BACKGROUND
gene duplication is considered a fundamental process in the generation of genes with novel functions and ultimately the evolution of biological diversity  <cit> .
a wide variety of evolutionary and comparative genomic analyses have been used to infer the origins and fates of newly created genes  <cit> .
while more limited in scope, molecular genetic and functional studies  <cit>  have provided new insights into the role gene duplication has played in generating novel functions.
in drosophila, comparative genomic analyses have revealed an enrichment of rapidly evolving or lineage specific gene families associated with reproduction  <cit> .
while the functional significance of this remains to be fully determined, the trend can be explained, in part, by creation of male-biased genes through retrotransposition.
recent studies have demonstrated that retrogenes in both drosophila  <cit>  and primates  <cit>  tend to acquire testis-specific expression.
however, we still have limited understanding of why new genes frequently acquire testis expression and whether testis-specific expression is obtained prior to, or following, acquisition of functionality.
nonetheless, several studies suggest that newly generated genes and gene families have had a functional impact on spermatogenesis and on the evolution of sperm  <cit> .
although new testis-specific genes might be expected to have gained functions in spermatogenesis, there are a limited number of characterized examples of such genes in drosophila.
one example is the strict paternal-effect gene msk <dig> , a gene created by retrotransposition prior to the divergence of the melanogaster subgroup  <cit> .
in wild-type eggs fertilized by sperm from k <dig> males, paternal chromosomes systematically fail to properly separate sister chromatids during the first zygotic division leading to lethality early in embryogenesis  <cit> .
interestingly, k <dig> is expressed only in primary spermatocytes where it presumably regulates aspects of spermatogenesis required for proper sperm function in the egg.
a second example is mojoless , created approximately 50mya through the retrotransposition of shaggy , a glycogen synthase kinase- <dig> encoding gene  <cit> .
rnai knockdown of mjl resulted in loss of the male germ line and male sterility.
although not yet ascribed a characterized function in the testis or sperm, a third gene, sdic, is a newly created gene encoding a protein present in the sperm tail  <cit> .
sdic is an unusual case of an x-linked chimeric gene specific to drosophila melanogaster that was created through the duplication of annexinx  and subsequent fusion with cdic, a cytoplasmic dynein gene.
the application of mass spectrometry  to the study of sperm has provided our first insights into the dynamic role new gene creation has played in shaping the constituents of the sperm proteome  <cit>  and has raised intriguing questions about how these new genes have impacted the molecular evolution of sperm.
for example, analysis of the drosophila sperm proteome revealed  <dig> novel sperm genes specific to d. melanogaster and a further  <dig> sperm genes created through retrotransposition during drosophila evolution  <cit> .
amongst these is the retrogene cg <dig>  which encodes s-lap  <dig>  examined in this study.
it is noteworthy that these new sperm components were found to be proteins across diverse functional classes, and both rapidly evolving  and highly conserved .
many proteolysis-related genes  have reproductive functions across diverse taxa, including insects  <cit>  and mammals  <cit> .
amongst these, proteolysis-related genes expressed in the drosophila accessory gland have been particularly well studied in terms of their mediation of a wide range of effects on females .
proteins with proteolytic activity are also found in the drosophila female reproductive tract and a subset of these are encoded by recently created genes  <cit> .
in both mammals and drosophila, the genes involved in reproductive proteolytic pathways or their targets have been demonstrated to evolve rapidly  <cit>  and it is hypothesized that this is due to coevolutionary forces associated with sexual conflict   <cit> .
these observations have been extended to the sperm proteome where an intensified signature of positive selection was observed for membrane and acrosomal sperm proteins that included a diverse set of metalloproteases and protease inhibitors  <cit> .
although well documented in reproductive tissues generally, only recently have proteolytic and related genes been shown to be present in spermatozoa  <cit> .
the functional significance and role in fertilization of these classes of proteins is unknown, but demonstration of their presence and activity in sperm is a necessary step towards more targeted studies.
here we present a detailed evolutionary and functional characterization of a family of eight drosophila sperm leucyl aminopeptidases that we have termed sperm-laps .
computational annotation places the s-laps in the m <dig> family of leucyl aminopeptidases  <cit> .
of the  <dig> annotated leucyl aminopeptidases in the drosophila genome, the s-laps are specifically expressed in the testis and all encode proteins incorporated in mature sperm  <cit> .
here we describe results of detailed comparative genomic analyses that demonstrate an expansion and diversification of the s-lap gene family during drosophila evolution.
we also provide  an independent proteomic analysis confirming the presence of s-laps in sperm,  a biochemical analysis of s-laps quantity and abundance in sperm,  data indicating that s-laps are expressed specifically in the testis and  evidence confirming s-lap enzymatic activity supporting their functional annotation.
this striking specificity of expression and cellular location suggests an unknown functional requirement for aminopeptidases in sperm.
furthermore, evolutionary diversification at the active site indicates that some s-laps have lost enzymatic activity and that the putative neofunctionalization of these non-enzymatic s-laps may be related to the subsequent expansion of the gene family.
these findings thus support a scenario where functional diversification within a gene family may promote further evolutionary changes in gene family composition through the selective retention of newly created genes.
