BACKGROUND
humans have two enzymes with α-galactosidase activity and an acidic ph optimum, α-n-acetylgalactosaminidase   and α-galactosidase a .
hereditary deficiency of each of the hydrolases causes a distinct lysosomal storage disorder in humans, schindler and fabry diseases, respectively  <cit> .
early studies suggested that both human enzymes were glycoforms with similar substrate specifities.
purified enzymes had similar physical properties, including subunit molecular mass , homodimeric structure, and amino acid sequences.
however, additional studies showed kinetic, structural, and immunologic differences proving that α-gal and α-naga were products of two different genes  <cit> .
the two genes differed in the number of exons  and also in the number, placement, and orientation of alu repeats.
exons  <dig> –  <dig> of the α-naga gene showed high similarity to the first six exons of α-gal gene.
because of the remarkable amino acid identity  and similarity  between the two genes and the similar intron placement, wang  <cit>  and co-workers suggested that a duplication event occurred during the evolution of both enzymes.
both enzymes belong to the glycoside hydrolase family  <dig> clan d  <cit> .
glycoside hydrolase family  <dig> clan d orthologs have been identified in a broad spectrum of prokaryotes and eukaryotes, including plants.
members of the family have a highly similar active site and share the same reaction mechanism.
the structures of chicken α-naga, human α-gal and rice α-gal have been determined by x-ray crystallography  <cit> .
chicken and human enzymes have a homodimeric quarternary structure whereas rice α-gal acts as a monomer.
the monomer units are composed of two distinct domains.
domain i contains the active site and adopts a  <dig> barrel structure, a domain fold observed commonly in glycosidases.
domain ii has eight antiparallel β strands, packed into two β sheets in a β sandwich fold containing a greek key motif  <cit> .
the physiological importance of both enzymes is evidenced by the severe presentation of α-naga and α-gal deficiencies in humans  <cit> .
our recent study on degradation of blood group a and b glycolipids in fabry cells indicated a high residual activity in fabry cells toward natural substrate glycolipid b-6- <dig>  <cit>  although α-galactosidase activity was completely absent when measured in vitro by routine procedures using artificial substrates.
we proposed that another enzyme, different from α-gal, contributes in vivo to hydrolysis of α-galactosides.
we suggested α-naga as the most likely candidate.
human α-naga is known to accept α-galactosides albeit with a high km  <cit> .
its activity must be inhibited when measuring α-gal in tissues with high α-naga activity  <cit> .
we investigated the phylogenesis of a single c. elegans α-gal and α-naga ortholog  to both human genes.
we present evidence suggesting that this gene has indeed evolved from the α-gal/α-naga ancestral gene before the duplication event which resulted in separate α-naga and α-gal genes in higher metazoans.
we further performed structural analysis of the gana- <dig> 3d model acquired by homology modeling.
we determined the spatial and temporal expression of the gene in transgenic worms using a translational reporter and examined the effect of rna interference  as a first step in the possible use of c. elegans as a model organism for schindler and fabry diseases.
RESULTS
cdna amplification and sequencing
the complete c. elegans genome  <cit>  contains only one open reading frame, designated gana- <dig>  that has sequence similar to human genes encoding α-gal and α-naga.
similar results were obtained from searching the available c. briggsae genome sequence  <cit> .
the gana- <dig> gene consists of  <dig> exons and  <dig> introns and is annotated as an ortholog of human α-naga.
several est clones for this open reading frame  have been reported and open-reading-frame sequence tag  is present in the worfdb database  <cit> .
available public database data are in agreement with our findings.
we verified the gene structure by sequencing the pcr products from genomic dna and cdna .
the analyzed region spanned the entire coding region and the 3' and 5' untranslated regions .
the 5' utr sl <dig> element suggests that the gene is either the only gene transcribed from the promoter or is the first gene in an operon including gana- <dig> and the two predicted downstream genes r07b <dig>  and r07b <dig> .
