialidases from other vertebrates.
an almost complete identity is observed among rep <dig>   <dig> and  <dig>  whereas rep <dig>   <dig> and  <dig> are more divergent, both in residue number and sequence, suggesting that duplication events occurred at different times during the evolution.
noteworthy, this repeat sequence is located between the evolutionary highly conserved sequence blocks gcqgsvsf and wlly.
the analysis of the multiple sequence alignment in this region confirmed the presence of a loop of variable size in neu <dig> vertebrate sialidases ranging from  <dig> residues, in the case of zebrafish neu <dig> - <dig>  to 41- <dig> residues for the mammalian enzymes.
interestingly, an insertion of  <dig> and  <dig> residues is located in the same region in human  <cit>  and mouse neu <dig>  <cit> , respectively.
using the crystal structure of hs neu <dig> as template  <cit> , a homology model of gg neu <dig> has been obtained, showing, as expected, the six-blade β-propeller architecture typical of sialidases  <cit> .
the analysis of the model revealed that the repeat sequence found in chicken and turkey neu <dig>  as well as the stretch of about  <dig> residues found in mammalian neu4s, are inserted at the opposite side of the active crevice.
this region represents the loop connecting the highly conserved first and second antiparallel β-strands of the fifth blade of the β-propeller.
the best model obtained for this loop region and based on ab initio structure prediction resulted in a long hairpin architecture with no particular secondary structure features .
interestingly the peptide chain is stabilized by several hydrogen bonds and hydrophobic interactions occurring between the first  <dig> and the last  <dig> residues .
these interactions create a sort of support for the long loop and isolate it from the core of gg neu <dig> structure.
this allows the exploration of new structural variants of the loop without impairing the protein biological activity.
along the hairpin chain  <dig> putative β-sheet regions have been also identified by secondary structure prediction and may contribute to stabilize this region.
in order to confirm that gg neu <dig> encodes for an active sialidase and the possible relevance of the loop, we expressed gg neu <dig> and gg neu3-del cdna in cos <dig>  a cell line that has been used extensively for the biochemical characterization of sialidase enzymes  <cit> .
studies on transiently transfected cells showed for both proteins a significant increase in sialidase activity, with an acidic ph optimum, comparable subcellular localization and membrane association, in agreement with data on human and mouse neu <dig>  <cit> .
interestingly gg neu <dig> and its deletion mutant revealed a peculiar hydrophobic feature when extracted with triton x- <dig> compared to mm neu <dig>  <cit> .
this feature probably does not depend on gg neu <dig> direct interaction with lipid bilayer, given its overall hydrophilic nature.
a possible explanation for this behavior of the protein could be found in different interaction of mouse and chicken neu <dig> with other component of the membrane.
overall, these data suggest that, in agreement with the rapid evolving loop hypothesis, the repeated region, at this stage of evolution, has apparently none or limited function at least for the biochemical and cellular properties considered herein.
it is intriguing to speculate about the fate of this loop region in sialidase evolution, particularly considering its increasing size, observed in neu <dig> proteins of higher vertebrates, and the unique repeat organization observed in chicken and turkey.
in this perspective, the long amino acid stretch found in chicken and turkey neu <dig>  could represent an intermediate step in the evolutive process leading toward novel domain relevant for the evolution of sialidases in this systematic group, as observed for lectin binding domain found in vibrio cholerae  <cit> .
concerning the repetitive nature of the loop found in gg neu <dig> it should be noted that it does not represent a unique case among sialidases.
indeed, the c-terminal domain of the trans-sialidase  from trypanosoma cruzi contains a variable number of a tandemly repeated 12-amino acid-long unit, unrelated to the one found in gg neu <dig>  termed sapa  that is not required for ts activity  <cit> .
although some studies indicate that the repetitive amino acid motif stabilizes the catalytic activity of the enzyme in blood, its function remains largely unknown  <cit> .