RESULTS
comparative genomics of the s-lap family in drosophila
gene ontology  analysis  <cit>  previously identified eight related leucylaminopeptidases of the m <dig> merops family with an overall average amino acid identity of  <dig> %  <cit> .
we examined their evolutionary relationships by first determining that the annotated aminopeptidase, granny-smith, was the closest related paralog to the s-lap gene family .
granny-smith has not been identified in drosophila sperm  <cit>  and has no reported function in spermatogenesis or reproduction.
phylogenetic analysis of d. melanogaster s-laps and granny-smith revealed two distinct s-lap clusters : cluster i includes s-laps 3- <dig> genes  and cluster ii includes s-laps 1- <dig> genes .
the average synonymous divergence levels amongst the s-lap genes  indicated that the expansion of the gene family was not recent in drosophila evolution.
all pairwise values exceeded  <dig>  with the exception of cg6372/cg <dig> , a finding consistent with a more recent gene duplication event resulting in cluster ii and our phylogenetic analyses .
to further estimate the evolutionary timing of s-lap gene family expansion, orthologs were identified using reciprocal "best-hit" blast searches, for each s-lap in the  <dig> available drosophila genomes.
this analysis determined that all s-laps have unique orthologs in most, if not all the drosophila genomes and that all have distinct orthologs in d. grimshawi, the most ancestrally diverged drosophila species for which genomic data is available .
therefore, s-lap gene family expansion either predates or occurred early in the evolution of the genus drosophila, an observation consistent with the observed levels of synonymous divergence.
related aminopeptidase genes in other insect taxa
a comprehensive search for related aminopeptidases was conducted in other insect species using reciprocal blast searches against a dataset combining all annotated refseq, non-refseq and ab initio gene sequences in anopheles gambiae, culex quinquefasciatus, aedes aegypti, tribolium castaneum, nasonia vitripennis, acyrthosiphon pisum, bombyx mori and apis mellifera.
this resulted in the identification of  <dig> aminopeptidases related to the s-lap family and  <dig> orthologs of granny-smith.
the number of identified s-lap related aminopeptidase genes varied between taxa, ranging from none in n. vitripennis to  <dig> in a. aegypti and a. gambiae.
copy number determination of related aminopeptidase genes in each species was also confirmed through the analysis of genome assembly sequences in each species .
s-laps: an expanded gene family in drosophila
phylogenetic analysis of the s-lap gene family, including related aminopeptidases from other taxa, revealed that s-lap cluster i and ii are related to single-copy genes in most insect taxa .
the  <dig> members of cluster i form a monophyletic group with a single related gene in all  <dig> mosquito species and the beetle, t. castaneum.
this is consistent with gene family expansion during early drosophila evolution.
furthermore, no cluster i related s-lap genes were detected in n. vitripennis, a. pisum, b. mori and a. mellifera.
the two more closely related drosophila members of cluster ii also form a monophyletic group with single-copy genes in other insect taxa with the exception of c. quinquefasciatus where two related aminopeptidases were found.
these observations are in contrast to granny-smith that has "one-to-one" orthology relationships in all taxa where an ortholog was identified.
it is also noteworthy that our analysis identified a set of seven aminopeptidases in non-drosophila taxa  distinct from either drosophila s-lap cluster i or ii.
this includes a single copy aminopeptidase gene across  <dig> insect taxa and  <dig> putative, additional paralogs in a. aegypti.
mechanisms of new s-lap gene creation
the age of s-lap clusters and the associated sequence divergence at these loci means that hallmarks associated with gene duplication mechanisms are often obscured, making gene duplication mechanisms difficult to infer.
however, two pairs of s-laps  are immediately adjacent in the genome and share common intron/exon structure suggesting they were created through tandem duplication.
s-lap  <dig> has previously been characterized as a retrotransposed gene  <cit> .
consistent with a retroposition event, s-lap  <dig> is intronless, unlike its likely progenitor s-lap  <dig>  and these genes reside in different genomic regions .
s-laps are expressed specifically in the testis
expression of each s-lap in d. melanogaster was characterized by rt-pcr using gene specific primers.
a high level of testis-specific expression for all eight s-laps was observed with no detectable expression in gonadectimized males , a finding consistent with previous microarray studies  <cit> .
the testis-specific expression of the s-lap family is in contrast to granny-smith, the closest related leucyl aminopeptidase in the genome, which is expressed broadly across a range of tissues, including  testis, ovary, tubule and salivary gland  <cit> .
confirmation and quantification of s-laps in drosophila sperm by 2d-gel
as part of an exhaustive analysis of sperm proteins from 2d-gels of varying isoelectric gradients, all eight s-laps were identified by both maldi-tof and tandem ms/ms analyses .
high confidence of s-lap protein identification was supported by,  multiple peptide assignments for each protein  as assigned by scaffold ;  mowse scores from maldi-tof measurements that exceeded statistical scoring requirements  <cit>  and  replicate spot assignments from multiple 2d-gels under varying separation conditions .
tubulins were also identified on 2d-gels at the expected molecular weight and isoelectric point positions .
whole sperm tandem ms
s-lap 1
r.fplwnfysk.a
r.llfaveper.i
s-lap 2
r.isvpsidlyatk.d
r.vpyysavaviglgk.e
s-lap 3
k.givlglyek.e
r.iaagigar.s
k.givlglyek.e
s-lap 4
k.qitdemgydlsndgr.g
k.qfqsagaltgdr.v
k.ifnnidpefr.s
k.pgdvvtlmnhk.s
k.glvvgvyqk.e
k.lelyespdyegwtr.g
s-lap 5
r.vaagvgar.a
r.etgikgelgk.g
s-lap 6
n.avtlddalggk.l
r.gtgmtttinpr.p
k.ttanavtlddalggk.l
s-lap 7
r.lplwqyyr.r
k.yglvpyltk.k
s-lap 8
k.hgvppyllk.d
r.lyqnidk.e
k.esglngelgvgr.l
*ms analysis from  <cit> .