although this region has not been reported as an operon, the physical distances between this cluster of three genes are suggestive of an operon  <cit> .
no alternative splicing was found using rt/pcr, a feature similar to both human and mouse orthologs.
rna editing was reported in the 3' utr of human α-gal, a finding that another group was unable to confirm  <cit> .
we noted no signs of rna editing in clones derived from the gana- <dig> cdna.
phylogenetic studies
we aligned gana- <dig> protein sequence with other melibiase family members  and constructed a phylogenetic tree .
the alignment showed a striking similarity of gana- <dig> to all other included sequences.
gana- <dig> had the highest sequence similarity with anopheles gambiae gal , the lowest similarity was observed with streptomyces avertimilis gal .
the results of our phylogenetic analysis are in accordance with generally accepted evolutionary concepts.
the analysis identified four main clades: animal nagas, animal gals, plant/lower organisms gals and the clade containing sequences of drosophila melanogaster, anopheles gambiae and caenorhabditis elegans.
the branch including c. elegans is positioned between higher animal gals and nagas and plant/lower organisms gals.
this position in the tree infers the evolutionary ancestrality of gana- <dig> gene to both animal gals and animal nagas.
however, this conclusion is not in complete agreement with the presence of pairs of genes in drosophila and anopheles genomes annotated as α-gals and α-nagas.
the presence of these genes in the caenorhabditis/drosophila/anopheles branch  could be due to low divergence of these sequences from a common ancestral gene or to independent gene duplication in the drosophila/anopheles ancestral organism.
it is also important to note that the phylogenetic analysis by maximum parsimony algorithm placed the caenorhabditis/drosophila/anopheles branch into the neighborhood of the animal nagas branch  <cit> .
in this case the computational algorithm probably favored the lower number of necessary sequence changes  between gana- <dig> and naga clade sequences.
in our opinion, the phylogenetic analysis provides evidence that the gana- <dig> evolved from a common ancestor of α-gal and α-naga enzymes.
however, the topology of the tree could also be explained by a loss of the second gene during the evolution of c. elegans.
in this case the enzyme found in c. elegans would probably be the ortholog of human α-naga and the lost gene would likely be the ortholog of human α-gal.
the likelihood of these two hypotheses depends on functional divergence of duplicated gene products and their dispensability for organism's metabolic pathways  <cit> .
homology modeling
the best squared root of mean square deviations value , found between gana- <dig> backbone atoms and the chicken α-naga template  <cit> , was  <dig>  Å. the structural model of the enzyme molecule has a two-domain structure .
domain i, which contains the predicted active site, adopts a  <dig> barrel structure which represents a common motif in many glycoside hydrolases.
less conserved is domain ii that has a β domain with β sandwich structure containing a greek key motif.
the active site pocket of gana- <dig> is formed by the same twelve amino acids   as in chicken α-naga.
this finding infers their identical function in catalytic reactions as described for chicken α-naga  <cit> .
d <dig> carboxyl starts the initial nucleophilic attack and d <dig> carboxylic oxygen serves as a subsequent donor and acceptor of the proton during the reaction cycle.
residues forming the "n-acetyl recognition loop" in the chicken α-naga  <cit>   have the closest contact with the n-acetyl moiety of the ligand.
these residues are completely conserved between human and chicken nagas, but in human α-gal serin  <dig> is replaced by glutamine and alanine  <dig> is replaced by leucine.
the replacements with bulkier residues apparently discriminate between α-gal and α-naga substrates.
while nagas can accommodate α-galactose and can have some α-gal activity, gals do not have α-naga activity and are not inhibited by n-acetylgalactosamine.
the corresponding residues of gana- <dig> in the naga recognition loop are s <dig> and a <dig> and are characteristic for nagas.
according to garman  <cit>  the key residue in the dimer interface in human α-gal is f <dig>  residues corresponding to this position in other orthologs can serve as predictive markers of the protein quartenary structure.