moreover, our data demonstrate that the insertion of a large loop in this region of the enzyme does not impair the 3d organization of sialidase, indicating that this portion of the enzyme could be exploited as insertional spot for artificial modifications of the protein.
we demonstrate that also neu <dig> from turkey contains the repeat region, whose length corresponds to the one of the chicken short allelic variant.
this is not surprising considering that turkey is a domestic bird strictly related to chicken in the evolutionary tree of the galliformes order.
besides chicken and turkey, the only other avian genome that has been sequenced belongs to the australian songbird zebra finch   <cit> .
we were surprised to discover that the zebra finch neu <dig> completely lacks the repeat region, showing instead a short loop of  <dig> residues, similar in length to the one found in cytosolic sialidase hs neu <dig>  this finding indicates that the repeat region is highly variable and is not a peculiar feature of all avian neu <dig> sialidases, suggesting three evolutionary hypotheses.
the repeat sequence has arisen: i) recently in the galliformes order; ii) in the galloanserae clade; or iii) in a common ancestor of both galloanserae and neoaves, and then has been lost in the latter at any point of the evolutionary tree leading to zebra finch  <cit> .
by considering the first two hypothesis we can not exclude that the repeat region is present only in breeding birds as a consequence of the high genetic pressure and selection applied by man to these species  <cit> .
the sequencing of additional avian genomes will allow to shed light on these evolutionary scenarios.
CONCLUSIONS
in this study we demonstrated that sialidase neu <dig> β-propeller contains a surface loop highly variable in both sequence and size connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice.
in the case of chicken and turkey, both belonging to the galliformes order, this loop is constituted by a number of repeats organized in tandem.
the removal of the repeat region does not seems to alter the evaluated properties of the gg neu <dig> enzyme.
our data indicate sialidase family as good model to study the evolutionary process based on the rapid diverging loop hypothesis  <cit> .
according to this theory, new random protein folding and architectures can emerge rapidly taking advantage of low constraint acting on surface loops at both sequence and structure level.
when a loop develops into a useful or stable structure, it becomes subjected to positive selection and the new variant could be retained and eventually originate a new protein with specific features.
this process may had occurred in sialidase protein family evolution leading to the membrane-bound enzyme forms, namely neu <dig> and neu <dig>  present today in higher organisms.
giving the peculiar features of its loop region, gallus gallus neu <dig> could be of particular interest in such evolutionary studies.
additional work is needed to unravel a possible role of the repeat region in the avian biology.

 large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for evolution of different structural variants within a protein family. residues present in these loops are, on average, expected to be involved in fewer intra-molecular interactions and the resulting lowered constraint on side-chain identity makes loop regions candidates for rapid sequence divergence and evolution. these loops can result in novel structural variants eventually leading to the acquisition of novel functions and/or protein domains. sialidases or neuraminidases are a family of glycohydrolytic enzymes that remove terminal sialic acid residues from various sialo-derivatives, such as glycoproteins, glycolipids and oligosaccharides. in mammals four enzymes have been identified: the lysosomal sialidase neu the soluble or cytosolic sialidase neu the membrane-associated sialidases neu and neu4; each one with different substrate specificity. the typical structure of sialidases is a β-propeller composed by four anti-parallel β-sheets organized in blades which compose a compact and stable structure due to hydrogen bonds between β-sheets. such a structure also results in several loops connecting the various β-sheets and not involved in the catalytic process. indeed, evidences collected so far from sialidases characterized in mammals, zebrafish, bacteria and viruses as well as from trypanosomal trans-sialidase have already demonstrated the presence of large and highly variable loops regions between β-sheets. taken together these evidences suggest that the sialidase protein family could effectively be studied to get deeper insights on the rapid evolving loop hypothesis mentioned above. following this intriguing scenario, we focused on the state of sialidase genes in another lineage in which genomic data has recently become available: avians and particularly chicken. chicken has been used as animal model in several research fields and the study of bird genome is seen as a key to better understand vertebrate evolution. efforts in avian genomics have led to the complete genome sequence of chicken in, and of turkey and zebra finch in. the analysis of the sialidase gene family in birds led us to the identification of a neu protein with a long amino acid repeat region that appears to be specific for chicken and strictly related avian species, such as turkey. here we describe the peculiar features of this region in gallus gallus neu our data support the occurrence of rapid evolving loops in the sialidase protein family.  