** 2d-gel protein spot locations presented in figure  <dig>
visual inspection of 2d-gels clearly indicated that both the s-laps and tubulins represent a substantial proportion of the total protein in sperm on 2d-gels .
we determined the relative abundance of each by comparing spot signal intensity for each class.
five 2d-gels  were processed, scanned and protein spot intensities calculated.
the results indicate an approximate 2-fold abundance of s-laps  over tubulins.
thus, we conclude that the tubulins and the s-lap family represent the major mass of protein in sperm.
s-lap enzymatic activity in purified sperm
s-lap activity in sperm extracts was directly measured using l-leu-amc, a sensitive and specific fluorogenic substrate for leucine aminopeptidase activity  <cit> .
negligible activity was observed in intact sperm suggesting that s-laps were not exposed on the sperm surface .
sonication of sperm in buffer alone resulted in measurable s-lap activity but with low protein yield .
addition of dtt resulted in both higher levels of soluble protein and measurable s-lap activity but with reduced specific activity .
however, while this establishes presence of leucylaminopeptidase activity, the relative contribution of each s-lap to the observed signal remains unclear.
*the percent values are calculated from maximal amount of extractable sperm protein.
‡ specific activity.
divergence at cation ligand and catalytic residues
biochemical and x-ray crystallography studies have defined  <dig> primary active site residues in m <dig> leucyl aminopeptidases involved in catalysis and binding of divalent cations, including a critical zn2+ ion binding region  <cit> .
these sites are highly conserved in leucyl aminopeptidases across animal taxa strongly suggesting their critical role in the coordination of the metal ions, water and a bicarbonate ion which coordinates a base attack on the peptide bond followed by breakdown of a tetrahedral diol intermediate with the terminal amino acid as the leaving group  <cit> .
intriguingly, the s-lap active site has diverged during drosophila evolution replaced in many cases by amino acids incapable of zn2+ binding or bicarbonate coordination .
in contrast, granny-smith contains the canonical active site residues.
furthermore, differences with likely functional relevance exist between the active site residues in s-lap clusters i and ii.
the two members of cluster ii retain a high level of amino acid identity with the m <dig> aminopeptidase consensus, including i)  <dig> out of  <dig> conserved amino acids amongst the tight zn2+ binding residues , ii) a conservative amino acid substitution  to a known m <dig> metal ligand amino acid at the fourth site and iii) a conservative amino acid substitution  to a known m <dig> metal ligand amino acid which contributes to divalent cation binding at the "loose" binding site .
this is in contrast to s-laps in cluster i that possess i) limited or no similarity to the m <dig> aminopeptidase consensus and ii) an abundance of substitutions in residues that do not bind metal ions or are not characterized as m <dig> lap metal ion ligands.
furthermore,  <dig> of the  <dig> s-laps in cluster i have undergone a substitution at lys <dig> which is essential for enzymatic activity  <cit> .
the  <dig> characterized functional residues of the active site are displayed on the s-lap protein alignment in additional file  <dig>  figure s <dig>
purifying selection on the s-lap gene family
given the rapid expansion of the s-lap gene family and the potential loss of catalytic activity amongst s-laps, we next sought to assess the role of positive selection in the functional diversification of the s-lap gene family.
to this end, we conducted codon based maximum likelihood analyses of selection for s-lap cluster i and ii using all drosophila s-lap orthologs.
this analysis resulted in strong evidence for purifying selection throughout the s-lap gene.
analysis of all cluster  <dig> sequences  resulted in a mean dn/ds estimate of  <dig> , significant evidence of negative selection  at  <dig> sites and no codons showing evidence of being under positive selection.
similarly, analysis of cluster ii  resulted in a mean dn/ds estimate of  <dig> , significant evidence of negative selection at  <dig> sites and no codons showing evidence of being under positive selection.
discussion
advances in comparative genomics have revealed that the creation of novel genes is a widespread process  <cit>  and that the composition of gene families associated with reproduction can vary dramatically between species  <cit> .
often, the expansion of gene families through gene creation results in functional diversification of gene family members, a process sometimes associated with rapid evolutionary changes at the dna sequence and spatio-temporal expression levels.
furthermore, it has also been well documented that new retrogenes often acquire testis expression, however a limited amount is known about the specific functional ramifications of these phenomenon.
to map specific functional consequences of gene family evolution, we have utilized comparative genomics to characterize the dynamic evolutionary history of the s-lap gene family and documented s-lap abundance and activity in spermatozoa.
gene family expansion and evolution
the recent availability of additional insect genome sequences has allowed the analysis of gene family evolution as it potentially relates to specific biological characteristics  <cit> .