phenylalanine or tyrosine is present in enzymes that act as homodimers while glycin indicates a monomeric structure  <cit> .
the equivalent residue to human α-gal f <dig> in gana- <dig> is lysine at position  <dig> which is suggestive of homodimeric structure due to its sterical properties.
the homology modeling showed that a groove opposing k <dig> is formed by residues t <dig>  l <dig>  d <dig>  m <dig>  i <dig>  v <dig> and v <dig> of the other monomer unit of gana- <dig>  in the case of chicken α-naga these residues are equivalent to s <dig>  y <dig>  e <dig>  q <dig>  n <dig>  p <dig> and s <dig> .
biochemical studies
standard bacteria/nematode separation protocol previously used by other authors  <cit>  while evaluating lysosomal enzyme activites is based on sucrose flotation approach.
we avoided standard sucrose flotation of worms because we could not exclude unpredictable artifacts caused by this compound, which is known to induce artificial lysosomal storage in eukaryotic cells and to alter lysosomal gene expression at concentrations significantly lower  <cit>  than those used in flotation protocols.
we found both α-gal and α-naga enzymatic activities in the homogenates from c. elegans n <dig> strain using 4-methylumbelliferyl  substrates.
the α-naga activity was dominant over the α-gal activity.
the activity of α-naga measured at 37°c was  <dig> nmol.mg- <dig> h- <dig> with mu-α-n-acetylgalactosaminide compared to the activity of α-gal with mu-α-galactopyranoside of  <dig> nmol.mg- <dig> h- <dig> .
in the assay of α-gal, the degradation of the mu-α-galactopyranoside was inhibited up to 95% in the presence of n-acetyl-d-galactosamine , whereas in the presence of d-galactose  the degradation of the same substrate was inhibited up to 75%.
in the assay of α-naga, the degradation of the mu-α-n-acetylgalactosaminide was inhibited up to 97% by d-galnac and up to 90% by d-gal.
no inhibition of α-naga and α-gal activity by d-glucose was observed.
according to published observations in human enzymes, d-galnac has no inhibitory effect on α-gal activity.
on the other hand, human α-naga activity is inhibited by both, d-galnac and d-gal  <cit> .
the model of gana- <dig> predicts only one active site per monomer of the enzyme.
if the enzyme had activity toward both substrates, mu-α-d-galactopyranoside and mu-α-n-acetylgalactosaminide, it is to be expected that d-galnac and d-gal would inhibit both activities.
the strong inhibitory effect of d-galnac on the α-gal activity, which is not present in human α-gal, supports the hypothesis that c. elegans has only one enzyme with both α-gal and α-naga activities.
nevertheless, these experiments were not conducted with the pure enzyme and thus do not provide absolute proof of this hypothesis.
rna interference
rna interference assays directed against the whole coding region of gana- <dig>  employing combination of microinjection and feeding approaches, did not reveal any abnormal morphological phenotypes.
nevertheless, measurement of α-gal and α-naga activities in four different experiments showed a simultaneous decrease of both enzymatic activities in rnai-treated worms  as compared with control animals.
in all rnai experiments, both α-gal and α-naga activities decreased proportionally, usually by tens of percent of activity of appropriate controls.
the activity of the control enzyme  did not differ between the rnai-treated nematodes and controls .
this finding supports the specificity of gana- <dig> rnai.
the differences between individual experiments are not surprising due to the well-known variability in the efficiency of rnai  <cit> .
the results of rnai experiments further support the hypothesis that gana- <dig> has both enzymatic activities.
both enzymatic activities were lower in rnai-treated and control worms cultured on the bacterial strain ht <dig>  <cit>  compared to a n <dig> strain cultured on the op <dig> strain.
rnai previously provided sufficient level of inhibition of structural lysosomal proteins for development of abnormal phenotypes in the worm  <cit> ; however, it is apparently not efficient enough for lysosomal catalytic proteins.