identification of sialidase genes in gallus gallus tblastn and blat searches conducted on chicken sequences using the four human sialidase proteins as queries resulted in four chicken genes representing the putative orthologs of human neu1- genes. the bioinformatic approach allowed us to recover the entire coding sequence of all chicken sialidases but neu for which only one est sequence is present in public sequence databases and no genomic sequence is available. the percentage of identity between human sialidases and the putative orthologous proteins in gallus gallus, as obtained from blastp comparison, together with the localization of the corresponding genes are given in table the unusual dimension of gg neu protein prompted us to a more detailed characterization of this member of the gene family. search in the entrez gene database for sequences annotated as neu in gallus gallus led to the identification of a mrna encoding a protein with high sequence identity to the middle and c-terminal region of human neu but lacking the n-terminal portion. the identification of chicken ests showing significant similarity to human neu in a tblastn search allowed us to extend on the 5' side the xm_ sequence and to assemble a cdna contig of bp. this sequence contains a bp orf encoding a amino acids protein with a calculated molecular mass of kda. a blastp search versus a non-redundant protein sequence database revealed that this novel protein shows the highest sequence identity to neu of different vertebrates including homo sapiens and mus musculus. according to the chicken gene nomenclature committee guidelines, the gene was named neu essential data of the putative orthologs of human sialidase genes identified in gallus gallus based on a bioinformatic approach n.d. = not determined; * = protein sequence translated from xr_ after mrna sequence correction based on comparison with ests sequences characterization of the neu gene in gallus gallus the gg neu gene is located in a bp region on chicken chromosome and, like its human counterpart, it is likely organized in exons. we also analyzed the genomic region surrounding neu in chicken and in man. conservation of synteny was observed between the two species with the exception of ppme ucp dnajb and paaf genes which in homo sapiens are located more distantly from neu than in their chicken counterparts. as expected, conserved synteny around the neu locus is observed also in zebra finch. pcr amplification on chicken gdna resulted in two different products, supporting the presence of two allelic isoforms of the gene which we named neu3*l and neu3*s, the latter encoding a polypeptide lacking amino acids belonging to a tandem repeat region described in detail below. as the neu3*l form was the one with a greatest est support, all further investigations were conducted on this variant. the presence of the two neu polymorphic variants in the chicken population was investigated by pcr amplification of genomic dna extracted from different individuals. our results demonstrated the presence of chickens homozygous for both alleles, as well as individuals that are heterozygous. an example of each genotype is shown in figure 1c. rt-pcr amplification with exon-spanning primers performed as described in methods revealed detectable transcripts in all tissue samples, in agreement with expression information available in biogps for the neu gene in human and mouse tissues, with both allelic variants expressed at a comparable level. neu protein analysis in gallus gallus and in other avian species a multiple sequence alignment of gg neu with human neu and neu as well as with neu from other vertebrates is shown in figure computational analysis of gg neu performed with motifscan and pfam revealed the presence of the typical sialidase signatures: a rip motif at residues 43- and three canonical asp boxes at residues 149- 222- 274- the protein is amino acid long, with a predicted molecular weight of kda, a theoretical pi of and a gravy value of -. the high level of sequence identity to human neu, whose 3d structure has been solved allowed the identification of amino acids possibly involved in the formation of the active site. all residues involved in the coordination of the competitive inhibitor dana in the active site of hs neu resulted conserved in gg neu interestingly, the multiple sequence alignment revealed the presence in gg neu of a polypeptide region absent in all other neu sialidases characterized so far, and responsible for the higher