this approach has been of great utility in our studies of drosophila sperm where gene creation has proven to be a widespread process and potentially a dynamic force in the molecular evolution of sperm  <cit> .
comparative genomic analyses of the sperm-specific s-laps in this study further extends this observation and demonstrates that the s-lap gene families are highly heterogeneous in composition across the insect genomes that have been sequenced to date.
this includes, but is not limited to, a dramatic expansion during the evolution of drosophila  and variable copy numbers across three mosquito species.
while some revision of the s-lap gene family evolution may result from the availability of additional insect genomes, these results clearly reiterate the fundamental importance of gene creation during the evolution of reproductive traits in drosophila.
leucyl aminopeptidases in drosophila sperm
aminopeptidases, a large family of related enzymes found throughout animal and plant kingdoms, have been described in many reproductive tissues including sperm and seminal fluids  <cit> .
our results extend these observations to include a newly recognized family, s-laps 1- <dig>  other than a role for a gpi-anchored aminopeptidase in the initiation of the acrosome activation in mussels   <cit> , little functional data exists for leucylaminopeptidases either in sperm or indeed other aspects of reproductive biology.
our results provide strong circumstantial data suggesting that s-laps may play an important role in sperm function:  s-laps are expressed exclusively during spermatogenesis;  they collectively represent the most abundant protein class in drosophila sperm by a 2: <dig> mass ratio compared to tubulin ;  bioinformatic analysis demonstrated s-laps as the most significantly over-represented protein domain within the proteome  <cit> ; and  analysis of stage specific expression during spermatogenesis  revealed that all  <dig> s-laps were upregulated specifically during the meiotic and post-meiotic stages of late spermatogenesis   <cit> , the developmental time window of spermiogenesis and sperm production.
further, we observed significant levels of leucyl aminopeptidase activity in both the soluble and insoluble fractions of purified sperm.
s-lap activity was recovered from the insoluble pellet in the presence of dtt.
furthermore, the two fractions differed in specific activities perhaps reflecting a partition of different s-laps that may be directly related to functional diversification between them .
in contrast to the rapid evolution often observed in genes associated with male reproduction, we observe a strong signal of purifying selection across all s-laps suggesting a constrained functional role in sperm.
this conservation is intriguing given that each cluster displays distinct patterns of substitutions at the active site consistent with functional diversification.
cluster i has undergone radical amino acid replacements in many of the canonical active site residues and all model studies of this class of enzyme suggest that these substitutions would be likely to render the catalytic site inactive.
this observation, in conjunction with the evolutionary conservation  of cluster i, suggests the possible evolutionary acquisition of important functions independent of enzymatic activity.
comparison of cluster i active site residues in drosophila and mosquito reveals that most active site substitutions occurred prior to the evolutionary expansion of cluster i, indicating that the cluster i progenitor gene had potentially lost enzymatic activity prior to the expansion of this cluster .
future studies are needed to determine if the mosquito orthologs of cluster i also function specifically in sperm.
in contrast, cluster ii s-laps more closely match the canonical m <dig> aminopeptidase active site and are likely to account for the enzymatic activity in purified sperm quantified in this study.
the amino acid substitution  in the catalytic site occurs at a residue not strictly required for enzymatic activity but instead is expected to decrease activity by  <dig> to 100-fold based on previous biochemical studies  <cit> .
inspection of the mosquito cluster ii active sites indicates that this substitution occurred more recently during drosophila evolution.
the other catalytic site, residue lys <dig>  is strictly conserved and has been shown essential for catalytic activity  <cit> .
it is possible that the cg6372/cg <dig> duplication event may have partially restored levels of enzymatic activity lost as a result of the arg413val substitution.
quantification of specific activity for each s-lap is the next necessary step in assessing the functional diversification of the gene family.
CONCLUSIONS
the presence of eight distinct s-laps and their extraordinarily high protein concentration in sperm strongly suggests that s-laps play a predominant  role in drosophila sperm biology.
however, our results suggest that this role  cannot be linked to enzymatic activity in cluster i s-laps where the canonical active site is clearly absent.
an outstanding question therefore remains: what is the acquired functionality of cluster i s-laps?
one testable possibility is that cluster i s-laps lost enzymatic activity but were retained, and subsequently diversified, following the acquisition of a structural function in sperm.
while such a scenario for sperm protein evolution is unprecedented, it has been well documented in other enzyme families such as the crystallin family of lens proteins that evolved from ubiquitously expressed metabolic enzymes .
although the precise evolutionary processes leading to the neofunctionalization of the crystallins are unknown, it did include the loss of enzymatic activity due to substitutions at critical active site residues, the expression of proteins at high concentrations and the subsequent establishment of their current structural and refractive roles in the eye lens  <cit> .
it is noteworthy that the s-laps share extensive sequence homology  to the bovine lens leucine aminopeptidase .
it will be of great interest to determine if, and to what extent, these two independent evolutionary processes parallel one another.
regardless, the s-lap gene family represents a unique example wherein loss of function  predates the dramatic expansion of a gene family.
as such, neofunctionalization may itself lead to a situation where gene family expansion is selectively favorable, an observation that complements the general model where gene duplication predates and facilitates the evolution of novel functionality.
finally, in recent years characterized functions of laps have expanded to include a range of other cellular processes .
it will be of considerable interest to investigate whether s-laps have similarly acquired diversified roles in sperm function and physiology.