expression of gana-1
to study the expression of gana- <dig> in c. elegans, we created transgenic worms with a reporter gene containing the entire coding region of gana- <dig> c-terminally tagged with green fluorescent protein  under the control of a  <dig> kb region of the gana- <dig> hypothetical promoter.
the presence of the gana-1::gfp transgene in the worms was confirmed on the level of genomic dna, cdna and protein.
however, no gfp signal was observed by fluorescence microscopy under the standard laboratory conditions.
as western blotting showed the presence of fusion protein of the expected size , we assumed that the absence of the gfp signal was caused by a ph-dependent quenching of gfp fluorescence, which has neutral to alkaline optimum   <cit> .
to study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product.
immunofluorescence detection of gfp fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression.
this transgene product was limited to body wall muscle cells   or found in a broader tissue distribution that included body wall muscle cells, intestinal cells and coelomocytes  .
this latter staining pattern is consistent with the gfp detection in nh4cl and concanamycin a  experiments  discussed below.
the expression of the transgene was observed in about 30% of the population which corresponded to usual expression efficiency of extrachromosomal array transgenes  <cit> .
the immunofluorescence staining protocol resulted in a significant decrease of inherent intestinal granular autofluorescence previously assigned to secondary lysosomes  <cit> .
the decrease of autofluorescence intensity together with its poorly defined emission spectra hampered co-localization study.
to confirm that the absence of the gfp signal was due to the quenching of fluorescence by low ph in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment  <cit>  to enhance the gfp emission.
soaking of gana-1::gfp transgenic animals in nh4cl or con a resulted in a distinct gfp signal in a vesicular compartment of endocytically active coelomocytes in a small proportion of worms .
the gfp signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used.
the first visible gfp signal was observed after  <dig> hour incubation in  <dig> mm nh4cl and within  <dig> hours of incubation in  <dig> nm con a. lower concentrations of both nh4cl and con a did not result in visible gfp signal even after prolonged incubation of up to  <dig> hours.
the reappearance of the gfp signal after treatment of the worms with compounds increasing the acidic compartment ph indirectly confirms lysosomal localization of the fusion protein.
the gfp signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining.
limited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies.
CONCLUSIONS
our findings showed that gana- <dig> is the only ortholog of human α-naga and α-gal in c. elegans.
based on phylogenetic and homology modeling analyses we speculate that gana- <dig> most probably developed from a hypothetical ancestral gene before the duplication event which gave rise to separate α-naga and α-gal genes.
we also speculate that gana- <dig> gene product has both α-naga and α-gal activities as detected in c. elegans homogenates.
importantly, both activities in the worm were inhibited by d-galactose and n-acetyl-d-galactosamine, which is a specific inhibitor of human α-naga and does not inhibit α-gal.
the gana-1::gfp fusion protein had a pattern of distribution that is compatible with lysosomal subcellular localization.
the lysosomal localization of the fusion protein was also supported by ph sensitive fluorescence of gfp that was detectable only after alkalization of the acidic cellular compartment.
not suprisingly, rnai of gana- <dig> yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states.
nevertheless, gana- <dig> rnai resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them.
it is possible that a deletion allele of gana- <dig> may provide more insight into the function of gana- <dig> and efforts are underway to isolate such alleles.
deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders.