molecular weight of this enzyme of avian origin. the analysis of gg neu sequence with the radar software showed that the region between positions and contains repeat units, starting with the mostly conserved sequence tspngdtq and repeated in a complex pattern. the complete organization of the repeat region within the neu polypeptide is shown in figure while the single repeat units are compared in figure 3a. in gg neu this region is composed by two blocks of repeats, separated by a stretch of unrelated residues. rep is preceded by another stretch of unrelated residues. there are almost identical motifs of residues, motifs of identical size but slightly different from those previously mentioned, and less similar motifs of and residues. although rep1- are peculiar of gg neu the last 2/ of rep result conserved also in neu proteins from other vertebrates as well as in mammalian neu and neu. to investigate the conservation of the repeat region among avian species we identified the putative neu gene both in turkey, meleagris gallopavo, and zebra finch, taeniopygia guttata. figure shows a portion of the mg neu polypeptide, identified as described in the methods section, together with the putative tg neu amino acid sequence, retrieved from the ncbi database. turkey neu presents the tandem repeat region whose length corresponds to the one found in gg neu3*s chicken allele. interestingly this region is absent in the tg neu polypeptyde, while the rest of the protein shows a high degree of sequence conservation. as shown in figure 3b, the chicken and turkey protein expansions are located in a region that is hypervariable in neu proteins of mammals, birds and teleosts. this hypervariable region is included between two highly conserved amino acid blocks and, while gg neu shows the largest insertion, its length increases from lower vertebrates to mammals neu3s. gg neu protein structure prediction prediction of gg neu structure, based on its alignment with human sialidase neu and performed using swiss-model, revealed, as expected, a high similarity with the neu structure. the predicted structure of the protein is the six blades β-propeller typical of sialidase, each blade being composed of four anti-parallel β-strands. the repeat region is located at the opposite side of the catalytic crevice and emerges as a long loop connecting two β-strands of the fifth blade of the β-propeller. this region is comprised between two highly conserved amino acid blocks, gcqgsvsf and wlly. based on gg neu structural model, the highly conserved svvsf block represents the first β-strand of the fifth blade of the β-propeller, followed by the expanded loop region containing the repeat units. the next highly conserved region is part of the seventh repeat unit organized in: i) wllyshp, as second β-strand of the fifth blade of the propeller; ii) tnrrhrr, as loop portion connecting the two anti-parallel β-strands and containing the essential catalytic residue arg- and; iii) dlg, as first portion of the third β-strand of the same blade. the ab initio structure prediction on the repeat region peculiar of gg neu, performed with rosetta and refined using i-tasser, resulted in a long hairpin architecture with no particular secondary structure features. several hydrogen bonds and hydrophobic interactions occur between the first and the last residues of this region. the putative β-sheets predicted in the repeat region by psipred are also indicated in figure 4d. expression and characterization of gg neu enzyme to demonstrate that the gg neu gene encodes for an active sialidase, the entire orf was subcloned in the mammalian expression vector pmt and the resulting construct was used to transiently transfect cos cells. crude homogenates from cells transfected with pmt21-gg neu3- myc or pmt alone were tested for sialidase activity using the artificial substrate 4-mu-neuac. transfection with pmt21-gg neu3-myc resulted in a -fold increase in the enzymatic activity detected in the crude extract. the enzyme showed a ph optimum at, similarly to bovine, human and mouse neu enzymes. fractionation by ultracentrifugation of crude extracts from transfected cos cells clearly demonstrated the association of gg neu with the particulate fraction, as about 85% of the activity detected in the homogenate was recovered in the pelleted material. this is in agreement with data available for the human and mouse counterparts. in order to study gg neu association to detergent resistant membranes, crude extracts were treated with triton x- as described in methods. immunoblotting analysis with anti-myc antibodies revealed a partial recovery of gg neu in drm fraction, identified by the presence of cav- a specific marker for these membrane domains. this finding is in agreement with the results already obtained for human and mouse neu. analysis of the hydrophilic/hydrophobic features of gg neu by aqueous-to-detergent repartition after triton x- treatment revealed the almost complete segregation of gg neu in the detergent phase together with cav- supporting