 gene duplication is considered a fundamental process in the generation of genes with novel functions and ultimately the evolution of biological diversity. a wide variety of evolutionary and comparative genomic analyses have been used to infer the origins and fates of newly created genes. while more limited in scope, molecular genetic and functional studies have provided new insights into the role gene duplication has played in generating novel functions. in drosophila, comparative genomic analyses have revealed an enrichment of rapidly evolving or lineage specific gene families associated with reproduction. while the functional significance of this remains to be fully determined, the trend can be explained, in part, by creation of male-biased genes through retrotransposition. recent studies have demonstrated that retrogenes in both drosophila and primates tend to acquire testis-specific expression. however, we still have limited understanding of why new genes frequently acquire testis expression and whether testis-specific expression is obtained prior to, or following, acquisition of functionality. nonetheless, several studies suggest that newly generated genes and gene families have had a functional impact on spermatogenesis and on the evolution of sperm. although new testis-specific genes might be expected to have gained functions in spermatogenesis, there are a limited number of characterized examples of such genes in drosophila. one example is the strict paternal-effect gene msk, a gene created by retrotransposition prior to the divergence of the melanogaster subgroup. in wild-type eggs fertilized by sperm from k males, paternal chromosomes systematically fail to properly separate sister chromatids during the first zygotic division leading to lethality early in embryogenesis. interestingly, k is expressed only in primary spermatocytes where it presumably regulates aspects of spermatogenesis required for proper sperm function in the egg. a second example is mojoless, created approximately 50mya through the retrotransposition of shaggy, a glycogen synthase kinase- encoding gene. rnai knockdown of mjl resulted in loss of the male germ line and male sterility. although not yet ascribed a characterized function in the testis or sperm, a third gene, sdic, is a newly created gene encoding a protein present in the sperm tail. sdic is an unusual case of an x-linked chimeric gene specific to drosophila melanogaster that was created through the duplication of annexinx and subsequent fusion with cdic, a cytoplasmic dynein gene. the application of mass spectrometry to the study of sperm has provided our first insights into the dynamic role new gene creation has played in shaping the constituents of the sperm proteome and has raised intriguing questions about how these new genes have impacted the molecular evolution of sperm. for example, analysis of the drosophila sperm proteome revealed novel sperm genes specific to d. melanogaster and a further sperm genes created through retrotransposition during drosophila evolution. amongst these is the retrogene cg which encodes s-lap examined in this study. it is noteworthy that these new sperm components were found to be proteins across diverse functional classes, and both rapidly evolving and highly conserved. many proteolysis-related genes have reproductive functions across diverse taxa, including insects and mammals. amongst these, proteolysis-related genes expressed in the drosophila accessory gland have been particularly well studied in terms of their mediation of a wide range of effects on females. proteins with proteolytic activity are also found in the drosophila female reproductive tract and a subset of these are encoded by recently created genes. in both mammals and drosophila, the genes involved in reproductive proteolytic pathways or their targets have been demonstrated to evolve rapidly and it is hypothesized that this is due to coevolutionary forces associated with sexual conflict. these observations have been extended to the sperm proteome where an intensified signature of positive selection was observed for membrane and acrosomal sperm proteins that included a diverse set of metalloproteases and protease inhibitors. although well documented in reproductive tissues generally, only recently have proteolytic and related genes been shown to be present in spermatozoa. the functional significance and role in fertilization of these classes of proteins is unknown, but demonstration of their presence and activity in sperm is a necessary step towards more targeted studies. here we present a detailed evolutionary and functional characterization of a family of eight drosophila sperm leucyl aminopeptidases that we have termed sperm-laps. computational annotation places the s-laps in the m family of leucyl aminopeptidases. of the annotated leucyl aminopeptidases in the drosophila genome, the s-laps are specifically expressed in the testis and all encode proteins incorporated in mature sperm. here we describe results of detailed comparative genomic analyses that demonstrate an expansion and diversification of the s-lap gene family during drosophila evolution. we also provide an independent proteomic analysis confirming the presence of s-laps in sperm, a biochemical analysis of s-laps quantity and abundance in sperm, data indicating that s-laps are expressed specifically in the testis and evidence confirming s-lap enzymatic activity supporting their functional annotation. this striking specificity of expression and cellular location suggests an unknown functional requirement for aminopeptidases in sperm. furthermore, evolutionary diversification at the active site indicates that some s-laps have lost enzymatic activity and that the putative neofunctionalization of these non-enzymatic s-laps may be related to the subsequent expansion of the gene family. these findings thus support a scenario where functional diversification within a gene family may promote further evolutionary changes in gene family composition through the selective retention of newly created genes.  