 humans have two enzymes with α-galactosidase activity and an acidic ph optimum, α-n-acetylgalactosaminidase and α-galactosidase a. hereditary deficiency of each of the hydrolases causes a distinct lysosomal storage disorder in humans, schindler and fabry diseases, respectively. early studies suggested that both human enzymes were glycoforms with similar substrate specifities. purified enzymes had similar physical properties, including subunit molecular mass, homodimeric structure, and amino acid sequences. however, additional studies showed kinetic, structural, and immunologic differences proving that α-gal and α-naga were products of two different genes. the two genes differed in the number of exons and also in the number, placement, and orientation of alu repeats. exons – of the α-naga gene showed high similarity to the first six exons of α-gal gene. because of the remarkable amino acid identity and similarity between the two genes and the similar intron placement, wang and co-workers suggested that a duplication event occurred during the evolution of both enzymes. both enzymes belong to the glycoside hydrolase family clan d. glycoside hydrolase family clan d orthologs have been identified in a broad spectrum of prokaryotes and eukaryotes, including plants. members of the family have a highly similar active site and share the same reaction mechanism. the structures of chicken α-naga, human α-gal and rice α-gal have been determined by x-ray crystallography. chicken and human enzymes have a homodimeric quarternary structure whereas rice α-gal acts as a monomer. the monomer units are composed of two distinct domains. domain i contains the active site and adopts a barrel structure, a domain fold observed commonly in glycosidases. domain ii has eight antiparallel β strands, packed into two β sheets in a β sandwich fold containing a greek key motif. the physiological importance of both enzymes is evidenced by the severe presentation of α-naga and α-gal deficiencies in humans. our recent study on degradation of blood group a and b glycolipids in fabry cells indicated a high residual activity in fabry cells toward natural substrate glycolipid b-6- although α-galactosidase activity was completely absent when measured in vitro by routine procedures using artificial substrates. we proposed that another enzyme, different from α-gal, contributes in vivo to hydrolysis of α-galactosides. we suggested α-naga as the most likely candidate. human α-naga is known to accept α-galactosides albeit with a high km. its activity must be inhibited when measuring α-gal in tissues with high α-naga activity. we investigated the phylogenesis of a single c. elegans α-gal and α-naga ortholog to both human genes. we present evidence suggesting that this gene has indeed evolved from the α-gal/α-naga ancestral gene before the duplication event which resulted in separate α-naga and α-gal genes in higher metazoans. we further performed structural analysis of the gana- 3d model acquired by homology modeling. we determined the spatial and temporal expression of the gene in transgenic worms using a translational reporter and examined the effect of rna interference as a first step in the possible use of c. elegans as a model organism for schindler and fabry diseases.  cdna amplification and sequencing the complete c. elegans genome contains only one open reading frame, designated gana- that has sequence similar to human genes encoding α-gal and α-naga. similar results were obtained from searching the available c. briggsae genome sequence. the gana- gene consists of exons and introns and is annotated as an ortholog of human α-naga. several est clones for this open reading frame have been reported and open-reading-frame sequence tag is present in the worfdb database. available public database data are in agreement with our findings. we verified the gene structure by sequencing the pcr products from genomic dna and cdna. the analyzed region spanned the entire coding region and the 3' and 5' untranslated regions. the 5' utr sl element suggests that the gene is either the only gene transcribed from the promoter or is the first gene in an operon including gana- and the two predicted downstream genes r07b and r07b. although this region has not been reported as an operon, the physical distances between this cluster of three genes are suggestive of an operon. no alternative splicing was found using rt/pcr, a feature similar to both human and mouse orthologs. rna editing was reported in the 3' utr of human α-gal, a finding that another group was unable to confirm. we noted no signs of rna editing in clones derived from the gana- cdna. phylogenetic studies we aligned gana- protein sequence with other melibiase family members and constructed a phylogenetic tree. the alignment showed a striking similarity of gana- to all other included sequences. gana- had the highest sequence similarity with anopheles gambiae gal, the lowest similarity was observed with streptomyces avertimilis gal. the results of our phylogenetic analysis are in accordance with generally accepted evolutionary concepts. the analysis identified four main clades: animal nagas, animal gals, plant/lower organisms gals and the clade containing sequences of drosophila melanogaster, anopheles gambiae and caenorhabditis elegans. the branch including c. elegans is positioned between higher animal gals and nagas and plant/lower organisms gals. this position in the