a strong association with cellular membranes. interestingly, this behavior differs from that of mm neu3-ha, tested as a control protein, which segregated in the aqueous phase. immunofluorescence localization was studied in cos cells transiently transfected with pmt21-gg neu3-myc at h post transfection. laser confocal microscopy analysis showed plasma membrane labelling together with intracellular tubular and vesicular structures, the latter being mainly concentrated in the juxtanuclear region of the cell. these observations are in agreement with the localization of human and mouse neu also performed in cos cells. co-localization experiments carried out using makers related to vesicular trafficking and protein recycling, such as tfr and eea are illustrated in figure. co-localization of gg neu with tfr and eea indicate that, beside its localization at the plasma membrane, the protein is present also in early and recycling endosomal compartments, as already reported for mm neu. gg neu mutant protein properties in order to evaluate the impact of the repeat region on the general properties of gg neu we carried out mutagenesis of the original protein as described in methods. this allowed us to generate a new protein of amino acids with a predicted mw of kda, lacking of the units contained in the repeat region described above. in detail, we performed a amino acids deletion from thr- to gly- leaving intact the seventh repeat unit and a amino acids spacer. the seventh repeat unit was maintained in the mutated construct since it contains fundamental structural elements and the enzyme catalytic residue arg-. overall gg neu3-del and the wild-type protein showed superimposable results in the biochemical and cellular studies we performed. indeed, the transfection with pmt21-gg neu3-del-myc resulted in a -fold increase of the enzymatic activity detected in the crude extract with a ph optimum at ph. fractionation of crude extracts from transfected cos cells after ultracentrifugation showed that about 80% of the activity detected in the homogenate was recovered in the pelleted material. the mutant protein repartition after treatments with triton x- and triton x- is also comparable with the results obtained with the wild-type protein. finally, the subcellular localization in cos transfected cells showed no significant differences in the distribution of the wild-type and mutant protein. discussion the insertion of loop of various lengths and not involved in catalytic activity, between two structurally conserved motifs, has been already observed in the ras gtpases protein family and could represent a mechanism for protein evolution and creation of novel protein domain. a preliminary survey of sialidase enzymes in vertebrates and mammals revealed the presence of a loop region which shows high sequence divergence and variable length in vertebrate neu and neu. moreover as suggested by the sialidase characterization in danio rerio, which posses orthologs of human neu gene but apparently no neu ortholog, evolution of this protein family has been complex, probably following the increasing complexity of sialoconjugates from microorganisms to higher vertebrates. to get better insight into evolution of the sialidase protein family, we decided to extend our studies to other vertebrates, based on the presence of rapid diverging loops in this class of enzymes. a bioinformatic analysis carried out on genomic and expressed sequences revealed that gallus gallus genome, similarly to mammals, contains four genes that, based on sequence identity and synteny analysis, encode the putative orthologs of neu neu neu and neu proteins. the comparison of the primary structure of neu from gallus gallus with its human counterpart confirmed the presence in the former of an amino acid insertion larger than the one found in all neu sialidases characterized so far. this large insertion may represent a rapid evolving loop and so a potentially important event for sialidase evolution. we thus decided to focus our interest on this member of the gallus gallus sialidase family. the gg neu gene is located on chicken chromosome in a region of conserved synteny with chromosome 11q harboring neu in homo sapiens. while in both species the gene is flanked on one side by the kcne pold chrdl xrra and spcs genes, an evolutionary rearrangement, probably an inversion of a kb region delimited by neu and kcne has separated gg neu from ppme in mammals. in chicken the neu gene is apparently expressed in an ubiquitous manner, like its mammalian counterparts. primary structure analysis of the gg neu polypeptide revealed that, besides the high identity score with the human and mouse enzymes, all sialidase canonical amino acid signatures rip motif and asp boxes) are present and conserved in topologically equivalent positions. the insertion identified in gg neu corresponds to a amino acid region mainly constituted by a peptide motif repeated in tandem and organized in six plus one highly conserved repeat sequences. the analysis of different