comparative genomics of the s-lap family in drosophila gene ontology analysis previously identified eight related leucylaminopeptidases of the m merops family with an overall average amino acid identity of %. we examined their evolutionary relationships by first determining that the annotated aminopeptidase, granny-smith, was the closest related paralog to the s-lap gene family. granny-smith has not been identified in drosophila sperm and has no reported function in spermatogenesis or reproduction. phylogenetic analysis of d. melanogaster s-laps and granny-smith revealed two distinct s-lap clusters: cluster i includes s-laps 3- genes and cluster ii includes s-laps 1- genes. the average synonymous divergence levels amongst the s-lap genes indicated that the expansion of the gene family was not recent in drosophila evolution. all pairwise values exceeded with the exception of cg6372/cg, a finding consistent with a more recent gene duplication event resulting in cluster ii and our phylogenetic analyses. to further estimate the evolutionary timing of s-lap gene family expansion, orthologs were identified using reciprocal "best-hit" blast searches, for each s-lap in the available drosophila genomes. this analysis determined that all s-laps have unique orthologs in most, if not all the drosophila genomes and that all have distinct orthologs in d. grimshawi, the most ancestrally diverged drosophila species for which genomic data is available. therefore, s-lap gene family expansion either predates or occurred early in the evolution of the genus drosophila, an observation consistent with the observed levels of synonymous divergence. related aminopeptidase genes in other insect taxa a comprehensive search for related aminopeptidases was conducted in other insect species using reciprocal blast searches against a dataset combining all annotated refseq, non-refseq and ab initio gene sequences in anopheles gambiae, culex quinquefasciatus, aedes aegypti, tribolium castaneum, nasonia vitripennis, acyrthosiphon pisum, bombyx mori and apis mellifera. this resulted in the identification of aminopeptidases related to the s-lap family and orthologs of granny-smith. the number of identified s-lap related aminopeptidase genes varied between taxa, ranging from none in n. vitripennis to in a. aegypti and a. gambiae. copy number determination of related aminopeptidase genes in each species was also confirmed through the analysis of genome assembly sequences in each species. s-laps: an expanded gene family in drosophila phylogenetic analysis of the s-lap gene family, including related aminopeptidases from other taxa, revealed that s-lap cluster i and ii are related to single-copy genes in most insect taxa. the members of cluster i form a monophyletic group with a single related gene in all mosquito species and the beetle, t. castaneum. this is consistent with gene family expansion during early drosophila evolution. furthermore, no cluster i related s-lap genes were detected in n. vitripennis, a. pisum, b. mori and a. mellifera. the two more closely related drosophila members of cluster ii also form a monophyletic group with single-copy genes in other insect taxa with the exception of c. quinquefasciatus where two related aminopeptidases were found. these observations are in contrast to granny-smith that has "one-to-one" orthology relationships in all taxa where an ortholog was identified. it is also noteworthy that our analysis identified a set of seven aminopeptidases in non-drosophila taxa distinct from either drosophila s-lap cluster i or ii. this includes a single copy aminopeptidase gene across insect taxa and putative, additional paralogs in a. aegypti. mechanisms of new s-lap gene creation the age of s-lap clusters and the associated sequence divergence at these loci means that hallmarks associated with gene duplication mechanisms are often obscured, making gene duplication mechanisms difficult to infer. however, two pairs of s-laps are immediately adjacent in the genome and share common intron/exon structure suggesting they were created through tandem duplication. s-lap has previously been characterized as a retrotransposed gene. consistent with a retroposition event, s-lap is intronless, unlike its likely progenitor s-lap and these genes reside in different genomic regions. s-laps are expressed specifically in the testis expression of each s-lap in d. melanogaster was characterized by rt-pcr using gene specific primers. a high level of testis-specific expression for all eight s-laps was observed with no detectable expression in gonadectimized males, a finding consistent with previous microarray studies. the testis-specific expression of the s-lap family is in contrast to granny-smith, the closest related leucyl aminopeptidase in the genome, which is expressed broadly across a range of tissues, including testis, ovary, tubule and salivary gland. confirmation and quantification of s-laps in drosophila sperm by 2d-gel as part of an exhaustive analysis of sperm proteins from 2d-gels of varying isoelectric gradients, all eight s-laps were identified by both maldi-tof and tandem ms/ms analyses. high confidence of s-lap protein identification was supported by, multiple peptide assignments for each protein as assigned by scaffold; mowse scores from maldi-tof measurements that exceeded statistical scoring requirements and replicate spot assignments from multiple 2d-gels under varying separation conditions. tubulins were also identified on 2d-gels at the expected molecular weight and isoelectric point positions. whole sperm tandem ms s-lap 1 r.fplwnfysk.a r.llfaveper.i s-lap 2 r.isvpsidlyatk.d r.vpyysavaviglgk.e s-lap 3 k.givlglyek.e r.iaagigar.s k.givlglyek.e s-lap 4 k.qitdemgydlsndgr.g k.qfqsagaltgdr.v k.ifnnidpefr.s k.pgdvvtlmnhk.s k.glvvgvyqk.e k.lelyespdyegwtr.g s-lap 5 r.vaagvgar.a r.etgikgelgk.g s-lap 6 n.avtlddalggk.l r.gtgmtttinpr.p k.ttanavtlddalggk.l s-lap 7 r.lplwqyyr.r k.yglvpyltk.k s-lap 8 k.hgvppyllk.d r.lyqnidk.e k.esglngelgvgr.l *ms analysis from. ** 2d-gel protein spot locations presented in figure visual inspection of 2d-gels clearly indicated that both the s-laps and tubulins represent a substantial proportion of the total protein in sperm on 2d-gels. we determined the relative abundance of each by comparing spot signal intensity for each class. five 2d-gels were processed, scanned and protein spot intensities calculated. the results indicate an approximate 2-fold abundance of s-laps over tubulins. thus, we conclude that the tubulins and the s-lap family represent the major mass of protein in sperm. s-lap enzymatic activity in purified sperm s-lap activity in sperm extracts was directly measured using l-leu-amc, a sensitive and specific fluorogenic substrate for leucine aminopeptidase activity. negligible activity was observed in intact sperm suggesting that s-laps were not exposed on the sperm surface. sonication of sperm in buffer alone resulted in measurable s-lap activity but with low protein yield. addition of dtt resulted in both higher levels of soluble protein and measurable s-lap activity but with reduced specific activity. however, while this establishes presence of leucylaminopeptidase activity, the relative contribution