tree infers the evolutionary ancestrality of gana- gene to both animal gals and animal nagas. however, this conclusion is not in complete agreement with the presence of pairs of genes in drosophila and anopheles genomes annotated as α-gals and α-nagas. the presence of these genes in the caenorhabditis/drosophila/anopheles branch could be due to low divergence of these sequences from a common ancestral gene or to independent gene duplication in the drosophila/anopheles ancestral organism. it is also important to note that the phylogenetic analysis by maximum parsimony algorithm placed the caenorhabditis/drosophila/anopheles branch into the neighborhood of the animal nagas branch. in this case the computational algorithm probably favored the lower number of necessary sequence changes between gana- and naga clade sequences. in our opinion, the phylogenetic analysis provides evidence that the gana- evolved from a common ancestor of α-gal and α-naga enzymes. however, the topology of the tree could also be explained by a loss of the second gene during the evolution of c. elegans. in this case the enzyme found in c. elegans would probably be the ortholog of human α-naga and the lost gene would likely be the ortholog of human α-gal. the likelihood of these two hypotheses depends on functional divergence of duplicated gene products and their dispensability for organism's metabolic pathways. homology modeling the best squared root of mean square deviations value, found between gana- backbone atoms and the chicken α-naga template, was Å. the structural model of the enzyme molecule has a two-domain structure. domain i, which contains the predicted active site, adopts a barrel structure which represents a common motif in many glycoside hydrolases. less conserved is domain ii that has a β domain with β sandwich structure containing a greek key motif. the active site pocket of gana- is formed by the same twelve amino acids as in chicken α-naga. this finding infers their identical function in catalytic reactions as described for chicken α-naga. d carboxyl starts the initial nucleophilic attack and d carboxylic oxygen serves as a subsequent donor and acceptor of the proton during the reaction cycle. residues forming the "n-acetyl recognition loop" in the chicken α-naga have the closest contact with the n-acetyl moiety of the ligand. these residues are completely conserved between human and chicken nagas, but in human α-gal serin is replaced by glutamine and alanine is replaced by leucine. the replacements with bulkier residues apparently discriminate between α-gal and α-naga substrates. while nagas can accommodate α-galactose and can have some α-gal activity, gals do not have α-naga activity and are not inhibited by n-acetylgalactosamine. the corresponding residues of gana- in the naga recognition loop are s and a and are characteristic for nagas. according to garman the key residue in the dimer interface in human α-gal is f residues corresponding to this position in other orthologs can serve as predictive markers of the protein quartenary structure. phenylalanine or tyrosine is present in enzymes that act as homodimers while glycin indicates a monomeric structure. the equivalent residue to human α-gal f in gana- is lysine at position which is suggestive of homodimeric structure due to its sterical properties. the homology modeling showed that a groove opposing k is formed by residues t l d m i v and v of the other monomer unit of gana- in the case of chicken α-naga these residues are equivalent to s y e q n p and s. biochemical studies standard bacteria/nematode separation protocol previously used by other authors while evaluating lysosomal enzyme activites is based on sucrose flotation approach. we avoided standard sucrose flotation of worms because we could not exclude unpredictable artifacts caused by this compound, which is known to induce artificial lysosomal storage in eukaryotic cells and to alter lysosomal gene expression at concentrations significantly lower than those used in flotation protocols. we found both α-gal and α-naga enzymatic activities in the homogenates from c. elegans n strain using 4-methylumbelliferyl substrates. the α-naga activity was dominant over the α-gal activity. the activity of α-naga measured at 37°c was nmol.mg- h- with mu-α-n-acetylgalactosaminide compared to the activity of α-gal with mu-α-galactopyranoside of nmol.mg- h-. in the assay of α-gal, the degradation of the mu-α-galactopyranoside was inhibited up to 95% in the presence of n-acetyl-d-galactosamine, whereas in the presence of d-galactose the degradation of the same substrate was inhibited up to 75%. in the assay of α-naga, the degradation of the mu-α-n-acetylgalactosaminide was inhibited up to 97% by d-galnac and up to 90% by d-gal. no inhibition of α-naga and α-gal activity by d-glucose was observed. according to published observations in human enzymes, d-galnac has no inhibitory effect on α-gal activity. on the other hand, human α-naga activity is inhibited by both, d-galnac and d-gal. the model of gana- predicts only one active site per monomer of the enzyme. if the enzyme had activity toward both substrates, mu-α-d-galactopyranoside and mu-α-n-acetylgalactosaminide, it is to be expected that d-galnac and d-gal would inhibit both activities. the strong inhibitory effect of d-galnac on the α-gal activity, which is not present in human α-gal, supports the hypothesis that c. elegans has only