chickens allowed us to identify a shorter allele of the gene characterized by the absence of a region encoding one of the repeats. these results demonstrate the presence of two alleles of gallus gallus neu gene. the repeat cluster that characterizes gg neu may derive from complex duplication events originated from rep as its last portion is conserved also in sialidases from other vertebrates. an almost complete identity is observed among rep and whereas rep and are more divergent, both in residue number and sequence, suggesting that duplication events occurred at different times during the evolution. noteworthy, this repeat sequence is located between the evolutionary highly conserved sequence blocks gcqgsvsf and wlly. the analysis of the multiple sequence alignment in this region confirmed the presence of a loop of variable size in neu vertebrate sialidases ranging from residues, in the case of zebrafish neu - to 41- residues for the mammalian enzymes. interestingly, an insertion of and residues is located in the same region in human and mouse neu, respectively. using the crystal structure of hs neu as template, a homology model of gg neu has been obtained, showing, as expected, the six-blade β-propeller architecture typical of sialidases. the analysis of the model revealed that the repeat sequence found in chicken and turkey neu as well as the stretch of about residues found in mammalian neu4s, are inserted at the opposite side of the active crevice. this region represents the loop connecting the highly conserved first and second antiparallel β-strands of the fifth blade of the β-propeller. the best model obtained for this loop region and based on ab initio structure prediction resulted in a long hairpin architecture with no particular secondary structure features. interestingly the peptide chain is stabilized by several hydrogen bonds and hydrophobic interactions occurring between the first and the last residues. these interactions create a sort of support for the long loop and isolate it from the core of gg neu structure. this allows the exploration of new structural variants of the loop without impairing the protein biological activity. along the hairpin chain putative β-sheet regions have been also identified by secondary structure prediction and may contribute to stabilize this region. in order to confirm that gg neu encodes for an active sialidase and the possible relevance of the loop, we expressed gg neu and gg neu3-del cdna in cos a cell line that has been used extensively for the biochemical characterization of sialidase enzymes. studies on transiently transfected cells showed for both proteins a significant increase in sialidase activity, with an acidic ph optimum, comparable subcellular localization and membrane association, in agreement with data on human and mouse neu. interestingly gg neu and its deletion mutant revealed a peculiar hydrophobic feature when extracted with triton x- compared to mm neu. this feature probably does not depend on gg neu direct interaction with lipid bilayer, given its overall hydrophilic nature. a possible explanation for this behavior of the protein could be found in different interaction of mouse and chicken neu with other component of the membrane. overall, these data suggest that, in agreement with the rapid evolving loop hypothesis, the repeated region, at this stage of evolution, has apparently none or limited function at least for the biochemical and cellular properties considered herein. it is intriguing to speculate about the fate of this loop region in sialidase evolution, particularly considering its increasing size, observed in neu proteins of higher vertebrates, and the unique repeat organization observed in chicken and turkey. in this perspective, the long amino acid stretch found in chicken and turkey neu could represent an intermediate step in the evolutive process leading toward novel domain relevant for the evolution of sialidases in this systematic group, as observed for lectin binding domain found in vibrio cholerae. concerning the repetitive nature of the loop found in gg neu it should be noted that it does not represent a unique case among sialidases. indeed, the c-terminal domain of the trans-sialidase from trypanosoma cruzi contains a variable number of a tandemly repeated 12-amino acid-long unit, unrelated to the one found in gg neu termed sapa that is not required for ts activity. although some studies indicate that the repetitive amino acid motif stabilizes the catalytic activity of the enzyme in blood, its function remains largely unknown. moreover, our data demonstrate that the insertion of a large loop in this region of the enzyme does not impair the 3d organization of sialidase, indicating that this portion of the enzyme could be exploited as insertional spot for artificial modifications of the protein. we demonstrate that also neu from turkey contains the repeat region, whose length corresponds to the one of the chicken short allelic variant. this is not surprising considering that turkey is a