of each s-lap to the observed signal remains unclear. *the percent values are calculated from maximal amount of extractable sperm protein. ‡ specific activity. divergence at cation ligand and catalytic residues biochemical and x-ray crystallography studies have defined primary active site residues in m leucyl aminopeptidases involved in catalysis and binding of divalent cations, including a critical zn2+ ion binding region. these sites are highly conserved in leucyl aminopeptidases across animal taxa strongly suggesting their critical role in the coordination of the metal ions, water and a bicarbonate ion which coordinates a base attack on the peptide bond followed by breakdown of a tetrahedral diol intermediate with the terminal amino acid as the leaving group. intriguingly, the s-lap active site has diverged during drosophila evolution replaced in many cases by amino acids incapable of zn2+ binding or bicarbonate coordination. in contrast, granny-smith contains the canonical active site residues. furthermore, differences with likely functional relevance exist between the active site residues in s-lap clusters i and ii. the two members of cluster ii retain a high level of amino acid identity with the m aminopeptidase consensus, including i) out of conserved amino acids amongst the tight zn2+ binding residues, ii) a conservative amino acid substitution to a known m metal ligand amino acid at the fourth site and iii) a conservative amino acid substitution to a known m metal ligand amino acid which contributes to divalent cation binding at the "loose" binding site. this is in contrast to s-laps in cluster i that possess i) limited or no similarity to the m aminopeptidase consensus and ii) an abundance of substitutions in residues that do not bind metal ions or are not characterized as m lap metal ion ligands. furthermore, of the s-laps in cluster i have undergone a substitution at lys which is essential for enzymatic activity. the characterized functional residues of the active site are displayed on the s-lap protein alignment in additional file figure s purifying selection on the s-lap gene family given the rapid expansion of the s-lap gene family and the potential loss of catalytic activity amongst s-laps, we next sought to assess the role of positive selection in the functional diversification of the s-lap gene family. to this end, we conducted codon based maximum likelihood analyses of selection for s-lap cluster i and ii using all drosophila s-lap orthologs. this analysis resulted in strong evidence for purifying selection throughout the s-lap gene. analysis of all cluster sequences resulted in a mean dn/ds estimate of, significant evidence of negative selection at sites and no codons showing evidence of being under positive selection. similarly, analysis of cluster ii resulted in a mean dn/ds estimate of, significant evidence of negative selection at sites and no codons showing evidence of being under positive selection. discussion advances in comparative genomics have revealed that the creation of novel genes is a widespread process and that the composition of gene families associated with reproduction can vary dramatically between species. often, the expansion of gene families through gene creation results in functional diversification of gene family members, a process sometimes associated with rapid evolutionary changes at the dna sequence and spatio-temporal expression levels. furthermore, it has also been well documented that new retrogenes often acquire testis expression, however a limited amount is known about the specific functional ramifications of these phenomenon. to map specific functional consequences of gene family evolution, we have utilized comparative genomics to characterize the dynamic evolutionary history of the s-lap gene family and documented s-lap abundance and activity in spermatozoa. gene family expansion and evolution the recent availability of additional insect genome sequences has allowed the analysis of gene family evolution as it potentially relates to specific biological characteristics. this approach has been of great utility in our studies of drosophila sperm where gene creation has proven to be a widespread process and potentially a dynamic force in the molecular evolution of sperm. comparative genomic analyses of the sperm-specific s-laps in this study further extends this observation and demonstrates that the s-lap gene families are highly heterogeneous in composition across the insect genomes that have been sequenced to date. this includes, but is not limited to, a dramatic expansion during the evolution of drosophila and variable copy numbers across three mosquito species. while some revision of the s-lap gene family evolution may result from the availability of additional insect genomes, these results clearly reiterate the fundamental importance of gene creation during the evolution of reproductive traits in drosophila. leucyl aminopeptidases in drosophila sperm aminopeptidases, a large family of related enzymes found throughout animal and plant kingdoms, have been described in many reproductive tissues including sperm and seminal fluids. our results extend these observations to include a newly recognized family, s-laps 1- other than a role for a gpi-anchored aminopeptidase in the initiation of the acrosome activation in mussels, little functional data exists for leucylaminopeptidases either in sperm or indeed other aspects of reproductive biology. our results provide strong circumstantial data suggesting that s-laps may play an important role in sperm function: s-laps are expressed exclusively during spermatogenesis; they collectively represent the most abundant protein class in drosophila sperm by a 2: mass ratio compared to tubulin; bioinformatic analysis demonstrated s-laps as the most significantly over-represented protein domain within the proteome; and analysis of stage specific expression during spermatogenesis revealed that all s-laps were upregulated specifically during the meiotic and post-meiotic stages of late spermatogenesis, the developmental time window of spermiogenesis and sperm production. further, we observed significant levels of leucyl aminopeptidase activity in both the soluble and insoluble fractions of purified sperm. s-lap activity was recovered from the insoluble pellet in the presence of dtt. furthermore, the two fractions differed in specific activities perhaps reflecting a partition of different s-laps that may be directly related to functional diversification between them. in contrast to the rapid evolution often observed in genes associated with male reproduction, we observe a strong signal