one enzyme with both α-gal and α-naga activities. nevertheless, these experiments were not conducted with the pure enzyme and thus do not provide absolute proof of this hypothesis. rna interference rna interference assays directed against the whole coding region of gana- employing combination of microinjection and feeding approaches, did not reveal any abnormal morphological phenotypes. nevertheless, measurement of α-gal and α-naga activities in four different experiments showed a simultaneous decrease of both enzymatic activities in rnai-treated worms as compared with control animals. in all rnai experiments, both α-gal and α-naga activities decreased proportionally, usually by tens of percent of activity of appropriate controls. the activity of the control enzyme did not differ between the rnai-treated nematodes and controls. this finding supports the specificity of gana- rnai. the differences between individual experiments are not surprising due to the well-known variability in the efficiency of rnai. the results of rnai experiments further support the hypothesis that gana- has both enzymatic activities. both enzymatic activities were lower in rnai-treated and control worms cultured on the bacterial strain ht compared to a n strain cultured on the op strain. rnai previously provided sufficient level of inhibition of structural lysosomal proteins for development of abnormal phenotypes in the worm; however, it is apparently not efficient enough for lysosomal catalytic proteins. expression of gana-1 to study the expression of gana- in c. elegans, we created transgenic worms with a reporter gene containing the entire coding region of gana- c-terminally tagged with green fluorescent protein under the control of a kb region of the gana- hypothetical promoter. the presence of the gana-1::gfp transgene in the worms was confirmed on the level of genomic dna, cdna and protein. however, no gfp signal was observed by fluorescence microscopy under the standard laboratory conditions. as western blotting showed the presence of fusion protein of the expected size, we assumed that the absence of the gfp signal was caused by a ph-dependent quenching of gfp fluorescence, which has neutral to alkaline optimum. to study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product. immunofluorescence detection of gfp fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression. this transgene product was limited to body wall muscle cells or found in a broader tissue distribution that included body wall muscle cells, intestinal cells and coelomocytes. this latter staining pattern is consistent with the gfp detection in nh4cl and concanamycin a experiments discussed below. the expression of the transgene was observed in about 30% of the population which corresponded to usual expression efficiency of extrachromosomal array transgenes. the immunofluorescence staining protocol resulted in a significant decrease of inherent intestinal granular autofluorescence previously assigned to secondary lysosomes. the decrease of autofluorescence intensity together with its poorly defined emission spectra hampered co-localization study. to confirm that the absence of the gfp signal was due to the quenching of fluorescence by low ph in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment to enhance the gfp emission. soaking of gana-1::gfp transgenic animals in nh4cl or con a resulted in a distinct gfp signal in a vesicular compartment of endocytically active coelomocytes in a small proportion of worms. the gfp signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used. the first visible gfp signal was observed after hour incubation in mm nh4cl and within hours of incubation in nm con a. lower concentrations of both nh4cl and con a did not result in visible gfp signal even after prolonged incubation of up to hours. the reappearance of the gfp signal after treatment of the worms with compounds increasing the acidic compartment ph indirectly confirms lysosomal localization of the fusion protein. the gfp signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining. limited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies.  our findings showed that gana- is the only ortholog of human α-naga and α-gal in c. elegans. based on phylogenetic and homology modeling analyses we speculate that gana- most probably developed from a hypothetical ancestral gene before the duplication event which gave rise to separate α-naga and α-gal genes. we also speculate that gana- gene product has both α-naga and α-gal activities as detected in c. elegans homogenates. importantly, both activities in the worm were inhibited by d-galactose and n-acetyl-d-galactosamine, which is a specific inhibitor of human α-naga and does not inhibit α-gal. the gana-1::gfp fusion protein had a pattern of distribution that is compatible with lysosomal subcellular localization. the lysosomal localization of the fusion protein was also supported by ph sensitive fluorescence of gfp that was detectable only after alkalization of the acidic cellular compartment. not suprisingly, rnai of gana- yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states. nevertheless, gana- rnai resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them. it is possible that a deletion allele of gana- may provide more insight into the function of gana- and efforts are underway to isolate such alleles. deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders. 