domestic bird strictly related to chicken in the evolutionary tree of the galliformes order. besides chicken and turkey, the only other avian genome that has been sequenced belongs to the australian songbird zebra finch. we were surprised to discover that the zebra finch neu completely lacks the repeat region, showing instead a short loop of residues, similar in length to the one found in cytosolic sialidase hs neu this finding indicates that the repeat region is highly variable and is not a peculiar feature of all avian neu sialidases, suggesting three evolutionary hypotheses. the repeat sequence has arisen: i) recently in the galliformes order; ii) in the galloanserae clade; or iii) in a common ancestor of both galloanserae and neoaves, and then has been lost in the latter at any point of the evolutionary tree leading to zebra finch. by considering the first two hypothesis we can not exclude that the repeat region is present only in breeding birds as a consequence of the high genetic pressure and selection applied by man to these species. the sequencing of additional avian genomes will allow to shed light on these evolutionary scenarios.  in this study we demonstrated that sialidase neu β-propeller contains a surface loop highly variable in both sequence and size connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice. in the case of chicken and turkey, both belonging to the galliformes order, this loop is constituted by a number of repeats organized in tandem. the removal of the repeat region does not seems to alter the evaluated properties of the gg neu enzyme. our data indicate sialidase family as good model to study the evolutionary process based on the rapid diverging loop hypothesis. according to this theory, new random protein folding and architectures can emerge rapidly taking advantage of low constraint acting on surface loops at both sequence and structure level. when a loop develops into a useful or stable structure, it becomes subjected to positive selection and the new variant could be retained and eventually originate a new protein with specific features. this process may had occurred in sialidase protein family evolution leading to the membrane-bound enzyme forms, namely neu and neu present today in higher organisms. giving the peculiar features of its loop region, gallus gallus neu could be of particular interest in such evolutionary studies. additional work is needed to unravel a possible role of the repeat region in the avian biology. 
these loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. giving the peculiar organization of the loop region identified in gg neu this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution. these data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. the repeat region is located at the same position as the roughly amino acid loop characteristic of mammalian neu based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved β-strands of the fifth blade of the sialidase β-propeller. a good model to study this hypothesis is represented by sialidase enzymes. gallus gallus genome contains one neu gene encoding a protein with a unique amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. indeed, the structure of sialidases is a β-propeller composed by anti-parallel β-sheets connected by loops that suit well with the rapid evolving loop hypothesis. finally, we found that the general enzymatic properties and subcellular localization of gg neu are not influenced by the deletion of the repeat sequence. in this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice. these features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases.
 large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. these loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. a good model to study this hypothesis is represented by sialidase enzymes. indeed, the structure of sialidases is a β-propeller composed by anti-parallel β-sheets connected by loops that suit well with the rapid evolving loop hypothesis. these features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases.  gallus gallus genome contains one neu gene encoding a protein with a unique amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. the repeat region is located at the same position as the roughly amino acid loop characteristic of mammalian neu based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved β-strands of the fifth blade of the sialidase β-propeller. moreover this loop is highly variable in sequence and size in neu sialidases from other vertebrates. finally, we found that the general enzymatic properties and subcellular localization of gg neu are not influenced by the deletion of the repeat sequence.  in this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice. these data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. giving the peculiar organization of the loop region identified in gg neu this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution. rapid evolving loopssialidasebirdscomparative genomicspeptide tandem repeat 