of purifying selection across all s-laps suggesting a constrained functional role in sperm. this conservation is intriguing given that each cluster displays distinct patterns of substitutions at the active site consistent with functional diversification. cluster i has undergone radical amino acid replacements in many of the canonical active site residues and all model studies of this class of enzyme suggest that these substitutions would be likely to render the catalytic site inactive. this observation, in conjunction with the evolutionary conservation of cluster i, suggests the possible evolutionary acquisition of important functions independent of enzymatic activity. comparison of cluster i active site residues in drosophila and mosquito reveals that most active site substitutions occurred prior to the evolutionary expansion of cluster i, indicating that the cluster i progenitor gene had potentially lost enzymatic activity prior to the expansion of this cluster. future studies are needed to determine if the mosquito orthologs of cluster i also function specifically in sperm. in contrast, cluster ii s-laps more closely match the canonical m aminopeptidase active site and are likely to account for the enzymatic activity in purified sperm quantified in this study. the amino acid substitution in the catalytic site occurs at a residue not strictly required for enzymatic activity but instead is expected to decrease activity by to 100-fold based on previous biochemical studies. inspection of the mosquito cluster ii active sites indicates that this substitution occurred more recently during drosophila evolution. the other catalytic site, residue lys is strictly conserved and has been shown essential for catalytic activity. it is possible that the cg6372/cg duplication event may have partially restored levels of enzymatic activity lost as a result of the arg413val substitution. quantification of specific activity for each s-lap is the next necessary step in assessing the functional diversification of the gene family.  the presence of eight distinct s-laps and their extraordinarily high protein concentration in sperm strongly suggests that s-laps play a predominant role in drosophila sperm biology. however, our results suggest that this role cannot be linked to enzymatic activity in cluster i s-laps where the canonical active site is clearly absent. an outstanding question therefore remains: what is the acquired functionality of cluster i s-laps? one testable possibility is that cluster i s-laps lost enzymatic activity but were retained, and subsequently diversified, following the acquisition of a structural function in sperm. while such a scenario for sperm protein evolution is unprecedented, it has been well documented in other enzyme families such as the crystallin family of lens proteins that evolved from ubiquitously expressed metabolic enzymes. although the precise evolutionary processes leading to the neofunctionalization of the crystallins are unknown, it did include the loss of enzymatic activity due to substitutions at critical active site residues, the expression of proteins at high concentrations and the subsequent establishment of their current structural and refractive roles in the eye lens. it is noteworthy that the s-laps share extensive sequence homology to the bovine lens leucine aminopeptidase. it will be of great interest to determine if, and to what extent, these two independent evolutionary processes parallel one another. regardless, the s-lap gene family represents a unique example wherein loss of function predates the dramatic expansion of a gene family. as such, neofunctionalization may itself lead to a situation where gene family expansion is selectively favorable, an observation that complements the general model where gene duplication predates and facilitates the evolution of novel functionality. finally, in recent years characterized functions of laps have expanded to include a range of other cellular processes. it will be of considerable interest to investigate whether s-laps have similarly acquired diversified roles in sperm function and physiology. 
the evolutionary diversification of gene families through gene creation is a dynamic process believed to be critical to the evolution of functional novelty. to assess putative functional activities of s-laps, we demonstrated that all s-laps are specifically expressed in the testis, confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, determined that they represent a major portion of the total protein in sperm and identified aminopeptidase enzymatic activity in sperm extracts using lap-specific substrates. functionally significant divergence at the canonical m active site indicates that the largest phylogenetic group of s-laps lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family. previous identification of a closely related family of eight annotated metalloprotease genes of the m merops family in the drosophila sperm proteome led us to hypothesize that this gene family may have experienced such a diversification during insect evolution. comparative genomic and phylogenetic analyses revealed the dramatic expansion of the s-lap gene family during drosophila evolution and copy number heterogeneity in the genomes of related insects. this finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function.
 the evolutionary diversification of gene families through gene creation is a dynamic process believed to be critical to the evolution of functional novelty. previous identification of a closely related family of eight annotated metalloprotease genes of the m merops family in the drosophila sperm proteome led us to hypothesize that this gene family may have experienced such a diversification during insect evolution.  to assess putative functional activities of s-laps, we demonstrated that all s-laps are specifically expressed in the testis, confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, determined that they represent a major portion of the total protein in sperm and identified aminopeptidase enzymatic activity in sperm extracts using lap-specific substrates. functionally significant divergence at the canonical m active site indicates that the largest phylogenetic group of s-laps lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family.  comparative genomic and phylogenetic analyses revealed the dramatic expansion of the s-lap gene family during drosophila evolution and copy number heterogeneity in the genomes of related insects. this finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function. spermproteomicsgene duplicationgene familyproteasespermatogenesistestis 