both α-gal and α-naga enzymatic activities were detected in c. elegans mixed culture homogenates. phylogenetic analyses and homology modeling of gana- based on the 3d structure of chicken α-naga, rice α-gal and human α-gal suggest a close evolutionary relationship of gana- to both human α-gal and α-naga. immunofluorescence detection of the fusion protein using polyclonal anti-gfp antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes. the aim of this study was to identify and characterize c. elegans orthologs to both human lysosomal luminal proteins α-gal and α-naga. blastp searches for orthologs of human α-gal and α-naga revealed a single c. elegans gene with homology to both human genes – gana- we cloned and sequenced the complete gana- cdna and elucidated the gene organization. gana- is a single c. elegans ortholog of both human α-gal and α-naga proteins. the gfp signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with concanamycin a or nh4cl, agents that increase the ph of the cellular acidic compartment. human α-galactosidase a and α-n-acetylgalactosaminidase are presumed to share a common ancestor. a gana-1::gfp fusion protein expressed from a transgene, containing the complete gana- coding region and kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. however, α-gal activity on an artificial substrate was completely inhibited by the α-naga inhibitor, n-acetyl-d-galactosamine. inhibition of gana- by rna interference resulted in a decrease of both α-gal and α-naga activities measured in mixed stage culture homogenates but did not cause any obvious phenotype. caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with lsds. phylogenetic, homology modeling, biochemical and gfp expression analyses support the hypothesis that gana- has dual enzymatic activity and is localized in an acidic cellular compartment.
 human α-galactosidase a and α-n-acetylgalactosaminidase are presumed to share a common ancestor. deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders – fabry and schindler diseases. caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with lsds. the aim of this study was to identify and characterize c. elegans orthologs to both human lysosomal luminal proteins α-gal and α-naga.  blastp searches for orthologs of human α-gal and α-naga revealed a single c. elegans gene with homology to both human genes – gana- we cloned and sequenced the complete gana- cdna and elucidated the gene organization. phylogenetic analyses and homology modeling of gana- based on the 3d structure of chicken α-naga, rice α-gal and human α-gal suggest a close evolutionary relationship of gana- to both human α-gal and α-naga. both α-gal and α-naga enzymatic activities were detected in c. elegans mixed culture homogenates. however, α-gal activity on an artificial substrate was completely inhibited by the α-naga inhibitor, n-acetyl-d-galactosamine. a gana-1::gfp fusion protein expressed from a transgene, containing the complete gana- coding region and kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. the gfp signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with concanamycin a or nh4cl, agents that increase the ph of the cellular acidic compartment. immunofluorescence detection of the fusion protein using polyclonal anti-gfp antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes. inhibition of gana- by rna interference resulted in a decrease of both α-gal and α-naga activities measured in mixed stage culture homogenates but did not cause any obvious phenotype.  gana- is a single c. elegans ortholog of both human α-gal and α-naga proteins. phylogenetic, homology modeling, biochemical and gfp expression analyses support the hypothesis that gana- has dual enzymatic activity and is localized in an acidic cellular compartment. 